We report on the response of reaction center (RC) from Rhodobacter sphaeroides (an archetype of membraneproteins) to the exposure at high temperature. The RCs have been solubilized in aqueous solution of thedetergent N,N-dimethyldodecylamine-N-oxide (LDAO). Changes in the protein conformation have beenprobed by monitoring the variation in the absorbance of the bacteriochlorine cofactors and modification inthe efficiency of energy transfer from tryptophans to cofactors and among the cofactors (throughfluorescence measurements). The RC aggregation taking place at high temperature has been investigated bymeans of dynamic light scattering. Two experimental protocols have been used: (i) isothermal kinetics, inwhich the time evolution of RC after a sudden increase of the temperature is probed, and (ii) T-scans, inwhich the RCs are heated at constant rate. The analysis of the results coming from both the experimentsindicates that the minimal kinetic scheme requires an equilibrium step and an irreversible process. Theirreversible step is characterized by a activation energy of 205±14 kJ/mol and is independent from thedetergent concentration. Since the temperature dependence of the aggregation rate was found to obey to thesame law, the aggregation process is unfolding-limited. On the other hand, the equilibrium process betweenthe native and a partially unfolded conformations was found to be strongly dependent on the detergentconcentration. Increasing the LDAO content from 0.025 to 0.5 wt.% decreases the melting temperature from49 to 42 °C. This corresponds to a sizeable (22 kJ/mol at 25 °C) destabilization of the native conformationinduced by the detergent. The nature of the aggregates formed by the denatured RCs depends on thetemperature. For temperature below 60 °C compact aggregates are formed while at 60 °C the clusters are lessdense with a scaling relation between mass and size close to that expected for diffusion-limited aggregation.The aggregate final sizes formed at different temperatures indicate the presence of an even number ofproteins suggesting that these clusters are formed by aggregation of dimers.

Anchored, biotinylated phospholipids forming the capturing layers in an electrolytegated organic field-effect transistor (EGOFET) allow label-free electronic specific detection at a concentration level of 10 nM in a high ionic strength solution. The sensing mechanism is based on a clear capacitive effect across the PL layers involving the charges of the target molecules.

An organic field-effect transistor (OFET) integrating bacteriorhodopsin (bR) nanoassembled lamellae is proposed for an in-depth study of the proton translocation processes occurring as the bioelectronic device is exposed either to light or to low concentrations of general anesthetic vapors. The study involves the morphological, structural, electrical, and spectroscopic characterizations necessary to assess the functional properties of the device as well as the bR biological activity once integrated into the functional biointerlayer (FBI)-OFET structure. The electronic transduction of the protons phototranslocation is shown as a current increase in the p-type channel only when the device is irradiated with photons known to trigger the bR photocycle, while Raman spectroscopy reveals an associated C=C isomer switch. Notably, higher energy photons bring the cis isomer back to its trans form, switching the proton pumping process off. The investigation is extended also to the study of a PM FBI-OFET exposed to volatile general anesthetics such as halothane. In this case an electronic current increase is seen upon exposure to low, clinically relevant, concentrations of anesthetics, while no evidence of isomer-switching is observed. The study of the direct electronic detection of the two different externally triggered proton translocation effects allows gathering insights into the underpinning of different bR molecular switching processes. © 2014 American Chemical Society.

A general method to obtain the efficient entrapment of mixtures of glycoenzymes in calcium alginate hydrogel is proposed in this paper. As a proof of principle, three glycoenzymes acting in series (trehalase, glucose oxidase, and horseradish peroxidase) have been coimmobilized in calcium alginate beads. The release of the enzymes from the hydrogel mesh (leakage) is avoided by exploiting the enzyme's aggregation induced by the concanavalin A. The aggregation process has been monitored by dynamic light scattering technique, while both enzyme encapsulation efficiency and leakage have been quantified spectrophotometrically. Obtained data show an encapsulation efficiency above 95% and a negligible leakage from the beads when enzyme aggregates are larger than 300 nm. Operational stability of "as prepared" beads has been largely improved by a coating of alternated shells of polycation poly(diallyldimethylammonium chloride) and of alginate. As a test for the effectiveness of the overall procedure, analytical bioassays exploiting the enzyme-containing beads have been developed for the optical determination of glucose and trehalose, and limit of detection values of 0.2 and of 40 ?M, respectively, have been obtained.

To satisfy the demand for fast and smart analytical systems a great interest has been focused on the study and development of novel bio-sensing devices. Electronic transduction can open new perspectives for point-of-care diagnosis actuated by fast, sensitive, selective and reliable biosensors. Recently our group demonstrated the feasibility of the coupling of a biological recognition element to an organic field-effect device [3, 4]. As a further step, investigations on different deposition techniques have been developed, to improve the adhesion and the homogeneity of the biological element ontothe organic semiconductor.

Biosystems integration into an organic field-effect transistor (OFET)structure is achieved by spin coating phospholipid or protein layersbetween the gate dielectric and the organic semiconductor. Anarchitecture directly interfacing supported biological layers to theOFET channel is proposed and, strikingly, both the electronic propertiesand the biointerlayer functionality are fully retained. Theplatform bench tests involved OFETs integrating phospholipids andbacteriorhodopsin exposed to 1-5% anesthetic doses that revealdrug-induced changes in the lipid membrane. This result challengesthe current anesthetic action model relying on the so far providedevidence that doses much higher than clinically relevant ones(2.4%) do not alter lipid bilayers' structure significantly. Furthermore,a streptavidin embedding OFET shows label-free biotin electronicdetection at 10 parts-per-trillion concentration level, reachingstate-of-the-art fluorescent assay performances. These examplesshow how the proposed bioelectronic platform, besides resultingin extremely performing biosensors, can open insights into biologicallyrelevant phenomena involving membrane weak interfacialmodifications.

Bright field microscopy and atomic force microscopy techniques are used to investigate morphological properties of synthetic eumelanin, obtained by oxidation of l-DOPA solution, deposited on glass and mica substrates. Deposits of eumelanin are characterized by aggregates with different shape and size. On a micrometric scale, filamentous as well as granular structures are present on glass and mica substrates, with a larger density on the former than on the latter. On a nanometric scale, filamentous aggregates, several microns long and about 100. nm wide and high, and granular aggregates, ~50. nm high and 100. nm wide, are found on both substrates, whereas point-like deposits less than 10. nm high and less than 50. nm wide are found on mica substrate. Dynamic light scattering measurements and atomic force microscopy images support the evidence that eumelanin presents only nanometric point-like aggregates in aqueous solution, whereas such nanoaggregates organize themselves according to granular and filamentous structures when deposition occurs, as a consequence of interactions with the substrate surface. © 2014 Elsevier Ltd.

Determination of phenolic derivatives is very important in medical, food and environmental samplesbecause of their relevant significance in health care and pollution monitoring. Tyrosinase-based biosensorsare promising tools for this purpose because of several advantages with respect to currently useddetection methods.A key aspect in the development of a biosensor is the effective immobilization of the enzyme. In thiswork, ordered tyrosinase films on an optical transparent support were immobilized by a "layer-by-layer"(LbL) assembly, alternating the enzyme with the polycation polymer poly(dimethyldiallylammoniumchloride). As confirmed by UV-vis spectroscopy, the LbL deposition allowed a high loading of enzyme.The immobilized tyrosinase functionality was proven and its kinetic parameters were spectrophotometricallydetermined. The prepared biosensor was used to optically detect the o-diphenolic compoundl-3,4-dihydroxyphenyl-alanine (l-DOPA) and exhibited good repeatability and time stability. The sensingproperties of the system were studied by means of both absorption and fluorescence spectroscopy.The bioassay based on the absorbance measurements gave a LOD of 23M and a linear response up to350M. The bioassay based on the fluorescence measurements gave a LOD of 3Mand a linear responsein the range of tens of micromolar (the exact value depends on the number of mushroom tyrosinaselayers). Biosensor sensitivity could be modulated varying the number of the immobilized enzyme layers.

A totally innovative electrolyte-gated field effect transistor, embedding a phospholipid filmat the interface between the organic semiconductor and the gating solution, is described.The electronic properties of OFETs including a phospholipid film are studied in both purewater and in an electrolyte solution and compared to those of an OFET with the organicsemiconductor directly in contact with the gating solution. In addition, to investigate therole of the lipid layers in the charge polarization process and quantify the field-effectmobility, impedance spectroscopy was employed. The results indicate that the integrationof the biological film minimizes the penetration of ions into the organic semiconductorthus leading to a capacitive operational mode as opposed to an electrochemical one. TheOFETs operate at low voltages with a field-effect mobility in the 103 cm2 V1 s1 rangeand an on/off current ratio of 103. This achievement opens perspectives to the developmentof FET biosensors potentially capable to operate in direct contact with physiological fluids.

Organic field-effect transistors including a functional biorecognition interlayer, sandwiched between the dielectric and the organic semiconductor layers, have been recently proposed as ultrasensitive label-free biosensors capable to detect a target molecule in the low pM concentration range. The morphology and the structure of the stacked bilayer formed by the protein biointerlayer and the overlying organic semiconductor is here investigated for different protein deposition methods. X-ray scattering techniques and scanning electron microscopy allow us to gather key relevant information on the interface structure and to assess target analyte molecules capability to percolate through the semiconducting layer reaching the protein deposit lying underneath. Correlations between the structural and morphological data and the device analytical performances are established allowing us to gather relevant details on the sensing mechanism and further improving sensor performances, in particular in terms of sensitivity and selectivity. © 2014 American Chemical Society.

A new optical biosensor for trehalose determination has been realized immobilizing three glycoenzymes on a transparent support. Trehalase, glucose oxidase and horseradish peroxidase have been alternated with layers of Concanavalin A by a "layer-by-layer" (LbL) deposition. The driving force of this assembly is the biospecific complexation between Concanavalin A and sugar residues in the glycoenzymes. As confirmed by UV-vis spectroscopy, the LbL deposition allowed a high ordinate architecture with high loading of enzymes. After the assembly, the functionality of immobilized enzymes was spectrophotometrically proven, demonstrating also that they can act in series catalyzing cascade reactions. The prepared biosensor was used to optically detect trehalose, giving a LOD of 10 ?M and a linear response up to 4 mM, and it showed also good time stability. The trehalose content in a real sample (eyewash) was successfully determined by the biosensor. © 2014 Elsevier B.V.

A simple and time-saving wet method to endow the surface of organic semiconductor films with carboxyl functional groups is presented. A thin layer of poly(acrylic acid) (pAA) is spin-coated directly on the electronic channel of an electrolyte-gated organic FET (EGOFET) device and cross-linked by UV exposure without the need for any photo-initiator. The carboxyl functionalities are used to anchor phospholipid bilayers through the reaction with the amino-groups of phosphatidyl-ethanolamine (PE). By loading the membranes with phospholipids carrying specific functionalities, such a platform can be easily implemented with recognition elements. Here the case of biotinylated phospholipids that allow selective streptavidin electronic detection is described. The surface morphology and chemical composition are monitored using SEM and XPS, respectively, during the whole process of bio-functionalization. The electronic and sensing performance level of the EGOFET biosensing platform is also evaluated. Selective analyte (streptavidin) detection in the low pM range is achieved, this being orders of magnitude lower than the performance level obtained by the well assessed surface plasmon resonance assay reaching the nM level, at most.

The detailed action mechanism of volatile general anesthetics is still unknown despite their effect has been clinically exploited for more than a century. Long ago it was also assessed that the potency of an anesthetic molecule well correlates with its lipophilicity and phospholipids were eventually identified as mediators. As yet, the direct effect of volatile anesthetics at physiological relevant concentrations on membranes is still under scrutiny. Organic field-effect transistors (OFETs) integrating a phospholipid (PL) functional bio inter-layer (FBI) are here proposed for the electronic detection of archetypal volatile anesthetic molecules such as diethyl ether and halothane. This technology allows to directly interface a PL layer to an electronic transistor channel, and directly probe subtle changes occurring in the bio-layer. Repeatable responses of PL FBI-OFET to anesthetics are produced in a concentration range that reaches few percent, namely the clinically relevant regime. The PL FBI-OFET is also shown to deliver a comparably weaker response to a non-anesthetic volatile molecule such as acetone. © 2012 Elsevier B.V.