We demonstrate the direct bioconjugation of hydrogen-bonded organic semiconductors with two different complex functional proteins in an aqueous environment. The representative semiconductors are epindolidione and quinacridone, materials used in devices in the form of vacuum-evaporated polycrystalline films. First, these molecules in thin films react spontaneously with N -hydroxysuccinimide functionalized linkers: disuccinimidyl suberate and succinimidyl biotinate. The suberate linker is then used to covalently bind the Rhodobacter sphaeroides reaction centre (RC), the key photoenzyme for conversion of light into electrical charges in photosynthetic bacteria. Similarly, the biotin linker is used to bridge streptavidin to the surface of the hydrogen-bonded semiconductor film. Multiple-reflection infrared spectroscopy, water contact angle measurements, and atomic force microscopy are used to verify surface functionalization. The presence and functional integrity of the immobilized proteins are demonstrated by specific experiments: a charge recombination kinetics assay in the case of the RC, and photoluminescence measurements for quantum dot-labelled streptavidin. As key results of our work, we have shown that upon bioconjugation, the semiconductors preserve their favourable electrical properties: as evidenced by photoconductor devices operating under water sensitized by the RC, and thin film transistor measurements before and after bioconjugation. These are enabling steps for using hydrogen-bonded semiconductors as platforms for multifunctional bioelectronics device

Abstract: This paper describes the evaluation of the oxidative potential of atmospheric aerosol, for the PM2.5 and PM10 size fractions, using the DTT assay. This is an indicator related with oxidative stress of particulate matter (PM) leading to potential health effects. Measured DTT activity has been correlated with aerosol concentrations and with the content of carbonaceous species on collected samples. The oxidative potential of PM associated to natural sources during Saharan Dust and sea-spray advection events is investigated and compared with non-event days.

ABSTRACT The development and characterization of a novel bioactive polymer based on the immobilization of glucose oxidase enzyme (GOx) in a polyvinyl alcohol (PVA) film showing antibacterial activity is presented. The PVA-GOx composite material was extensively characterized by UV-vis, X-ray Photoelectron (XPS) spectroscopy and by Fourier Transform Infrared (FTIR) spectroscopy to verify the preservation of enzyme structural integrity and activity. The antimicrobial activity of this composite material against Escherichia coli and Vibrio alginolyticus was assessed. Furthermore the lysozyme-like activity of PVA-GOx was highlighted by a standard assay on Petri dishes employing Micrococcus lysodeikticus cell walls. The findings from this study have implications for future investigations related to the employment of PVA-GOx system as a composite material of pharmaceutical and technological interest.

Ultrasounds are used in many industrial, medical and research applications. Properties and function of proteins are strongly influenced by the interaction with the ultrasonic waves and their bioactivity can be lost because of alteration of protein structure. Surprisingly, to the best of our knowledge no study was carried out on Integral Membrane Proteins (IMPs), which are responsible for a variety of fundamental biological functions. In this work, the photosynthetic Reaction Center (RC) of the bacterium Rhodobacter sphaeroides has been used as a model for the study of the ultrasound-induced IMP denaturation. Purified RCs were suspended in i) detergent micelles, in ii) detergent-free buffer and iii) reconstituted in liposomes, and then treated with ultrasound at 30W and 20kHz at increasing times. The optical absorption spectra showed a progressive and irreversible denaturation in all cases, resulting from the perturbation of the protein scaffold structure, as confirmed by circular dichroism spectra that showed progressive alterations of the RC secondary structure. Charge recombination kinetics were studied to assess the protein photoactivity. The lifetime for the loss of RC photoactivity was 32min in detergent micelles, ranged from 3.8 to 6.5min in the different proteoliposomes formulations, and 5.5min in detergent-free buffer. Atomic force microscopy revealed the formation of large RC aggregates related to the sonication-induced denaturation, in agreement with the scattering increase observed in solution.

Curcumin is a natural hydrophobic polyphenol found in the powdered rhizomes of Curcuma longa. Due to its capacity to interfere with many signalling pathways, it has been shown that curcumin has potential beneficial pharmacological effects including antioxidant, anti-inflammatory, anticarcinogenic properties. However, the use of curcumin is fairly restricted because of its poor water solubility, low bioavailability, inadequate tissue absorption and degradation at alkaline pH. In the present contribution, we first verified the anti-proliferative effects of natural curcuminoids towards two different cell lines derived from an ovarian and a breast adenocarcinoma cancer. Later, curcuminoids were successfully encapsulated into reconstituted oil bodies. Once encapsulated into the triacylglycerol cores of the reconstituted oil bodies, curcumin, the most hydrophobic and active of the three curcuminoids, was better stabilized in comparison with albumin stabilization. Oil body encapsulated curcuminoids showed the same effects on cancer cell viability as the free drug, confirming the great potential of natural oil bodies as micro/nano-capsules in drug delivery applications.

Abstract Deep eutectic solvents (DESs) are emerging as a new class of green solvents with the potential to replace organic solvents in several fundamental and applied processes. In this work, we offer an unprecedented characterization of the behavior of the bacterial photosynthetic reaction center (RC) from Rhodobacter sphaeroides in a series of choline chloride based DESs. RC is a membrane-spanning three-subunit pigment protein complex that, upon illumination, is capable of producing a stable charge-separated state. Thus, it represents the ideal model for carrying out basic studies of protein solvent interactions. Herein, we first report that, in many DES mixtures investigated, RC (a) is stable, (b) is capable of generating the charge-separated state, and (c) is even able to perform its natural photocycle. It proved, indeed, to be effective in reducing quinone molecules to quinol by withdrawing electrons from cytochrome c. As an example of biotechnological application, a photoelectrochemical cell based on DES-dissolved RC has also been designed and successfully employed to generate photocurrents arising from the reduction of the electron-donor ferrocenemethanol.

The development of an amperometric biosensor for herbicide detection, using bacterial reaction centers (RC) as biorecognition element, is presented. RC immobilization on gold screen printed electrodes was achieved by LIFT, a powerful physisorption-based immobilization technique that enhances the intimate contact between the protein and the electrode surface. As a result, stable photocurrents driven by direct electron transfer at the donor side were observed, both in the presence and in the absence of a quinone substrate in solution. The addition of quinone UQ(0) increased the photocurrents, while the UQ(0)-free system showed higher sensitivity to the herbicide terbutryn, a model inhibitor, acting as photocurrent attenuator. In spite of its simple design, the performances achieved by our mediatorless device are comparable or superior to those reported for analogous RC-based photoelectrochemical cells, in terms of both terbutryn sensing and photocurrent generation.

Abstract View references (51) Exposure to atmospheric particulate matter (PM) leads to adverse health effects although the exact mechanisms of toxicity are still poorly understood. Several studies suggested that a large number of PM health effects could be due to the oxidative potential (OP) of ambient particles leading to high concentrations of reactive oxygen species (ROS). The contribution to OP of specific anthropogenic sources like road traffic, biomass burning, and industrial emissions has been investigated in several sites. However, information about the OP of natural sources are scarce and no data is available regarding the OP during Saharan dust outbreaks (SDO) in Mediterranean regions. This work uses the a-cellular DTT (dithiothreitol) assay to evaluate OP of the water-soluble fraction of PM2.5 and PM10 collected at an urban background site in Southern Italy. OP values in three groups of samples were compared: standard characterised by concentrations similar to the yearly averages; high carbon samples associated to combustion sources (mainly road traffic and biomass burning) and SDO events. DTT activity normalised by sampled air volume (DTTV), representative of personal exposure, and normalised by collected aerosol mass (DTTM), representing source-specific characteristics, were investigated. The DTTV is larger for high PM concentrations. DTTV is well correlated with secondary organic carbon concentration. An increased DTTV response was found for PM2.5 compared to the coarse fraction PM2.5-10. DTTV is larger for high carbon content samples but during SDO events is statistically comparable with that of standard samples. DTTM is larger for PM2.5 compared to PM10 and the relative difference between the two size fractions is maximised during SDO events. This indicates that Saharan dust advection is a natural source of particles having a lower specific OP with respect to the other sources acting on the area (for water-soluble fraction). OP should be taken into account in epidemiological studies to evaluate the potential health risks associated to ROS in regions affected by high pollution events due to Saharan dust advection.

Photosynthetic reaction center (RC) is the minimal nanoscopic photoconverter in the photosynthetic membrane that catalyzes the conversion of solar light to energy readily usable for the metabolism of the living organisms. After electronic excitation the energy of light is converted into chemical potential by the generation of a charge separated state accompanied by intraprotein and ultimately transmembrane proton movements. We designed a system which fulfills the minimum structural and functional requirements to investigate the physico/chemical conditions of the processes: RCs were reconstituted in closed lipid vesicles made of selected lipids entrapping a pH sensitive indicator, and electron donors (cytochrome c2 and K4[Fe(CN)6]) and acceptors (decylubiquinone) were added to sustain the photocycle. Thanks to the low proton permeability of our preparations, we could show the formation of a transmembrane proton gradient under illumination and low buffering conditions directly by measuring proton-related signals simultaneously inside and outside the vesicles. The effect of selected ionophores such as gramicidin, nigericin and valinomycin was used to gain more information on the transmembrane proton gradient driven by the RC photochemistry.

This work demonstrates that lipid-detergent mixed micelles can be employed successfully in order to achieve and modulate the transfer of bio-active hydrophobic compounds into lipid carriers by means of a simple and bio-safe procedure. In our specific investigation, liposome preparations incorporating mixtures of natural carotenoids with high lycopene content were developed and characterized, aiming to obtain formulations of potential nutraceutical and pharmaceutical interest. The starting material was a solvent-free high-quality lycopene rich oleoresin (LRO) obtained by extracting a freeze-dried tomato matrix with supercritical carbon dioxide (SC-CO2). Mixed micelles containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and cholate were loaded with LRO antioxidants by means of two slightly different procedures, which surprisingly resulted in significant differences in both quality and quantity of incorporated carotenoids. In particular, the selective incorporation of (all-E)-lycopene was achieved by extracting the oleoresin with a pre-formed cholate/POPC micelle suspension whilst (Z)-isomers were preferentially integrated when treating a POPC/LRO mixed film with cholate. The micelle to vesicle transition (MVT) method was employed in order to produce vesicles of well-defined lamellarity and size. Visible and infrared (IR) spectroscopy as well as Dynamic Light Scattering (DLS) and Transmission Electron Microscopy (TEM) measurements allowed the extensive characterization of LRO-loaded micelles and liposomes. The antioxidant potential of preparations was assessed by measuring the radical scavenging activity towards the coloured radical cation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonate) (ABTS). Important information about the reliability of different approaches for antioxidant capacity evaluation of micelle and liposome preparations was gained and the successful incorporation of LRO antioxidant power in a bio-deliverable water-dispersed form was demonstrated.

The use of fluorescent nanocrystals (NCs) as probes for bioimaging applications has emerged as an advantageous alternative to conventional organic fluorescent dyes. Therefore their toxicological evaluation and intracellular delivery are currently a primary field of research. In this work, hydrophobic and highly fluorescent CdSe@ZnS NCs were encapsulated into the lipid bilayer of liposomes by the micelle-to-vesicle transition (MVT) method. The obtained aqueous NC-liposome suspensions preserved the spectroscopic characteristics of the native NCs. A systematic study of the in vitro toxicological effect on HeLa cells of these red emitting NC-liposomes was then carried out and compared to that of empty liposomes. By using liposomes of different phospholipid composition, we evaluated the effect of the lipid carrier on the cytotoxicity towards HeLa cells. Surprisingly, a cell proliferation and death study along with the MTT test on HeLa cells treated with NC-liposomes have shown that the toxic effects of NCs, at concentrations up to 20 nM, are negligible compared to those of the lipid carrier, especially when this is constituted by the cationic phospholipid DOTAP. In particular, obtained data suggest that DOTAP has a dose- and time-dependent toxic effect on HeLa cells. In contrast, the addition of PEG to the liposomes does not alter significantly the viability of the cells. In addition, the ability of NC-liposomes to penetrate the HeLa cells was assessed by fluorescence and confocal microscopy investigation. Captured images show that NC-liposomes are internalized into cells through the endocytic pathway, enter early endosomes and reach lysosomes in 1 h. Interestingly, red emitting NCs co-localized with endosomes and were positioned at the limiting membrane of the organelles. The overall results suggest that the fluorescent system as a whole, NCs and their carrier, should be considered for the development of fully safe biological applications of CdSe@ZnS NCs, and provide essential indications to define the optimal experimental conditions to use the proposed system as an optical probe for future in vivo experiments.

Aptamers are chemically produced oligonucleotides, able to bind a variety of targets such as drugs, proteins and pathogens with high sensitivity and selectivity. Therefore, aptamers are largely employed for producing label-free biosensors (aptasensors), with significant applications in diagnostics and drug delivery. In particular, the anti-thrombin aptamers are biomolecules of high interest for clinical use, because of their ability to recognize and bind the thrombin enzyme. Among them, the DNA 15-mer aptamer (TBA), has been widely explored around the possibility of using it in aptasensors. This paper proposes a microscopic model of the electrical properties of TBA and of the aptamer-thrombin complex, combining information from both structure and function, following the issues addressed in an emerging branch of electronics known as proteotronics. The theoretical results are compared and validated with measurements reported in the literature. Finally, the model suggests resistance measurements as a novel tool for testing aptamer-target affinity.

Osmotic shock was used as a tool to obtain cardiolipin (CL) enriched chromatophores of Rhodobacter sphaeroides. After incubation of cells in iso- and hyper-osmotic buffers both chromatophores with a physiological lipid profile (Control) and with an almost doubled amount of CL (CL enriched) were isolated. Spectroscopic properties, reaction centre (RC) and reducible cytochrome (cyt) contents in Control and CL enriched chromatophores were the same. The oxidoreductase activity was found higher for CL enriched than for Control chromatophores, raising from 60 ± 2 to 93 ± 3 mol cyt c s−1 (mol total cyt c)−1. Antymicin and myxothiazol were tested to prove that oxidoreductase activity thus measured was mainly attributable to the cyt bc1 complex. The enzyme was then purified from BH6 strain yielding a partially delipidated and almost inactive cyt bc1 complex, although the protein was found to maintain its structural integrity in terms of subunit composition. The ability of CL in restoring the activity of the partially delipidated cyt bc1 complex was proved in micellar systems by addition of exogenous CL. Results here reported indicate that CL affects oxidoreductase activity in the bacterium Rhodobacter sphaeroides both in chromatophores and in purified cyt bc1 complex.

Photosynthetic reaction centres are membrane-spanning proteins, found in several classes of autotroph organisms, where a photoinduced charge separation and stabilization takes place with a quantum efficiency close to unity. The protein remains stable and fully functional also when extracted and purified in detergents thereby biotechnological applications are possible, for example, assembling it in nano-structures or in optoelectronic systems. Several types of bionanocomposite materials have been assembled by using reaction centres and different carrier matrices for different purposes in the field of light energy conversion (e.g., photovoltaics) or biosensing (e.g., for specific detection of pesticides). In this review we will summarize the current status of knowledge, the kinds of applications available and the difficulties to be overcome in the different applications. We will also show possible research directions for the close future in this specific field.

Human olfactory 17-40 and Bacteriorhodopsin are two protein receptors that received particular attention in electronics, due to the possibility of implementing nano-biodevices able to detect odours and light and thus useful for medical and green energy harvesting applications. Some recent experiments concerning the electrical responses of these receptors are reviewed. Data are interpreted in the framework of a new science exploiting the complexity in biology and biomedical engineering called proteotronics. In particular, the single protein is modelled as an impedance network whose topological properties affect the electrical response as measured by experiments.

Semiquinone oscillations induced by light pulses in the presence of exogenous electron donors are a valuable source of information on the kinetics and thermodynamics of ubiquinone chemistry relevant to the QB site of the photosynthetic reaction center (RC). In previous attempts to achieve the quantitative interpretation of data, the ubiquinone concentration was considered constant during the experiment since it was much bigger than that of RC. In this work, we extended existing models to low ubiquinone concentrations revealing several hidden processes taking place during the ubiquinone photoreduction and enabling the evaluation of the ubiquinone binding constant KQ at the QB site. The proposed approach provides for the first time the evaluation of KQ without any preliminary treatment of ubiquinone extraction from the binding site, thereby better preserving its native structure.

Liposomes represent a versatile biomimetic environment for studying the interaction between integral membrane proteins and hydrophobic ligands. In this paper, the quinone binding to the QB-site of the photosynthetic reaction centers (RC) from Rhodobacter sphaeroides has been investigated in liposomes prepared with either the zwitterionic phosphatidylcholine (PC) or the negatively charged phosphatidylglycerol (PG) to highlight the role of the different phospholipid polar heads. Quinone binding (KQ) and interquinone electron transfer (LAB) equilibrium constants in the two type of liposomes were obtained by charge recombination reaction of QB-depleted RC in the presence of increasing amounts of ubiquinone-10 over the temperature interval 6-35 °C. The kinetic of the charge recombination reactions has been fitted by numerically solving the ordinary differential equations set associated with a detailed kinetic scheme involving electron transfer reactions coupled with quinone release and uptake. The entire set of traces at each temperature was accurately fitted using the sole quinone release constants (both in a neutral and a charge separated state) as adjustable parameters. The temperature dependence of the quinone exchange rate at the QB-site was, hence, obtained. It was found that the quinone exchange regime was always fast for PC while it switched from slow to fast in PG as the temperature rose above 20 °C. A new method was introduced in this paper for the evaluation of constant KQ using the area underneath the charge recombination traces as the indicator of the amount of quinone bound to the QB-site.