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Here the feasibility of layers based on the conducting polymer polyaniline (PANI) as component of glucose biosensors using glucose oxidase (GOx) as enzyme and [Os(bpy)2(4-aminomethylpyridine)Cl]PF6 (OsCmplx) as electrochemical mediator, is evaluated. Particularly, PANI was employed to obtain a nanostructured macroporous material (m-PANI) around polystyrene nanoparticles taken as template and the mediator was co-immobilized during the polymerizing procedure. The GOx biosensor based on OsCmplx modified m-PANI provides a linear response to glucose concentration in the range 5 up to 65 mM with a sensitivity of 3.54 µA/mM/cm2 (on a projected geometric area=0.07 cm2), an LOD of 0.8 mM and a good precision (% RSD≤7, n=5); the biosensor is stable showing a decrease of 10% to the value of the sensitivity after 15 days of use and of about 50% after 40 days. [ABSTRACT FROM AUTHOR]

The reversible assembly of β-cyclodextrin-functionalizedgold NPs (β-CD Au NPs) is studied on mixed self-assembled monolayer(SAM), formed by coadsorptionof redox-active ferrocenylalkylthiols and n-alkanethiolson gold surfaces. The surface coverage and spatial distribution ofthe β-CD Au NPs monolayer on the gold substrate are tuned bythe self-assembled monolayer composition. The binding and releaseof β-CD Au NPs to and from the SAMs modified surface are followedby surface plasmon resonance (SPR) spectroscopy. The redox state ofthe tethered ferrocene in binary SAMs controls the formation of thesupramolecular interaction between ferrocene moieties and β-CD-cappedAu NPs. As a result, the potential-induced uptake and release of β-CDAu NPs to and from the surface is accomplished. The competitive bindingof β-CD Au NPs with guest molecules in solution shifted theequilibrium of the complexation–decomplexation process involvingthe supramolecular interaction with the Fc-functionalized surface.The dual controlled assembly of β-CD Au NPs on the surface enabledto use two stimuli as inputs for logic gate activation; the couplingbetween the localized surface plasmon, associated with the Au NP,and the surface plasmon wave, associated with the thin metal surface,is implemented as readout signal for “AND” logic gateoperations. [ABSTRACT FROM AUTHOR]

Self-assembled monolayers (SAMs) provide a convenient, flexible and simple system to tailor the interfacial properties of metals, metal oxides and semiconductors. Monomolecular films prepared by self-assembly are attractive for several exciting applications because of the unique possibility of making the selection of different types of terminal functional groups and as emerging tools for nanoscale observation of biological interactions. The tenability of SAMs as platforms for preparing biosurfaces is reviewed and critically discussed. The different immobilization approaches used for anchoring proteins to SAMs are considered as well as the nature of SAMs; particular emphasis is placed on the chemical specificity of protein attachment in view of preserving protein native structure necessary for its functionality. Regarding this aspect, particular attention is devoted to the relation between the immobilization process and the electrochemical response (i.e. electron transfer) of redox proteins, a field where SAMs have attracted remarkable attention as model systems for the design of electronic devices. Strategies for creating protein patterns on SAMs are also outlined, with an outlook on promising and challenging future directions for protein biochip research and applications. [ABSTRACT FROM AUTHOR]

Abstract: A novel approach towards a quick, easy and reliable solution to the quite complex problems connected with the Amadori''s compounds and furosine formation in several foodstuff is here proposed and based on enzymatic reactions, either as far as L-lysine release after partial hydrolysis from proteic material of the food matrix (bioavailable L-lysine), or as far as the determination of total and free L-lysine is concerned. The contribution proposes the use of specific L-lysine sensitive bioelectrodes for the evaluation of the possible heat damage through the rapid measure of L-lysine consumption. The L-lysine biosensor has been realized by immobilizing the enzyme L-lysine-α-oxidase (LOx) on an amperometric H2O2 sensitive electrode. The bioelectrode-enzymatic reaction arrangement allows to perform determinations of L-lysine and bioavailable L-lysine in foodstuff from different sources and after different thermal treatments in a very rapid inexpensive and reliable way in the range 1.0–33 ppm, with a limit of detection LOD=0.5 ppm, so that a considerable number of samples can be easily monitored quickly and with very low costs. [Copyright &y& Elsevier]

Microperoxidase-11 (MP-11) was first soft landed onto the gold surface of a screen-printed electrode. Intact protein deposition was verified by time-of-flight secondary ion mass spectrometry. The coupling of soft landing with electrochemical techniques allowed unique information to be obtained about the deposition features. A full characterization of the direct electron-transfer properties was performed by modeling data obtained from cyclic voltammetry experiments; calculated values of kinetic electron-transfer constant, formal redox potential, and reorganization energy allow us to hypothesize the mechanism involved in soft landing immobilization and demonstrate the different conformation of the enzyme deposited from two different charged species. The strong interaction between MP11 and the gold surface and long-term stability of the functionalized electrode characterizes the peculiar features of this procedure, which enhance its power with respect to the existing immobilization procedure and ensure its suitability for those practical applications that could benefit from an unmediated bridgeless bioeletrochemical electron transfer (e.g., biosensor transducers or electrode elements in biofuel cells). [ABSTRACT FROM AUTHOR]

Abstract: A novel bioelectrochemical method for the direct determination of D(−) L(+) lactic acid and of L(−) malic acid in wines is presented. Multienzymatic biosensors were realized for the selective determination of the three analytes: D(−) and L(+) lactic acid were measured by a trienzymatic biosensor based on the catalytic activities of the enzymes L(+) lactate oxidase (LOD), D(−) lactate dehydrogenase (D-LDH) and horseradish peroxidase (HRP); L(−) malic acid was measured by a bienzymatic electrode, realized by coupling the enzymes L(−) malic dehydrogenase (L-MDH) and horseradish peroxidase (HRP). In both cases the enzymes were immobilized on an oxygen selective Clark electrode. The simultaneous determination of the two organic acids can be accomplished either in batch or in a flow injection analysis apparatus using the same biosensors as detectors. The analytical performance of the method, tested in standard aqueous solutions and on real samples of wines, showing high repeatability, short response times and reduced cost of analysis, suggest that the experimental approach here described could be followed to monitor the progress of malolactic fermentation. [Copyright &y& Elsevier]

Abstract: This article presents a bioelectrochemical system for the determination of pesticides by enzyme inhibition data. Measurements are performed by electrochemically monitoring the inhibition of the catalytic activity of the enzyme alkaline phosphatase (ALP) either in the presence or in the absence of pesticides, and particularly of Malathion and of 2,4-dichlorophenoxyacetic acid (2,4-D), representatives of the general classes of organophosphorous and organochlorinated agents. ALP catalytic activity was determined by using two different analytical configurations: (a) an amperometric ALP based biosensors using 3-indoxyl phosphate as the enzyme substrate; (b) a system allowing the voltammetric determination of the electroactive products of the ALP catalyzed reactions by using two different substrates: phenyl phosphate (PP) and ascorbate-2-phosphate (A-2-P). Studies of cyclic voltammetry and amperometry were performed first to define the optimal experimental conditions of the electrochemical measurements. For the optimal experimental conditions (Tris–0.1 M HCl aqueous buffer solution at pH 8.0, incubation time of 30–60 min) a detection limit of 0.5–6 μgl−1 is obtained, with a response, which is linear over 1–2 decades of concentration. [Copyright &y& Elsevier]

This work aimed to develop an easy-to-use smartphone-based electrochemical biosensor to quickly assess a coffee blend's total polyphenols (Phs) content at the industrial and individual levels. The device is based on a commercial carbon-based screen-printed electrode (SPE) modified with multi-walled carbon nanotubes (CNTs) and gold nanoparticles (GNPs). At the same time, the biological recognition element, Laccase from Trametes versicolor, TvLac, was immobilized on the sensor surface by using glutaraldehyde (GA) as a cross-linking agent. The platform was electrochemically characterized to ascertain the influence of the SPE surface modification on its performance. The working electrode (WE) surface morphology characterization was obtained by scanning electron microscopy (SEM) and Fourier-transform infrared (FT-IR) imaging. All the measurements were carried out with a micro-potentiostat, the Sensit Smart by PalmSens, connected to a smartphone. The developed biosensor provided a sensitivity of 0.12 μA/μM, a linear response ranging from 5 to 70 μM, and a lower detection limit (LOD) of 2.99 μM. Afterward, the biosensor was tested for quantifying the total Phs content in coffee blends, evaluating the influence of both the variety and the roasting degree. The smartphone-based electrochemical biosensor's performance was validated through the Folin–Ciocâlteu standard method. [ABSTRACT FROM AUTHOR]

A catalase-based (NAF/MWCNTs) nanocomposite film modified glassy carbon electrode for hydrogen peroxide (H2O2) detection was developed. The developed biosensor was characterized in terms of its bioelectrochemical properties. Cyclic voltammetry (CV) technique was employed to study the redox features of the enzyme in the absence and in the presence of nanomaterials dispersed in Nafion® polymeric solution. The electron transfer coefficient, α, and the electron transfer rate constant, ks, were found to be 0.42 and 1.71 s−1, at pH 7.0, respectively. Subsequently, the same modification steps were applied to mesoporous graphite screen-printed electrodes. Also, these electrodes were characterized in terms of their main electrochemical and kinetic parameters. The biosensor performances improved considerably after modification with nanomaterials. Moreover, the association of Nafion with carbon nanotubes retained the biological activity of the redox protein. The enzyme electrode response was linear in the range 2.5–1150 μmol L−1, with LOD of 0.83 μmol L−1. From the experimental data, we can assess the possibility of using the modified biosensor as a useful tool for H2O2 determination in packaged beverages. [ABSTRACT FROM AUTHOR]

The aim of this work is to show how the integration of gold nanoparticles (AuNPs) into multi-wall-carbon-nanotubes (MWCNTs) based screen-printed electrodes and into graphene-based screen-printed electrodes (GPHs) could represent a potential way to further enhance the electrochemical properties of those electrodes based on nanoparticles. Laccase from Trametes versicolor (TvL) was immobilized over MWCNTs and GPH previously modified with AuNPs (of 5 and 10 nm). The characterization of the modified electrode surface has been carried out by cyclic voltammetry. The results showed that the use of AuNPs for modification of both graphene and MWCNTs screen-printed electrode surfaces would increase the electrochemical performances of the electrodes. MWCNTs showed better results than GPH in terms of higher electroactive area formation after modification with AuNPs. The two modified nanostructured electrodes were successively proven to efficiently immobilize the TvL; the electrochemical sensing properties of the GPH- and MWCNT-based AuNPs-TvL biosensors were investigated by choosing 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic-acid diammonium salt (ABTS), catechol and caffeic acid as laccase mediators; and the kinetic parameters of the laccase biosensor were carefully evaluated. [ABSTRACT FROM AUTHOR]

This work presents the realization of a label-free electrochemical immunosensor for the quick, cheap, and straightforward determination of atrazine. This biodevice is based on developing a technological platform where a gold screen printed electrode (Au-SPE) surface was modified by the electrodeposition of a highly porous gold layer. As an internal probe redox, a Prussian Blue thin layer (PB) was then electrosynthetized onto the modified Au-SPE. Atrazine antibody (Ab-ATZ) was immobilized using G protein-functionalized magnetic nanoparticles (MNPs@protG) to ensure the correct orientation of the antibody to enhance the immunoaffinity. Under optimum experimental conditions, the electrochemical characterization of the developed immunosensor displays a linearity range towards atrazine within 0.05–1.5 ng/mL, a LOD of 0.011 ng/mL good reproducibility and stability. The immunosensor was tested in the analysis of spiked drinking water samples with a mean recovery ranging from 95.7 to 108.4%. The overall good analytical performances of this immunodevice suggest its application for the screening and monitoring of atrazine in real matrices. [ABSTRACT FROM AUTHOR]

In this work data are reported on the possibility for detection of environmental contaminants endowed with toxic effects by specifically designed electrochemical biosensors. A new bienzymatic inhibition biosensor based on the combined catalytic activity of the enzymes alkaline phosphatase and glucose oxidase is proposed and discussed for the direct determination of 2,4-dichlorophenoxyacetic acid (2,4-D), one of the most powerful and diffused defoliants, which also is endowed with estrogenic properties. A discussion on the different approaches followed to manage the experimental data obtained by enzymatic inhibition biosensors is also presented. [ABSTRACT FROM AUTHOR]

In this study, the ability of an unmodified ESI Z-spray source to carry out ambient ion soft landing (AISL) and microdroplet reactions (MR) was tested. To this aim, heme proteins were ionized and landed onto the working area of multi-walled carbon nanotubes electrodes. The voltammetric and spectroscopic analyses of the modified electrodes evidenced a strong interaction of unfolded structures of the heme proteins with the carbon nanotubes surface. Moreover, the possibility to use the ESI Z-spray source to study microdroplet reactions was attempted by exploring the accelerated acid-catalyzed decomposition of carbohydrates. The study demonstrated that a commercial ESI Z-spray source can provide successful modifications of surfaces by AISL, and represents a suitable platform for the study of accelerated microdroplet reactions. [Display omitted] • Ions and microdroplets deposition by using a commercial ESI Z-spray source. • Ambient ion soft landing of redox heme proteins. • Accelerated carbohydrate dehydration reaction in charged microdroplets. [ABSTRACT FROM AUTHOR]

Calix[n]arenes, as the 'third-generation' host molecules, are cyclic oligomer, featuring a unique three-dimensional structure with a hydrophobic cavity, allowing the interaction with many size-matched guest molecules, and a surface which can be functionalised at both the upper and lower rims with several functional groups to tailor the recognition properties towards a specific class of analytes for various applications. In the last two decades, due to their configuration, unique molecule recognition function and aggregation properties, calixarenes and their derivatives have attracted considerable attention as a new versatile scaffold for highly sensitive detection and site-oriented immobilisation in biosensor development. In this review, the development of sensors featuring calixarene-based transducers tuned for binding many analytes, ranging from simple (i.e., amino acids and neurotransmitters) to complex (i.e., enzymes and antibodies) organic molecules, was described. The rational design, the synthetic strategies fine-tuned to prepare these complex structures and their grafting in surface modification were included. [ABSTRACT FROM AUTHOR]

One of the main problems in the development of immunosensors is to overcome the complexity of binding antibodies to the sensor surface. Most immobilizing methods lead to a random orientation of antibodies with a lower binding site density and immunoaffinity. In order to control the orientation of antibody immobilization, several resorc[4]arene derivatives were designed and synthesized. After the spectroscopic characterization of resorc[4]arene self‐assembled monolayers (SAMs) onto gold films, the surface coverage and the orientation of insulin antibody (Ab‐Ins) were assessed by a surface plasmon resonance (SPR) technique and compared with a random immobilization method. Experimental results combined with theoretical studies confirmed the dipole–dipole interaction as an important factor in antibody orientation and demonstrated the importance of the upper rim functionalization of resorcarenes. Accordingly, resorcarene 5 showed a major binding force towards Ab‐Ins thanks to the H‐bond interactions with the amine protein groups. Based on these findings, the resorcarene‐based immunosensor is a powerful system with improved sensitivity providing new insight into sensor development. [ABSTRACT FROM AUTHOR]

• It is proposed the poly(vinyl alcohol) for punctual and in-situ EIS analysis. • An electrochemical cell is built in order to place the self-consistent disk of PVA. • The conductivity of PVA is tested. • EIS spectra are obtained employing PVA instead of traditional solution. • PVA polymer electrolyte membranes perform likely electrolytic solutions. The development of an electrochemical cell based on poly(vinyl alcohol) (PVA) hydrogel as polymer electrolyte for electrochemical impedance spectroscopy analysis (EIS) is discussed. PVA hydrogel is prepared with different cycles of freeze/thawing (F/T) employing distillated and mineral water as solutions. The PVA-disks obtained are compared for their conductivities in order to employ it in an appropriate cell for diagnostic purposes, reducing the invasiveness of the analysis on the sample. The test of the self-consistent disk of PVA is carried out on a modern sample of copper and on an archaeological bronze coin, obtaining an alternative of the classical electrochemical cell for EIS measurements on ancient metals. [ABSTRACT FROM AUTHOR]

In this study, polythiophene copolymers have been used as modifier for electrode surfaces in order to allow the immobilization of active pyrroloquinoline quinone dependent glucose dehydrogenase (PQQ-GDH) and to simultaneously improve the direct electrical connection of the enzyme with the electrode. Polymer films are electrosynthesized in aqueous solution without the need of surfactants onto carbon nanotubes modified gold electrodes from mixtures of 3-thiopheneacetic acid (ThCH 2 CO 2 H) and 3-methoxythiophene (ThOCH 3 ) using a potentiostatic pulse method. Polythiophene deposition significantly improves the bioelectrocatalysis of PQQ-GDH: the process starts at − 200 mV vs. Ag/AgCl and allows well-defined glucose detection at 0 V vs. Ag/AgCl with high current density. Several parameters of the electro-polymerization method have been evaluated to maximize the anodic current output after enzyme coupling. The polymer deposited by this new procedure has been morphologically and chemically characterized by different methods (SEM, EDX, FT-IR, UV–Vis, XPS and Raman spectroscopy). The bioelectrocatalytic response towards increasing glucose concentrations exhibits a dynamic range extending from 1 μM to 2 mM. The low applied potential allows to avoid interferences from easily oxidizable substances such as uric acid and ascorbic acid. Short and long-term stability has been evaluated. Finally, the PQQ-GDH electrode has been coupled to a bilirubin oxidase (BOD)- and carbon nanotube-based cathode in order to test its performance as anode of a biofuel cell. The promising results suggest a further investigation of this kind of polymers and, in particular, the study of the interaction with other enzymes in order to employ them in building up biosensors and biofuel cells. [ABSTRACT FROM AUTHOR]

This paper presents a new direct electron transfer based-miniaturized glucose/oxygen enzymatic fuel cell (EFC) whose operating ability has been tested in real saliva samples. The bioanode and biocathode are a graphene working electrode and a graphite counter electrode localized on the same screen printed electrode (SPE) modified with poly(vinyl alcohol) N -methyl-4(4′-formylstyryl)pyridinium methosulfate acetal (PVA-SbQ)/cellobiose dehydrogenase from Corynascus Thermophilus ( Ct CDH) C291Y/AuNPs and with Trametes Hirsuta laccase ( Th Lac)/AuNPs, respectively. In order to optimize the bioanode, several CDH immobilization procedures were adopted, such as drop-casting, use of Nafion membrane or PVA-SbQ photopolymer. The photopolymer showed the best performance in terms of stability and reliability. As biocathode a partially optimized laccase electrode was employed with the variant that the used nanomaterials allowed to reduce the overpotential of O 2 /H 2 O redox reaction catalyzed by Trametes Hirsuta Laccase ( Th Lac), drop-casted onto the gold nanoparticles (AuNPs) modified SPE. The performances of bioanode and biocathode were tested separately, initially immobilizing the two enzymes onto separated graphene SPEs. An efficient direct electron transfer was achieved for both elements, obtaining an apparent heterogeneous electron transfer rate constant ( k s ) of 0.99 ± 0.05 s −1 for Ct CDH C291Y and 5.60 ± 0.05 s −1 for Th Lac. Both electrodes were then assembled in a two compartment EFC obtaining a maximal power output of 5.16 ± 0.15 μW cm −2 at a cell voltage of 0.58 V and an open circuit voltage (OCV) of 0.74 V. Successively, the bioanode and biocathode were assembled in a non-compartmentalized EFC and a remarkable 50% decrease of the maximum power output at the value of 2.15 ± 0.12 μW cm −2 at cell voltage of 0.48 V and an OCV of 0.62 V at pH 6.5 was registered. In order to reduce the cell dimensions in view of its possible integration in biomedical devices, the bioanode and biocaythode were realized by immobilization of both enzymes onto the same SPE. The so miniaturized EFC delivered a maximal power output of 1.57 ± 0.07 μW cm 2 and 1.10 ± 0.12 μW cm −2 with an OCV of 0.58 V and 0.41 V in a 100 μM glucose solution and in human saliva, respectively. [ABSTRACT FROM AUTHOR]

The immobilization procedure is one of the key factors affecting the electron transfer based biosensors performance. It must assure both the enzyme retention activity and an efficient communication between the redox center of the enzyme and the electrode surface. In this regard, hydrogels are attractive materials due to their three-dimensional hydrophilic networks and their high-water concentration, promoting the biomolecule long-term stability and providing a suitable scaffold for trapping. To this aim, we have synthesized a gelling tripeptide Fluorenylmethyloxycarbonyl-triphenylalanine (FmocPhe 3 ) produced starting from Fmoc-phenylalanine and diphenylalanine by the catalytic activity of Lipase in water and characterized its performance as a new immobilization support for biosensors. For this purpose, we have evaluated the main bioelectrochemical properties of Trametes versicolor Laccase (TvL) immobilized within the hydrogel and a hydrogel-nanocomposite structure in the presence of gold nanoparticles (AuNPs). Either the Fmoc and the benzyl rings inside the tripeptide structure contributed to the hydrogel network formation by π-π stacking interactions, which promoted the physical entrapment of Laccase as well as the interaction with the carbon-based electrode surface. Furthermore, the π-electrons flow throughout the tripeptide based hydrogel allowed a good electron transfer between the immobilized enzyme and the electrode. The obtained results showed a significative increase in the hydrogel nanocomposite-based graphite biosensor performance with respect to those obtained with the hydrogel-based graphite biosensor. The experimental results, in terms of analytical performance and costs, assessed the possible use of FmocPhe 3 based hydrogels in the development of electrochemical biosensors. [ABSTRACT FROM AUTHOR]

In this work, we have developed for the first time a method to make novel gold and platinum hybrid bimetallic nanostructures differing in shape and size. Au-Pt nanostructures were prepared by electrodeposition in two simple steps. The first step consists of the electrodeposition of nanocoral Au onto a gold substrate using hydrogen as a dynamic template in an ammonium chloride solution. After that, the Pt nanostructures were deposited onto the nanocoral Au organized in pores. Using Pt (II) and Pt (IV), we realized nanocoral Au decorated with Pt nanospheres and nanocoral Au decorated with Pt nanoflowers, respectively. The bimetallic nanostructures showed better capability to electrochemically oxidize hydrogen peroxide compared with nanocoral Au. Moreover, Au-Pt nanostructures were able to lower the potential of detection and a higher performance was obtained at a low applied potential. Then, glucose oxidase was immobilized onto the bimetallic Au-Pt nanostructure using cross-linking with glutaraldehyde. The biosensor was characterized by chronoamperometry at +0.15 V vs. Ag pseudo-reference electrode (PRE) and showed good analytical performances with a linear range from 0.01 to 2.00 mM and a sensitivity of 33.66 µA/mM cm 2 . The good value of K m app (2.28 mM) demonstrates that the hybrid nanostructure is a favorable environment for the enzyme. Moreover, the low working potential can minimize the interference from ascorbic acid and uric acid as well as reducing power consumption to effect sensing. The simple procedure to realize this nanostructure and to immobilize enzymes, as well as the analytical performances of the resulting devices, encourage the use of this technology for the development of biosensors for clinical analysis. [ABSTRACT FROM AUTHOR]

The establishment of a polythiophene-supported electron transfer of PQQ-dependent glucose dehydrogenase (PQQ-GDH) at multiwalled carbon nanotubes is reported. For this purpose, thiol-functionalized MWCNTs are deposited on a gold electrode, which is further modified by on-top electropolymerization of different thiophene monomers. The enzyme is covalently bound to such an electrode by activating the carboxy groups of the polymer. The presence of the polythiophene copolymers allows for electrochemical wiring of PQQ-GDH that can subsequently transfer the electrons from the glucose oxidation to the electrode. Bioelectrocatalysis starts just at −0.2 V vs. Ag/AgCl and the anodic current reaches high values already at 0 V vs. Ag/AgCl. In order to improve the catalytic response, different parameters in the electropolymerization process are evaluated and buffer effects are investigated. The modified electrode surface is characterized by SEM-EDX, FTIR, UV–vis and XPS. The bioelectrocatalytic response towards increasing glucose concentrations is measured at 0 V vs. Ag/AgCl, showing a dynamic range extending from 1 μM to 500 μM. [ABSTRACT FROM AUTHOR]

This work reports the first employment of a gel polymer-based electrochemical cell to perform electrochemical impedance analysis of archaeological remains. The patinas of three Roman coins are studied through of electrochemical techniques (especially Electrochemical Impedance Spectroscopy, EIS) along with Electron Scanning Microscopy (SEM-EDS) and X-ray diffraction (XRD), in order to validate the results obtained with jellified electrolyte. SEM-EDS and XRD analyses reveal the composition of the patinas suggesting that the original alloys were made of Cu and Cu–Sn–Pb. Furthermore, EIS results make one able to assess some characteristics of coins, as for instance, the presence of a noble patina . This outcome is corroborated by the SEM-EDS and XRD analysis, suggesting that the method based on the jellified electrolyte on archaeological samples can be employed. Moreover, supporting studies are carried out using Voltammetry of Microparticles (VMP), which is able to detect the presence of some specific corrosion products (cuprite). So far, EIS is not a common technique in the Cultural Heritage field despite it can provide useful information about the conditions of conservation, i.e., corrosion processes. Here we can show the advantages of developing a gel-based system for EIS measurement which allows carrying out a punctual and quick analysis on the ancient bronze roman coins. [ABSTRACT FROM AUTHOR]

In this work, we developed an impedimetric label-free immunosensor for the detection of 2,4-Dichlorophenoxy Acetic Acid (2,4-D) herbicide either in standard solution and spiked real samples. For this purpose, we prepared by electropolymerization a conductive polymer poly-(aniline- co -3-aminobenzoic acid) (PANABA) then we immobilized anti-2,4-D antibody onto a nanocomposite AuNPs-PANABA-MWCNTs employing the carboxylic moieties as anchor sites. The nanocomposite was synthesized by electrochemical polymerization of aniline and 3-aminobenzoic acid, in the presence of a dispersion of gold nanoparticles, onto a multi-walled carbon nanotubes-based screen printed electrode. Aniline-based copolymer, modified with the nanomaterials, allowed to enhance the electrode conductivity thus obtaining a more sensitive antigen detection. The impedimetric measurements were carried out by electrochemical impedance spectroscopy (EIS) in faradic condition by using Fe(CN) 6 3-/4- as redox probe. The developed impedimetric immunosensor displayed a wide linearity range towards 2,4-D (1–100 ppb), good repeatability (RSD 6%), stability and a LOD (0.3 ppb) lower than herbicide emission limits. [ABSTRACT FROM AUTHOR]

In this research, we developed a direct-flow surface plasmon resonance (SPR) immunosensor for ampicillin to perform direct, simple, and fast measurements of this important antibiotic. In order to better evaluate the performance, it was compared with a conventional amperometric immunosensor, working with a competitive format with the aim of finding out experimental real advantages and disadvantages of two respective methods. Results showed that certain analytical features of the new SPR immunodevice, such as the lower limit of detection (LOD) value and the width of the linear range, are poorer than those of a conventional amperometric immunosensor, which adversely affects the application to samples such as natural waters. On the other hand, the SPR immunosensor was more selective to ampicillin, and measurements were more easily and quickly attained compared to those performed with the conventional competitive immunosensor. [ABSTRACT FROM AUTHOR]

In this paper we report on a facile, cost effective and environmental friendly green synthesis method of gold and silver nanoparticles (NPs) by using quercetin as reducing agent. The obtained NPs were characterized by transmission electron microscopy (TEM), energy dispersive spectroscopy (EDS), dynamic light scattering (DLS) and UV-Vis spectroscopy and parameters such as pH, ionic strength and temperature, effectively affecting shape and size of NPs, have been carefully studied and optimized. The obtained results showed that the synthesized NPs were circular in shape with an average diameter of 5 and 8 nm for the AuNPs and the AgNPs, respectively. The 'green' NPs, showing increased electroactive areas (AEA) and electronic transfer rate constants ( k 0), were successively used to fabricate a novel third generation lactose biosensor based on cellobiose dehydrogenase from Trametes villosa ( TvCDH). The TvCDH/AuNPs based lactose biosensor revealed the best results showing very efficient DET and a detection limit for lactose of 3.5 mM, a large linear range from 10 to 300 mM, a high sensitivity (5.4 μA mM−1 cm−2) and long-term stability. [ABSTRACT FROM AUTHOR]

We report a new sensitive label-free electrochemical immunosensor to detect Vitamin D 3 (25-OHD 3) in untreated serum samples. To this aim, a graphite screen printed electrode (SPE) was modified using cysteamine (CYM) functionalized core-shell magnetic nanoparticles (Au@MNPs) then, the 25-OHD 3 antibody (AbD) was immobilized via glutaraldehyde crosslinking. The several steps involved in the immunosensor development and 25-OHD 3 analysis were monitored by using differential pulse voltammetry (DPV). The developed immunosensor showed a LOD of 2.4 ng mL−1 and a linear range between 7.4 and 70 ng mL−1. The effectiveness of the immunosensor in human serum analysis was assessed by comparing the results obtained with the chemiluminescence-immunoassay (CLIA) reference method. The high sensitivity and excellent agreement with the reference method suggest its potential use as a POCT to monitor hypovitaminosis 25-OHD levels. Scheme of assembly of the Au@MNPs-based 25(OH)D3 immunosensor. [Display omitted] •Cysteamine (CYM) functionalization of gold magnetic nanoparticles (AuMNPs) for the modification of graphite-SPE electrodes. •Development of site-directed antibody immobilization-based electrochemical immunosensor for quick and simple detection of Vitamin D 3. •Optimized nanostructured-based electrochemical immunosensor for Vitamin D 3 detection as POCT for fast hypovitaminosis D diagnosis. [ABSTRACT FROM AUTHOR]

Au nanocorals are grown on gold screen-printed electrodes (SPEs) by using a novel and simple one-step electrodeposition process. Scanning electron microscopy was used for the morphological characterization. The devices were assembled on a three-electrode SPE system, which is flexible and mass producible. The electroactive surface area, determined by cyclic voltammetry in sulphuric acid, was found to be 0.07 ± 0.01 cm 2 and 35.3 ± 2.7 cm 2 for bare Au and nanocoral Au, respectively. The nanocoral modified SPEs were used to develop an enzymatic glucose biosensor based on H 2 O 2 detection. Au nanocoral electrodes showed a higher sensitivity of 48.3 ± 0.9 μA/(mM cm 2 ) at + 0.45 V vs Ag | AgCl compared to a value of 24.6 ± 1.3 μA/(mM cm 2 ) at + 0.70 V vs Ag | AgCl obtained with bare Au electrodes. However, the modified electrodes have indeed proven to be extremely powerful for the direct detection of glucose with a non-enzymatic approach. The results confirmed a clear peak observed by using nanocoral Au electrode even in the presence of chloride ions at physiological concentration. Amperometric study carried out at + 0.15 V vs Ag | AgCl in the presence of 0.12 M NaCl showed a linear range for glucose between 0.1 and 13 mM. [ABSTRACT FROM AUTHOR]

In the last decades, in vitro diagnostic devices (IVDDs) became a very important tool in medicine for an early and correct diagnosis, a proper screening of targeted population, and also assessing the efficiency of a specific therapy. In this review, the most recent developments regarding different configurations of surface plasmon resonance affinity biosensors modified by using several nanostructured materials for in vitro diagnostics are critically discussed. Both assembly and performances of the IVDDs tested in biological samples are reported and compared. [ABSTRACT FROM AUTHOR]

This review presents recent advances in Au and Pt nanostructured-based electrochemical biosensors belonging to the third generation. We will list different routes to modify electrodes with Au and Pt nanoparticles and nanoporous films in more detail. Also, the advantages of such nanostructured electrodes on the electron transfer properties of hemoglobin, laccase, cytochrome c and peroxidase will be summarized. Future perspectives in this emerging field will be discussed in the conclusion part. [ABSTRACT FROM AUTHOR]

In this work, several theoretical aspects involved in the first-generation inhibition-based electrochemical biosensor measurements have been discussed. In particular, we have developed a theoretical-methodological approach for the characterization of the kinetic interaction between alkaline phosphatase (AlP) and 2,4-dichlorophenoxy acetic acid (2,4-D) as representative inhibitor studied by means of cyclic voltammetry and amperometry. Based on these findings, a biosensor for the fast, simple, and inexpensive determination of 2,4-D has been developed. The enzyme has been immobilized on screen-printed electrodes (SPEs). To optimize the biosensor performances, several carbon-based SPEs, namely graphite (G), graphene (GP), and multiwalled carbon nanotubes (MWCNTs), have been evaluated. AlP was immobilized on the electrode surface by means of polyvinyl alcohol with styryl-pyridinium groups (PVA-SbQ) as cross-linking agent. In the presence of ascorbate 2-phosphate (A2P) as substrate, the herbicide has been determined, thanks to its inhibition activity towards the enzyme catalyzing the oxidation of A2P to ascorbic acid (AA). Under optimum experimental conditions, the best performance in terms of catalytic efficiency has been demonstrated by MWCNTs SPE-based biosensor. The inhibition biosensor shows a linearity range towards 2,4-D within 2.1-110 ppb, a LOD of 1 ppb, and acceptable repeatability and stability. This analysis method was applied to fortified lake water samples with recoveries above 90 %. The low cost of this device and its good analytical performances suggest its application for the screening and monitoring of 2,4-D in real matrices. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

This work presents an inhibition-based biosensor for the fast, simple and inexpensive determination of atrazine. The method is based on the inhibition of the enzyme tyrosinase from mushrooms (Tyr), immobilized on screen printed electrodes (SPEs). To optimize the biosensor performances several carbon based SPEs: graphite (G), graphene (GP), and multiwalled carbon nanotubes (MWCNTs) and immobilization techniques (physical and chemical) have been tested. Tyrosinase was immobilized on the electrode surface by either polyvinyl alcohol with styrylpyridinium groups (PVA-SbQ) as cross-linking agent or Nafion membranes as physical entrapment or bovine serum albumin with glutaraldehyde as chemical immobilization. In the presence of catechol as substrate, atrazine can be determined thanks to its inhibition activity towards the enzyme which catalyzes the oxidation of catechol to o-quinone. Under optimum experimental conditions, the best performance in terms of catalytic efficiency has been demonstrated by MWCNTs screen printed electrode with PVA-SbQ as immobilization method. The developed inhibition biosensor displays a linearity range towards atrazine within 0.5–20 ppm, a LOD of 0.3 ppm and acceptable repeatability and stability. This analysis method was applied to spiked drinking water samples with recoveries close to 95% respect to measurements carried out in PBS buffer. The low cost of this device and its good analytical performances suggest its application for the screening and monitoring of atrazine in real matrices. [ABSTRACT FROM AUTHOR]

Novel methods to obtain Pt nanostructured electrodes have raised particular interest due to their high performance in electrochemistry. Several nanostructuration methods proposed in the literature use costly and bulky equipment or are time-consuming due to the numerous steps they involve. Here, Pt nanostructures were produced for the first time by one-step template-free electrodeposition on Pt bare electrodes. The change in size and shape of the nanostructures is proven to be dependent on the deposition parameters and on the ratio between sulphuric acid and chloride-complexes (i.e., hexachloroplatinate or tetrachloroplatinate). To further improve the electrochemical properties of electrodes, depositions of Pt nanostructures on previously synthesised Pt nanostructures are also performed. The electroactive surface areas exhibit a two order of magnitude improvement when Pt nanostructures with the smallest size are used. All the biosensors based on Pt nanostructures and immobilised glucose oxidase display higher sensitivity as compared to bare Pt electrodes. Pt nanostructures retained an excellent electrocatalytic activity towards the direct oxidation of glucose. Finally, the nanodeposits were proven to be an excellent solid contact for ion measurements, significantly improving the time-stability of the potential. The use of these new nanostructured coatings in electrochemical sensors opens new perspectives for multipanel monitoring of human metabolism. [ABSTRACT FROM AUTHOR]

In this work we have developed a new electrochemical DNA-based biosensor for the selective determination of the Hg 2+ ion by the use of different electrodes modified with polythymine, bearing methylene blue, as redox probe, in 3′ position. The determination of Hg 2+ can be employed with an excellent degree of selectivity by the use of DNA biosensors through the formation of the complex Thymine–Hg–Thymine (T–Hg–T): in fact, Hg 2+ tends to bind two thymines, generating a T–Hg–T complex with a formation constant higher than that one of the coupling Adenine–Thymine, which can be employed for a selective, fast and cost-effective Hg 2+ detection. The presence of the Hg 2+ in solution leads to the formation of T–Hg–T complex thus causing the “hairpin-like” folding of oligonucleotide, leading to an improved electronic exchange of methylene blue with the electrode surface due to the reduced distance and thus to an increase of the faradic current which is detected by means of square wave voltammetry (SWV). To test the feasibility of this kind of biosensor to be applied to the analysis of Hg 2+ we have developed several biosensors configuration by modifying the electrochemical sensor transducer: (a) Au electrode; (b) Au screen-printed electrode (SPE). The proposed system, allows the determination of Hg 2+ in the range 0.2–100 nM (0.05–20 ppb), with a sensitivity 0.327 µA/nM, LOD 0.1 nM (0.02 ppb), LOQ 0.2 nM (0.05 ppb) and RSD ≤4.3% when Au electrode is used as electrochemical transducer; on the other hand, in the case of Au SPE the linear range is 0.2–50 nM (0.05–10 ppb), with a sensitivity 0.285 µA/nM, while LOD and LOQ are the same as previously and RSD is ≤3.8%. This enabled the detection of mercury in real samples (waters and fishes) with good accuracy (recoveries 92–101% on waters and 92–107% on fishes, respectively) and reproducibility (RSD ≤9.6% for measurements on waters and ≤8.8% on fishes, respectively). [ABSTRACT FROM AUTHOR]

A detailed comparison was made of the analytical features of a new Surface Plasmon Resonance (SPR) immunodevice for triazine pesticide determination with those of two other amperometric (conventional and screen-printed) immunosensors and the advantages and disadvantages of the SPR method were thoroughly investigated. For conventional amperometric and screen-printed devices, "competitive" assays were used; conversely, the SPR transduction technique allowed a "direct" measurement format to be used. As far as the main analytical data are concerned, the SPR method does not seem to offer substantial advantages. Nevertheless the measurement time is much shorter and the measurement itself much easier to perform. Lastly several applications and recovery tests were carried out on bovine milk samples, before and after spiking, to check for triazine pesticides in the samples, obtaining satisfactory results. [ABSTRACT FROM AUTHOR]

The present work describes the development and optimization of electrochemical biosensors for specific determination of the biogenic polyamine spermine (Spm) and spermidine (Spmd) whose assessment represents a novel important analytical tool in food analysis and human diagnostics. These biosensors have been prepared using novel engineered enzymes: polyamine oxidase (PAO) endowed with selectivity towards Spm and Spmd and spermine oxidase (SMO) characterized by strict specificity towards Spm. The current design entails biosensors in which the enzymes were entrapped in poly(vinyl alcohol) bearing styrylpyridinium groups (PVA-SbQ), a photocrosslinkable gel, onto an electrode surface. Screen-printed electrodes (SPEs) were used as electrochemical transducers for enzymatically produced hydrogen peroxide, operating at different potential vs Ag/AgCl according to the material of the working electrode (WE): +700 mV for graphite (GP) or −100 mV for Prussian blue (PB)-modified SPE, respectively. Biosensor performances were evaluated by means of flow injection amperometric (FIA) measurements. The modified electrodes showed good sensitivity, long-term stability and reproducibility. Under optimal conditions, the PAO biosensor showed a linear range 0.003-0.3 mM for Spm and 0.01-0.4 mM for Spmd, while with the SMO biosensor, a linear range of 0.004-0.5 mM for Spm has been obtained. The main kinetic parameters apparent Michaelis constant ( K), turnover number ( K) and steady-state current ( I) were determined. The proposed device was then applied to the determination of biogenic amines in blood samples. The results obtained were in good agreement with those obtained with the GC-MS reference method. [ABSTRACT FROM AUTHOR]

Abstract: In mammalian secretions, such as milk, lactoferrin is an important iron-binding glycoprotein, present in large quantities; it is also contained in powdered milks for babies sold in drugstores. Our team tested the feasibility of constructing a new immunosensor for lactoferrin analysis based on surface plasmon resonance (SPR) using two different devices and operating in batch or flow mode. The new method proposed is not competitive but “direct” and has the advantage of halving the measurement time which was deemed to be too long as is often the case in previous research when competitive immunological methods were used. Lastly, detailed comparison was made of the analytical features of new devices with those of classical or screen-printed immunosensor methods and the advantages and disadvantages of the new SPR method investigated. Lastly several applications and comparisons were carried out on cow and goat milk and on different types of dried milk for babies. [Copyright &y& Elsevier]

Abstract: This work has been focused on the development of novel optical (Surface Plasmon Resonance) and electrochemical based biosensors for the determination of 25-OH vitamin D (25OHD) which is an important factor involved in avoiding both skeletal damage and a variety of pathological conditions, and to evaluate their potential use in clinical practice. Different approaches to the determination of vitamin D using affinity based biosensors, are described herein; firstly, an immunosensor based on SPR transduction was realized for direct determination of vitamin D, obtaining a LOD of 2μg/ml which unfortunately is too far from the needs in clinical analysis. In order to enhance the sensitivity, the vitamin D was modified with gold nanoparticles (AuNPs): the binding of 25OHD with AuNPs determines the amplification of SPR signal, allowing to lower the LOD down to 1μg/ml, doubling the sensitivity. An alternative SPR method, based on the indirect determination of vitamin D by means of Vitamin D Binding Protein (VDBP), led to a further sensitivity increase reaching a LOD of 45ng/ml which is really close to the fixed accomplishment. Finally, an electrochemical transduced biosensor has been realized, based on the reaction of vitamin D with 4-ferrocenylmethyl-1,2,4-triazoline-3,5-dione (FMTAD): once derivatized, the determination of 25OHD was possible in the range 20–200ng/ml with a LOD of 10ng/ml. The latter proposed system fits the requirement of determining vitamin D in a concentration range which is of significance for clinical applications; moreover, since a screen printed electrode has been used, this opens the possibility to miniaturize the sensor and developing a portable and easy-to-automate point-of-care testing device. The proposed devices provide an improvement with respect to traditional methods that are time and reagents consuming and require radioactive compounds, pretreatment procedures and expensive instrumentation. [Copyright &y& Elsevier]

Abstract: Within the framework of research carried out by our team aimed at developing new immunological methods to determine proteins such as immunoglobulins G in different biological matrixes, for instance, serum and milk, tests were performed on several immunosensors based on different transducer types, i.e. amperometric (classical or screen-printed) electrodes for hydrogen peroxide. Lastly the feasibility of constructing immunosensors based on surface plasmon resonance (SPR) was investigated. “Competitive” immunological procedures were used in the first two cases. Conversely, the surface plasmon resonance transduction technique allowed a “direct” measurement. Applications were performed on human serum, powdered milks for babies and particularly on several animal milks, in the case of buffalo milk seeking a routine control method to identify possible inflammatory affections in the animals. [Copyright &y& Elsevier]

Abstract: In this work three different immobilization methods (physico-chemical, electrostatic and covalent) of fungal laccase from Trametes versicolor (TvL) on multi-walled carbon nanotubes electrodes (MWCNTs) have been characterized and compared. To this aim, in particular, the following immobilization agents were used: polyazetidine prepolymer (PAP), Nafion solution and succinimide-carbodiimide (EDC-NHS). The comparison of these procedures has been realized by evaluating the enzymatic activity of the resulting bioelectrodes and their catalytic efficiency for oxygen reduction in the presence of the 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonate) diammonium salt (ABTS) as non-phenolic redox mediator and catechol as phenolic one. Another aspect taken into account was the diffusion evaluation of several mediators as ABTS, dopamine, catechol and caffeic acid across the immobilizing layer at the pH range where the enzyme shows the maximum activity. The experimental results put in evidence the better performances of TvL-PAP-MWCNTs biosensor in terms of bioelectrochemical and diffusion properties, allowing us to asses that PAP is the best immobilizing agent thanks to its good permeability to mediators and the ability to keep the enzyme bioelectrochemical properties. [Copyright &y& Elsevier]

Abstract: The protein ERp57 (also known as PDIA3) is a widely distributed protein, mainly localized in the endoplasmic reticulum, where it acts as disulfide isomerase, oxidoreductase and chaperone, in concert with the lectins calreticulin (CRT) and calnexin. The ERp57/CRT complex has been detected on the cell surface and previous studies have suggested its involvement in programmed cell death. Although the ERp57-CRT complex has been characterized, little is known about its role in different cellular compartments as well as inhibitors of this interaction. We focused on the kinetic, extent and stability of the ERp57-CRT complex, using the surface plasmon resonance spectroscopy, investigating the possible role as inhibitor of the antibiotic vancomycin. Equilibrium thermodynamic data suggested that vancomycin may hinder the interaction between the two proteins and could interfere with the ERp57 conformational changes that stabilize the complex. Furthermore, by means of confocal microscopy, we evaluated the effect of the in vivo administration of vancomycin on the ERp57/CRT complex on the surface of HeLa cells. The model presented here could be used for the search of other specific inhibitors/interactors of ERp57, which can be extremely helpful to understand the biological pathways where the protein is involved and to modulate its activity. [Copyright &y& Elsevier]

In this work, we have characterized a diamine oxidase (DAO) from Lathyrus sativus and evaluated its use, for the first time, as biocatalytic component of an electrochemical biosensor for the determination of biogenic amines index in wine and beer samples. Firstly, DAO was electrokinetically characterized free in solution by means of a platinum electrode and then immobilized by using polyazetidine prepolimer on the surface of screen-printed electrodes constituted of two gold working electrodes. The amperometric measurements were carried out by using a flow system at a fixed potential of +600 mV vs the internal silver pseudo reference in phosphate buffer solution (0.1 mol l, pH = 7.4). The analysis of wine and beer samples were performed in flow injection system using the dual channel transducer providing simultaneous detection of sample and blank signal, and the resulting signal (after subtraction of the blank signal) was referred to that of putrescine. The results were compared with those obtained using a modified reference method based on gas chromatography-mass spectrometry analysis on the same samples. The results obtained in the analysis of Italian wines shows the better suitability of DAO-based biosensor in the determination of the biogenic amines (BAs) index expressed as putrescine equivalent in both red and white wines, being less efficient in beer samples where it underestimates by about 50% the BAs content. [ABSTRACT FROM AUTHOR]

Polyamine oxidases are FAD-dependent enzymes catalyzing the oxidation of polyamines at the secondary amino groups. Zea mays PAO (ZmPAO) oxidizes the carbon on the endo-side of the N5-nitrogen of spermidine (Spd) and spermine (Spm). The structure of ZmPAO revealed that the active site is formed by a catalytic tunnel in which the N5 atom of FAD lies in close proximity to the K300 side chain, the only active-site residue conserved in all PAOs. A water molecule, (HOH309), is hydrogen-bound to the amino group of K300 and mutation of this residue results in a 1400-fold decrease in the rate of flavin reduction. The structural studies on the catalytically impaired ZmPAO-K300M mutant described here show that substrates are bound in an 'out-of-register' mode and the HOH309 water molecule is absent in the enzyme-substrate complexes. Moreover, K300 mutation brings about a 60 mV decrease in the FAD redox potential and a 30-fold decrease in the FAD reoxidation rate, within a virtually unaltered geometry of the catalytic pocket. Taken together, these results indicate that the HOH309-K300 couple plays a major role in multiple steps of ZmPAO catalytic mechanism, such as correct substrate binding geometry as well as FAD reduction and reoxidation kinetics. The coordinates of the ZmPAO-K300M mutant in the free state and in complex with Spd and Spm have been deposited in the Protein Data Bank () with the PDB codes , and , respectively Structural bases governing the catalytic mechanism of polyamine oxidation by Zea Mays polyamine oxidase were revealed by studying the crystal structure of its K300M mutant. The data demonstrate that a water molecule (HOH309) hydrogen-bound to the amino group of the K300 side chain is involved in the correct positioning of the substrates within the active site, modulates the FAD redox potential and participates in the FAD reoxidation reaction. [ABSTRACT FROM AUTHOR]

Abstract: Laccases and other multicopper oxidases are reported to be able to carry out direct electron transfer reactions when immobilized onto electrode surface. This allows detailed research of their electron transfer mechanisms. We have recently characterized the kinetic properties of four laccases in homogenous solution and immobilized onto an electrode surface with respect to a set of different redox mediators. In this paper we report the direct electron transfer of four purified laccases from Trametes hirsuta (ThL), Trametes versicolor (TvL), Melanocarpus albomyces (r-MaL) and Rhus vernicifera (RvL), by trapping the proteins within an electrochemically inert polymer of tributylmethyl phosphonium chloride coating a gold electrode surface. In particular, we have characterized the steps involved in the laccases electron transfer mechanism as well as the factors limiting each step. During the voltammetric experiments, non-turnover Faradic signals with midpoint potential of about 790 and 400mV were observed for high potential laccases, ThL and TvL, corresponding to redox transformations of the T1 site and the T2/T3 cluster of the enzyme, respectively, whereas low redox potential laccases r-MaL and RvL shown a redox couple with a midpoint potential around 400mV. The electrocatalytic properties of these laccase modified electrodes for the reduction of oxygen have been evaluated demonstrating significative direct electron transfer kinetics. The biocatalytic activity of laccases was also monitored in the presence of a well known inhibitor, sodium azide. On the basis of the experimental results, a hypothesis about the electronic pathway for intramolecular electron transfer characterizing laccases has been proposed. [ABSTRACT FROM AUTHOR]

Abstract: Bioelectrochemical properties of Trametes versicolor Laccase (TvL) and Trametes hirsuta Laccase (ThL) immobilized by using polyazetidine prepolymer (PAP) onto multi-walled carbon nanotubes (MWCNTs) screen printed electrode (SPE) surface, have been studied with several redox mediators by cyclic voltammetry (CV). The efficient entrapment of laccase in the PAP layer was confirmed by determination of both kinetic parameters (maximum current and Michaelis–Menten apparent constant) and analytical performances by chronoamperometry. The Laccase-modified MWCNTs electrode provides an effective biosensor for determination of polyphenols and catecholamines in real matrices; performances of the considered biosensors for real samples analysis are also compared and discussed. [Copyright &y& Elsevier]

Bifunctional hydroxyl/thiol-functionalized fourth-generation polyamidoamine dendrimer (G4-PAMAM)-encapsulated Au nanoparticle (NP) was synthesized and immobilized on a mixed self-assembled monolayer (SAM)-modified gold surface. This modified surface was resistant to nonspecific adsorption of proteins having a wide range of molecular weight and isoelectric points. Part of the dendrimer thiol groups were converted to hydrazide functionalities providing an activated surface available to subsequently immobilize the receptor for developing a sensor surface to immunoaffinity reaction. Herein, the surface plasmon resonance (SPR) detection of insulin was obtained by means of a competitive immunoassay principle. The resulting Au NP dendrimer-modified surface provided an assay with high stability, significantly enhanced sensitivity, and a detection limit for analyzing insulin of 0.5 pM. The SPR detection of insulin was amplified due to the changes in the dielectric properties of the matrixes, occurring upon the biorecognition processes on the sensor surface, through the coupling of the localized plasmon of the NPs with the surface plasmon wave. The developed sensor chip was used to analyze insulin in human serum samples from healthy and diabetic patients. The results showed good correlation to the reference method. The specificity and the improved sensitivity of this biosensing platform could have significant implications for the detection of a wide range of molecules and biomarkers in complex biological media. [ABSTRACT FROM AUTHOR]

Abstract: In this work we have developed and characterized the use of Laccases from Trametes versicolor (TvL) and Trametes hirsuta (ThL) as biocatalytic components of electrochemical biosensors for the determination of polyphenol index in wines. Polyazetidine prepolimer (PAP) was used as immobilizing agent, multi-walled and single-walled carbon nanotubes screen-printed electrodes as sensors (MWCNTs-SPE and SWCNTs-SPE) and gallic acid as standard substrate. The amperometric measurements were carried out by using a flow system at a fixed potential of −100mV vs. silver/silver chloride electrode in Britton–Robinson buffer 0.1molL−1, pH 5. The results were compared with those obtained with the Folin–Ciocalteau reference method. The results obtained in the analysis of twelve Italian wines put in evidence the better suitability of ThL-MWCNTs-based biosensor in the determination of the polyphenol index in wines. This biosensor shows fast and reliable amperometric responses to gallic acid with a linear range 0.1–18.0mgL−1 (r 2 =0.999). The influence of the interferences on both spectrophotometric and electrochemical measurements have been carefully evaluated. [Copyright &y& Elsevier]

Abstract: The electrochemical studies of laccase–mediator systems are aimed at understanding the mechanism of their redox transformation and their efficiency in both homogeneous and heterogeneous reactions; this topic has paramount application spanning from bleaching of paper pulp and the enzymatic degradation of lignin to the biosensors and biofuel cell development. In this paper four different laccases from Trametes hirsuta (ThL), Trametes versicolor (TvL), Melanocarpus albomyces (r-MaL) and Rhus vernicifera (RvL) were characterized from both biochemical and electrochemical points of view. Two of them (TvL and ThL) are high redox potential and two (RvL and r-MaL) are low redox potential laccases. The outline of this work is focused on the determination of catalytic and bioelectrochemical properties of these four enzymes in homogenous solution as well as immobilized onto electrode surface in the presence of a set of different redox mediators. The results measured in the homogenous reaction system correlated well with those measured with the immobilized enzymes. In addition, they are in good agreement with those reported with reference techniques, suggesting that the electrochemical methods employed in this work can be applied well in place of the traditional techniques commonly used for the kinetic characterization of laccases. These results are also discussed in terms of the known amino acid sequences and three-dimensional (3D) structures of the laccases. [Copyright &y& Elsevier]

A manganese peroxidase (MnP) isoenzyme from Panus tigrinus CBS 577.79 was produced in a benchtop stirred-tank reactor and purified to apparent homogeneity. The purification scheme involving ultrafiltration, affinity chromatography on concanavalin–A Sepharose, and gel filtration led to a purified MnP, termed “MnP II,” with a specific activity of 288 IU mg−1 protein and a final yield of 22%. The enzyme turned out to be a monomeric protein with molecular mass of 50.5 kDa, pI of 4.07, and an extent of N-glycosylation of about 5.3% of the high-mannose type. The temperature and pH optima for the formation of malonate manganic chelates were 45 °C and 5.5, respectively. MnP II proved to be poorly thermostable at 50 and 60 °C, with half-lives of 11 min and 105 s, respectively. Km values for H2O2 and Mn2+ were 16 and 124 μM, respectively. Although MnP II was able to oxidize veratryl alcohol and to catalyze the Mn2+-independent oxidation of several phenols, it cannot be assigned to the versatile peroxidase family. As opposed to versatile peroxidase oxidation, veratryl alcohol oxidation required the simultaneous presence of H2O2 and Mn2+; in addition, low turnover numbers and Km values higher than 300 μM characterized the Mn2+-independent oxidation of substituted phenols. Kinetic properties and the substrate specificity of the enzyme markedly differed from those reported for MnP isoenzymes produced by the reference strain P. tigrinus 8/18. To our knowledge, this study reports for the first time a thorough electrochemical characterization of a MnP from this fungus. [ABSTRACT FROM AUTHOR]

In this paper, we present a surface-plasmon-resonance-based immunosensor for the real-time detection of cortisol and cortisone levels in urine and saliva samples. The method proposed here is simple, rapid, economic, sensitive, robust, and reproducible thanks also to the special features of the polycarboxylate-hydrogel-based coatings used for the antibody immobilization. The sensor surface displays a high level of stability during repeated regeneration and affinity reaction cycles. The immunosensor shows high specificity for cortisol and cortisone; furthermore, no significant interferences from other steroids with a similar chemical structure have been observed. The suitability of the hydrogel coating for the prevention of nonspecific binding is also investigated. A good correlation is noticed between the results obtained by the proposed method and the reference liquid chromatography/tandem mass spectrometry method for the analysis of cortisol and cortisone in urine and saliva samples. Standard curves for the detection of cortisol and cortisone in saliva and urine are characterized by a detection limit less than 10 μg l−1, sufficiently sensitive for both clinical and forensic use. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Abstract: A highly stable functional composite film was prepared using polyazetidine prepolymer (PAP) with peroxidase from horseradish (HRP) and/or glucose oxidase (GOx). The good permeability of the PAP layer to classical electrochemical mediators, as evaluated by the determination of the diffusion coefficient of different redox molecules, is of great importance in view of the use of PAP as an immobilizing agent in second-generation biosensor development. Cyclic voltammetry of the HRP–PAP layer on a glassy carbon electrode (GCE) showed a pair of stable and quasi-reversible peaks for the HRP–Fe(III)/Fe(II) redox couple at about −370mV vs. Ag/AgCl electrode in pH 6.5 phosphate buffer. The electrochemical reaction of HRP entrapped in the PAP film exhibited a surface-controlled electrode process. This film and the successive modifications (HRP–PAP self-assembled monolayer (SAM) modified Au electrode) were used as a biological catalyst (hydrogen peroxide transducers) for glucose biosensors, after coupling to GOx. Both HRP/GOx–PAP and HRP/GOx–PAP SAM third generation biosensors were prepared and characterized. The use of PAP as immobilizing agent offers a biocompatible micro-environment for confining the enzyme and foreshadows the great potentiality of this immobilizing agent not only in theoretical studies on protein direct electron transfer but also from an applications point of view in the development of second- and third-generation biosensors. [Copyright &y& Elsevier]