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In questo articolo viene presentato il caso clinico di Monica, una donna di 32 anni che richiede una psicoterapia per un Disturbo di ansia sociale che sta mettendo a rischio il suo nuovo lavoro. L'ansia sociale è definita come una sindrome cognitivo-affettiva contraddistinta da apprensione e intensa paura che si manifesta in situazioni sociali in cui la persona è sottoposta alla valutazione oppure allo sguardo degli altri (Grimaldi, 2008; Leary & Kowalski, 1995). Gli approcci cognitivisti hanno studiato e approfondito le tematiche dell'ansia sociale, con la formulazione di diverse teorie e modelli (Beck & Emery, 1985; Clark & Wells, 1995; Hofmann, 2007; Moscovitch, 2009; Orazi & Mancini, 2011; Rapee & Heimberg, 1997; Rapee, 2010; Schlenker & Leary, 1982b; Stopa, 2009). A partire da questi modelli, sono stati implementati trattamenti efficaci per l'ansia sociale, tra cui i protocolli di trattamento della terapia cognitivo-comportamentale che sono ad oggi i più validati e statisticamente più efficaci. Tuttavia, questi modelli si focalizzano sui meccanismi di mantenimento, senza indicare come essi potrebbero svilupparsi, né specificare una base eziologica del disturbo (Wong et al., 2017), con ripercussioni sull'impostazione del trattamento. Il caso di Monica viene di seguito formulato facendo riferimento al Modello Cognitivo Integrato (Grimaldi, 2019) basato appunto sull'integrazione di modelli e ricerche provenienti da diversi ambiti al fine di costruire un modello psicopatologico che spieghi lo sviluppo e il mantenimento dei vari disturbi d'ansia sociale, e sulla base del quale mettere a punto un intervento terapeutico maggiormente efficace.

While the number and types of indoor air pollutants is rising, much is suspected but little is known about the impact of their potentially synergistic interactions, upon human health. Gases, particulate matter, organic compounds, but also allergens and viruses, fall within the ‘pollutant’ definition. Distinct populations, such as children and allergy and asthma sufferers are highly susceptible, while a low socioeconomic background is a further susceptibility factor; however, no specific guidance is available. We spend most of our time indoors; for children, the school environment is of paramount importance and potentially amenable to intervention. The interactions between some pollutant classes have been studied; for example, respiratory viral infection induces hyperresponsiveness to allergens, as well as irritants. However, a lot is missing in respect to understanding interactions between specific pollutants of different classes in terms of concentrations, timing, and sequence, to improve targeting and upgrade standards. SynAir-G is a European Commission-funded project aiming to reveal and quantify synergistic interactions between different pollutants affecting health, from mechanisms to real-life, focusing on the school setting. It will develop a comprehensive and responsive multipollutant monitoring system, advance environmentally friendly interventions, and disseminate the generated knowledge to relevant stakeholders in accessible and actionable formats.

IntroductionThe coronavirus disease 2019 (COVID-19) pandemic was accompanied by an overabundance of fake news increasing the risk of developing false memories (FMs). Previous studies have shown that the relationship between fake news and FMs could be mediated by some individual variables, including attitudinal biases. We explored the role of these variables in true memories (TMs) and FMs formation, with special emphasis on vaccine- and Green Pass (GP)-related topics. MethodWe set up a large online survey exploring several constructs including media usage, attitude toward vaccines and GP, perceived (PK) and objective knowledge (OK) about COVID-19-related information, fear of the disease, depression and anxiety symptoms, coping mechanisms, and reasoning skills. Then, we asked participants whether they remembered certain news (true or fake), providing confidence ratings. ResultsData from 289 respondents (198 females) from the general population were analyzed. Participants with positive attitude reported a greater fear that their loved ones contracted the COVID-19, a more frequent use of traditional media, and a higher PK when compared with respondents with negative attitude. On the whole sample, participants reported higher confidence levels when required to judge their memory of true than fake news; however, participants with positive attitude reported a higher confidence for both true and fake news. The relationship between attitude and TM confidence was mediated by the PK, whereas the relationship between attitude and FM confidence was probably affected by OK. ConclusionAttitude can modulate individual behaviors in the context of health issues. The PK and OK may interact with attitude in the memory formation.

African swine fever (ASF) is one of the most dangerous hemorrhagic infectious diseases that affect domestic and wild pigs. Currently, neither a vaccine nor effective treatments are available for this disease. As regards the degree of virulence, ASFV strains can be divided into high, moderate, or low virulence. The main detection methods are based on the use of the polymerase chain reaction (PCR). In order to prevent an uncontrolled spread of ASF, new on-site techniques that can enable the identification of an early-stage disease are needed. We have developed a specific immunological SPR-based assay for ASFV antigen detection directly in liquid samples. The developed assay allows us to detect the presence of ASFV at the dose of 103 HAD50/mL.

Odorant-binding proteins (OBPs) are a group of small and soluble proteins present in both vertebrates and insects. They have a high level of structural stability and bind to a large spectrum of odorant molecules. In the environmental field, benzene is the most dangerous compound among the class of pollutants named BTEX (benzene, toluene, ethylbenzene, and xylene). It has several effects on human health and, consequently, it appears to be important to monitor its presence in the environment. Commonly, its detection requires the use of very sophisticated and time-consuming analytical techniques (GC-MS, etc.) as well as the presence of specialized personnel. Here, we present the application of an odorant-binding protein (pOBP) isolated from pigs as a molecular recognition element (MRE) for a low-energy impedenziometric biosensor for outdoor and real-time benzene detection. The obtained results show that the biosensor can detect the presence of 64 pM (5 µg/m3) benzene, the limit value of exposure for human health set by the European Directive 2008/50/EC.

Detection of ciprofloxacin residues in milk by sensitive and rapid methods is of great interest due to its use in the treatment of dairy livestock health. Current analytical approaches to antibiotics detection, are laboratory-based methods and they are time-consuming and require trained personnel. To cope this problem, we propose an assay, based on fluorescence polarization principle, able to detect the presence of ciprofloxacin in diluted milk sample without any pre-treatment. The proposed method is based on the use of ciprofloxacin-protein conjugate labeled with near infrared fluorescence dye, which upon binding to specific antibody causes an increase of the fluorescence polarization emission signal. The developed assay allows for the detection of ciprofloxacin at a concentration of 1ppb, which represents an amount lower than the maximum residual limit (MRL) of ciprofloxacin in milk, as set by the European Union regulation (100 ppb).

In this paper we present the development of photonic integrated circuit (PIC) biosensors for the label-free detection of six emerging and endemic swine viruses, namely: African Swine Fever Virus (ASFV), Classical Swine Fever Virus (CSFV), Porcine Reproductive and Respiratory Syndrome Virus (PPRSV), Porcine Parvovirus (PPV), Porcine Circovirus 2 (PCV2), and Swine Influenza Virus A (SIV). The optical biosensors are based on evanescent wave technology and, in particular, on Resonant Rings (RRs) fabricated in silicon nitride. The novel biosensors were packaged in an integrated sensing cartridge that included a microfluidic channel for buffer/sample delivery and an optical fiber array for the optical operation of the PICs. Antibodies were used as molecular recognition elements (MREs) and were selected based on western blotting and ELISA experiments to ensure the high sensitivity and specificity of the novel sensors. MREs were immobilized on RR surfaces to capture viral antigens. Antibody–antigen interactions were transduced via the RRs to a measurable resonant shift. Cell culture supernatants for all of the targeted viruses were used to validate the biosensors. Resonant shift responses were dose-dependent. The results were obtained within the framework of the SWINOSTICS project, contributing to cover the need of the novel diagnostic tools to tackle swine viral diseases.

Background: The relationship binding body weight to psychological well-being is unclear. The present study aims at identifying the contribution, and specificity, of some dimensions (i.e., eating-related symptoms, body image disorders, eating habits, personality traits, and emotional difficulties) characterizing the psychological profile of obese adolescents (749 participants, 325 females; 58.3% normal-weight, 29.9% overweight, and 11.7% obese; mean age = 16.05, SD = 0.82). Methods: By introducing the scores obtained by standardized self-report tools into a generalized linear model, a factorial reduction design was used to detect the best fitting discriminant functions and the principal components explaining the higher proportion of the variance. Results: We found two discriminant functions correctly classifying 87.1% of normal-weight, 57.2% of overweight, and 68.2% of obese adolescents. Furthermore, two independent factors, explaining 69.68% of the total variance, emerged. Conclusions: The first factor, “Body Image Concerns”, included the drive for thinness, body dissatisfaction, and interpersonal distrust. The second factor, “Selective Depersonalization”, included a trend toward depersonalization and dissatisfaction with the torso. The neurophysiological implications of our findings will be discussed.

The purpose of this work is to provide an exhaustive overview of the emerging biosensor technologies for the detection of analytes of interest for food, environment, security, and health. Over the years, biosensors have acquired increasing importance in a wide range of applications due to synergistic studies of various scientific disciplines, determining their great commercial potential and revealing how nanotechnology and biotechnology can be strictly connected. In the present scenario, biosensors have increased their detection limit and sensitivity unthinkable until a few years ago. The most widely used biosensors are optical-based devices such as surface plasmon resonance (SPR)-based biosensors and fluorescence-based biosensors. Here, we will review them by highlighting how the progress in their design and development could impact our daily life.

The synthesis of two 5&prime, end (4-dimethylamino)azobenzene conjugated G-quadruplex forming aptamers, the thrombin binding aptamer (TBA) and the HIV-1 integrase aptamer (T30695), was performed. Their structural behavior was investigated by means of UV, CD, fluorescence spectroscopy, and gel electrophoresis techniques in K+-containing buffers and water-ethanol blends. Particularly, we observed that the presence of the 5&prime, (4-dimethylamino)azobenzene moiety leads TBA to form multimers instead of the typical monomolecular chair-like G-quadruplex and almost hampers T30695 G-quadruplex monomers to dimerize. Fluorescence studies evidenced that both the conjugated G-quadruplexes possess unique fluorescence features when excited at wavelengths corresponding to the UV absorption of the conjugated moiety. Furthermore, a preliminary investigation of the trans-cis conversion of the dye incorporated at the 5&prime, end of TBA and T30695 showed that, unlike the free dye, in K+-containing water-ethanol-triethylamine blend the trans-to-cis conversion was almost undetectable by means of a standard UV spectrophotometer.

We have developed and characterized a surface plasmon resonance (SPR) biosensor in a plastic optical fiber (POF) to detect traces of naphthalene (NAPTHA) in water. As previously developed for perfluorinated compounds (PFAs), the gold surface of the plasmonic POF sensor has been chemically modified through the formation of specific reactive groups and functionalized with antibodies able to specifically recognize the NAPTHA. The obtained limit of detection value (0.76ng/mL) is lower than the maximum residue limit value of naphthalene (0.13µg/mL) referred as the water environmental quality standards.

L-glutamate (Glu) is the major excitatory transmitter in mammalian brain. Inadequate concentration of Glu in the brain correlates to mood disorder. In industry, Glu is used as a flavour enhancer in food and in foodstuff processing. A high concentration of Glu has several effects on human health such as hypersensitive effects, headache and stomach pain. The presence of Glu in food can be detected by different analytical methods based on chromatography, or capillary electrophoresis or amperometric techniques. We have isolated and characterized a glutamate-binding protein (GluB) from the Gram-positive bacteria Corynebacterium glutamicum. Together with GluC protein, GluD protein and the cytoplasmic protein GluA, GluB permits the transport of Glu in/out of cell. In this study, we have investigated the binding features of GluB as well as the effect of temperature on its structure both in the absence and in the presence of Glu. The results have showed that GluB has a high affinity and selectivity versus Glu (nanomolar range) and the presence of the ligand induces a higher thermal stability of the protein structure. (C) 2020 Elsevier B.V. All rights reserved.

Human heparanase (HPSE) is an enzyme that degrades the extracellular matrix. It is implicated in a multiplicity of physiological and pathological processes encouraging angiogenesis and tumor metastasis. The protein is a heterodimer composed from a subunit of 8 kDa and a subunit of 50 kDa. The two protein subunits are non-covalently associated. The cloning and expression of the two protein subunits in Escherichia coli, and their subsequent purification to homogeneity under native conditions result in the production of an active heparanase enzyme. The substrate specificity of the HPSE was studied by docking of a putative substrate that is a designed oligosaccharide with the minimum recognition backbone, with the additional 2-N-sulfate and 6-O-sulfate groups at the non-reducing GlcN and a fluorogenic tag at the reducing extremity GlcN. To develop a quantitative fluorescence assay with this substrate would be extremely useful in studies on heparanase, since the heparanase cleavage of fluorogenic tag would result in a measurable response. This article is protected by copyright. All rights reserved

Background The use of media device, such as smartphone and tablet, is currently increasing, especially among the youngest. Adolescents spend more and more time with their smartphones consulting social media, mainly Facebook, Instagram and Twitter because. Adolescents often feel the necessity to use a media device as a means to construct a social identity and express themselves. For some children, smartphone ownership starts even sooner as young as 7 yrs, according to internet safety experts. Material and methods We analyzed the evidence on media use and its consequences in adolescence. Results In literature, smartphones and tablets use may negatively influences the psychophysical development of the adolescent, such as learning, sleep and sigh. Moreover, obesity, distraction, addiction, cyberbullism and Hikikomori phenomena are described in adolescents who use media device too frequently. The Italian Pediatric Society provide action-oriented recommendations for families and clinicians to avoid negative outcomes. Conclusions Both parents and clinicians should be aware of the widespread phenomenon of media device use among adolescents and try to avoid psychophysical consequences on the youngest.

In this study we developed an optical fiber biosensor able to detect the presence of naphthalene in sea-water. With this aim, we designed and produced an antibody specific for the naphthalene molecule. The capability of the antibody to bind to naphthalene was characterized by ELISA tests. A surface plasmon resonance (SPR) sensor platform was realized by sputtering a gold layer on a modified plastic optical fiber (POF). The gold surface was derivatizated and functionalized with the produced antibody by using the EDC/NHS amino-coupling immobilization protocol. The obtained results indicated that the POF-biosensor is able to sense the presence of naphthalene in a sea-water solution. The limit of detection (LOD) value was calculated to be 0.76 ng/mL, a value lower than the maximum residue limit value of naphthalene (0.13 µg/mL) referred as the water environmental quality standards (EQS). In addition, to the high sensitivity of the assay, it is remarkable to point out the possibility to monitor the presence of naphthalene in a real sea water solution by exploiting a simple experimental setup with a remote sensing capability offered by the POF-biosensor.

The structure and stability of the fluorescent protein monomeric Kusabira Orange (mKO), a GFP-like protein, was studied under different pressure levels and in different chemical environments. At different pH values (between pH 7.4 and pH 4.0) and under a pressure up to 600 MPa (at 25 °C), mKO did not show significant fluorescence spectral changes, indicating a structural stability of the protein. In more extreme chemical conditions (at pH 4.0 in the presence of 0.8 M guanidine hydrochloride), a marked reduction of mKO fluorescence intensity emission was observed at pressures above 300 MPa. This fluorescence emission quenching may be due to the loss of the intermolecular bonds and, consequently, to the destructuration of the mKO chromophore structure. Since the electrostatic and hydrophobic interactions as well as the salt bridges present in proteins are usually perturbed under high pressure, the reduction of mKO fluorescence intensity emission is associated to the perturbation of the protein salt bridges network., Highlights • Effect of pressure on the protein structure and function by fluorescence spectroscopy. • mKO as a model system under high-pressure perturbation. • Pressure effect on its structure and stability.

Documento redatto dagli autori nell'ambito del progetto Interdipartimentale Foresight S&T internazionale del CNR costituito con protocollo AMMCNT-CNR no. 0049037 in data 14/07/2015 a firma del Presidente. Il documento è stato presentato e discusso nella riunione del CE del progetto in data 23/01/2018.

A novel surface plasmon resonance (SPR) optical fiber biosensor, able to bind perfluorooctanoate and per-fluorooctanesulfonate compounds, is presented. In the first step, an ad hoc antibody compound has been designed, produced and tested by ELISA, then, in the second step, the gold surface of a plastic optical fiber sensor has been derivatizated and functionalized with this new bio-receptor, able to bind target analytes with high affinity and selectivity. The experimental data have shown that the developed SPR optical fiber biosensor makes it possible to detect these compounds. One advantage of this approach stems from the possibility to monitor the perfluorinated compounds in the environment exploiting the remote sensing capability offered by the optical fibers. The measurements were performed in laboratory, also exploiting matrices mimicking the real environment. The limit of detection of the assay was 0.21 ppb, a value that is lower than the maximum residue limit fixed by the European Union regulations.

The synthesis and the structural characterization of azobenzene-conjugated GQs were formed. Firstly, we synthesized the R 4-(dimethylamino)azobenzene glycerol derivative (4-DMAzo). Trans-cis photo-isomerisation of 4-DMAzo can be induced by violet light, suitable to be used on DNA systems (1). Furthermore, the two hydroxyl groups of the glycerol linker inserted on one benzene ring are used to convert the azobenzene derivative into the appropriate phosphoramidite building block, thus allowing to the synthesis of 4-DMAzo 5'-end conjugated TBA and T30695. TBA forms a chair-like monomolecular and antiparallel GQ (2), whereas parallel-stranded monomers of T30695-GQs stack at 5'-ends to form very stable GQ dimers (3). We have explored by CD, CD melting, UV fluorescence and Electrophoresis Mobility Shift Assay (EMSA) techniques, the folding properties of the two G-quadruplex forming aptamers. From the collected data it appeared that the conjugated sequences are able to fold into GQs very similar to that of the corresponding unmodified aptamers. CD melting analyses evidenced that the difference between the melting temperatures of conjugated DMAzo-TBA-GQ and TBA is∼-6°C whereas that of DMAzo-T30695-GQ and T30695 was ∼+10°C. Furthermore, cis-trans photo-isomerisation of DMAzo moiety conjugated at 5'-end GQs, was preliminarily explored by UV-Vis spectroscopy. Spectra of free DMAzo, of 5'-DMAzo-TBA and 5'-DMAzo-T30695 were performed before and after irradiation by LED light at 435 nm. We also explored the fluorescence proprieties of the two conjugated GQ sequences in comparison to that of TBA and T30695 (4). In particular, steady-state emission spectra were acquired at 25 °C using buffered phosphate solution at pH 7.0 or ethanol/water as solvents. The preliminary results show that the intrinsic fluorescence of the GQs depends on the presence of DMAzo moiety at 5'-end as well as the medium used as solvent.

In this work, a "bio-electronic nose" for vapour phase detection of odorant molecules based on surface acoustic wave (SAW) resonators is presented. The biosensor system is composed of an array of five SAW resonators coated with three types of odorant-binding proteins (OBPs): the wild-type OBP from bovine (wtbOBP), a double-mutant of the OBP from bovine (dmbOBP), and the wild-type OBP from pig (wtpOBP). High resolution deposition of OBPs onto the active area of SAW resonators was implemented through laser-induced forward transfer (LIFT). The resonant frequency shifts of the SAW resonators after the deposition of the biomolecules confirmed the immobilisation of the proteins onto the Al/Au inter-digital transducers (IDTs). In addition, a low increase of insertion losses with a limited degradation of Q-factors is reported. The "bio-electronic nose" fabricated by LIFT is tested in nitrogen upon exposure to separated concentrations of R-(-)-1-octen-3-ol (octenol) and R-(-)-carvone (carvone) vapours. The "bio-electronic nose" showed low detection limits for the tested compounds (i.e. 0.48 ppm for the detection of octenol, and 0.74 ppm for the detection of carvone). In addition, the bio-sensing system was able to discriminate the octenol molecules from the carvone molecules, making it pertinent for the assessment of food contamination by moulds, or for the evaluation of indoor air quality in buildings.

In recent years, air pollution has been a subject of great scientific and public interests for the strong impact on human health. Air pollution is due to the presence in the atmosphere of polluting substances, such as carbon monoxide, sulfur and nitrogen oxides, particulates and volatile organic compounds (VOCs), derived predominantly from various combustion processes. Benzene is a VOC belonging to group-I carcinogens with a toxicity widely demonstrated. The emission limit values and the daily exposure time to benzene (TLV-TWA) are 5?g/m3 (0.00157 ppm) and 1.6mg/m3 (0.5 ppm), respectively. Currently, expensive and time-consuming analytical methods are used for detection of benzene. These methods require to perform a few preliminary steps such as sampling, and matrices pre-treatments. In addition, it is also needed the support of specialized personnel. Recently, single-walled carbon nanotube (SWNTs) gas sensors with a limit detection (LOD) of 20 ppm were developed for benzene detection. Other innovative bioassay, called bio-report systems, were proposed. They use a whole cell (Pseudomona putida or Escherichia coli) as molecular recognition element and exhibit a LOD of about 10 ?M. Here, we report on the design of a highly sensitive fluorescence assay for monitoring atmospheric level of benzene. For this purpose, we used as molecular recognition element the porcine odorant-binding protein (pOBP). 1-Aminoanthracene was selected as extrinsic fluorescence probe for designing a competitive fluorescence resonance energy transfer (FRET) assay for benzene detection. The detection limit of our assay was 3.9?g/m3, a value lower than the actual emission limit value of benzene as regulated by European law.

Over the last few decades the development of new technologies, the fabrication of new materials, and the introduction of nanotechnologies created new trends in a series of advances that produced innovations in biological sensing devices with a wide range of application from health, security, defense, food, and medicine, to the environment. Specificity, low cost, rapidity, sensitivity, and multiplicity are some of the reasons for their growth, and their commercial success is expected to increase in the next future. Biosensors are devices in which the recognition part of the target molecule is accomplished by biological macromolecules such as proteins, enzymes, antibodies, aptamers, etc. These biomolecules are able to bind to the target molecules with high selectivity and specificity. The interaction between the target molecule and the specific biomolecule is reflected as a change of the biomolecule structural features. The extent of this change is strictly related to the biosensor response. Fluorescence spectroscopy, due to its sensitivity, is often used as the principal technique to monitor biological interactions, and thus the biosensor response as well. Both the intrinsic ultraviolet fluorescence of protein, arising from aromatic amino acids (tryptophan, tyrosine, and phenylalanine), and extrinsic fluorescent labels emitting in the visible region of the spectrum together allow for very flexible transduction of the analyte recognition, suitable for many different applications. This chapter focuses special attention on enzymes as practically unmatched recognition elements for biosensors and emphasizes the potential advantages of customized biosensor devices using apo- or holo forms of enzymes also isolated from thermophile sources.

The MariaBox project, funded by the European Commission (Contract 614088), is developing an autonomous, analytical device, based on novel biosensors, for monitoring chemical and biological pollutants in sea water. The device, currently at first prototype level, is suitable for installation in free floating devices, buoys or ships. The main, high-level user requirements for the system are for the device to be of high-sensitivity, portable and capable of repeating measurements over a long time, allowing long-term deployment at sea. The first phase of the project, “user requirements collection”, was dedicated to understanding the current needs of water monitoring through institutions in different areas of Europe, at different location types. The locations in which the research was focused are also the ones in which the field validation of MariaBox will take place: a sea water lagoon in Spain with different characteristics of water salinity and natural environment, currently exploited for shellfish farming; a natural site in Ireland (Galway bay); the Skagerrak, a sea arm between Norway, Denmark and Sweden; and a harbour area close to industrial facilities in Cyprus. The analytes of interest for the monitoring institutions are coherent to the list of pollutants to be monitored by the European Commission. A first trade-off between end-user requirements and engineering feasibility, available time and final system cost had to be made. On one hand, the MariaBox device is intended to be portable and its size must be such as to allow permanent positioning inside sea buoys. Several modules are required to achieve the aim of monitoring, in an autonomous and unattended way, all selected target analytes for a period of several months. Energy expenditure is a relevant constraint. Nevertheless, being a device deployed at sea, energy scavenging is a real option to guarantee the system autonomy or, at least, to keep the system operating at a minimum level, that is, to maintain the biosensors and the biochemical reactants in a controlled and safe environment. This controlled environment is managed by the analytical core unit of MariaBox, that acts as a cooler or heater depending on the external temperatures (from −10°C up to +50°C). The system design has been confronted with all of these challenging requirements. The device is intended to provide not only scientific data but also early warnings in relation to both algal blooming and production of toxins, as well as to chemical, man-made pollutants (heavy metals, camphechlor, naphthalene, PFOS). This means that considering the European thresholds for pollutants is not enough. The MariaBox detection capacity has to be under the legal threshold, so as to enable early warnings and the possibility to prevent the pollution with adequate countermeasures. The first MariaBox prototype has been recently produced and is currently being tested and validated in the lab. Within 2017, the device will be replicated and 4 replicas will be installed in the 4 selected pilot locations.

Over the last few decades the development of new technologies, the fabrication of new materials, and the introduction of nanotechnologies created new trends in a series of advances that produced innovations in biological sensing devices with a wide range of application from health, security, defense, food, and medicine, to the environment. Specificity, low cost, rapidity, sensitivity, and multiplicity are some of the reasons for their growth, and their commercial success is expected to increase in the next future. Biosensors are devices in which the recognition part of the target molecule is accomplished by biological macromolecules such as proteins, enzymes, antibodies, aptamers, etc. These biomolecules are able to bind to the target molecules with high selectivity and specificity. The interaction between the target molecule and the specific biomolecule is reflected as a change of the biomolecule structural features. The extent of this change is strictly related to the biosensor response. Fluorescence spectroscopy, due to its sensitivity, is often used as the principal technique to monitor biological interactions, and thus the biosensor response as well. Both the intrinsic ultraviolet fluorescence of protein, arising from aromatic amino acids (tryptophan, tyrosine, and phenylalanine), and extrinsic fluorescent labels emitting in the visible region of the spectrum together allow for very flexible transduction of the analyte recognition, suitable for many different applications. This chapter focuses special attention on enzymes as practically unmatched recognition elements for biosensors and emphasizes the potential advantages of customized biosensor devices using apo- or holo forms of enzymes also isolated from thermophile sources.

The promising direction of the development of a modern glucometer is the construction of sensing element on the basis of stained (dyed) protein which changes its fluorescence upon glucose binding. One of the proteins that can be used for this purpose is the D -trehalose/ D -maltose-binding protein (TMBP) from the thermophilic bacteria Thermococcus litoralis . We investigated the physical–chemical properties of the protein and evaluated its stability to the denaturing action of GdnHCl and heating. It was confirmed that TMBP is an extremely stable protein. In vivo , the intrinsic ligands of TMBP are trehalose and maltose, but TMBP can also bind glucose. The dissociation constant of the TMBP–glucose complex is in the range of 3–8 mM. The binding of glucose does not noticeably change the intrinsic fluorescence of the TMBP. To register protein-glucose binding, we used the fluorescence of the thiol-reactive dye BADAN attached to TMBP. Because the fluorescence of BADAN attached to the cysteine Cys182 of TMBP does not change upon glucose binding, the mutant forms ТМВР/C182S/X_Cys were created. In these mutant proteins, Cys182 is replaced by Ser, removing intrinsic binding site of BADAN and a new dye binding sites were introduced. The largest increase (by 1.4 times) in the intensity of the dye fluorescence was observed upon TMBP/C182S/A14C-BADAN–Glc complex formation. The dissociation constant of this complex is 3.4 ± 0.1 mM. We consider TMBP/C182S/A14C mutant form with attached fluorescent dye BADAN as a good basis for further research aimed to develop of series of TMBP mutant forms with different affinities to glucose labeled with fluorescent dyes.

Patulin is a toxic secondary metabolite of a number of fungal species belonging to the genera Penicillium and Aspergillus. One important aspect of the patulin toxicity in vivo is an injury of the gastrointestinal tract including ulceration and inflammation of the stomach and intestine. Recently, patulin has been shown to be genotoxic by causing oxidative damage to the DNA, and oxidative DNA base modifications have been considered to play a role in mutagenesis and cancer initiation. Conventional analytical methods for patulin detection involve chromatographic analyses, such as HPLC, GC, and, more recently, techniques such as LC/MS and GC/MS. All of these methods require the use of extensive protocols and the use of expensive analytical instrumentation. In this work, the conjugation of a new derivative of patulin to the bovine serum albumin for the production of polyclonal antibodies is described, and an innovative competitive immune-assay for detection of patulin is presented. Experimentally, an important part of the detection method is based on the optical technique called surface plasmon resonance (SPR). Laser beam induced interactions between probe and target molecules in the vicinity of gold surface of the biochip lead to the shift in resonance conditions and consequently to slight but easily detectable change of reflectivity.

d -serine is an amino acid that has an important role in the regulation of N-methyl- d -aspartate (NMDA) signalling in the central nervous system. In fact, it acts as an endogenous co-agonist molecule for the NMDA receptor activation. Since an abnormal body level of d -amino acids is usually associated to different neurological diseases, it appears evident that it is of high interest to develop a fast and specific biosensor for the determination of d -serine. In this work, we report on the design and development of a surface acoustic wave (SAW) biosensor to detect the level of d -serine. The SAW-biosensor is based on the utilization of an inactivated form of the d -serine dehydratase isolated from Saccharomyces cerevisiae , a biomolecule that is able to specifically bind d -serine. In particular, we produced a PLP-reduced d -serine dehydratase biomolecule that was still able to bind d -serine and not to transform it into the product. The binding event between the “inactivated” d -serine dehydratase and d -serine was monitored by SAW delay-lines.

Background/Purpose: Laparoscopic fundoplication (LF) represents the gold standard for surgical treatment for pediatric patients with gastroesophageal reflux disease (GERD). Methods: We report the results of long-term outcome of 36 patients who had undergone LF from January to December 1998, with a follow-up longer than 10 years (range 11–12 years). The patients were invited, by phone, to undergo a clinical follow-up. All patients underwent the modified European Society for Pediatric Gastroenterology Hepatology and Nutrition (ESPGHAN)’s Roma III questionnaire; however, only 22 out of 36 patients accepted to be controlled in a day hospital setting, and 10 out of 36 accepted to undergo a telephonic questionnaire. Our study is focused on the data of these 32 patients. Results: Twenty-eight out of 32 (87.5%) patients had completely recovered; 4 out of 32 patients (12.5%) had a mild persistent GER; 9 out of 32 patients (28%) referred a mild dysphagia; 21 out of 32 (66%) patients could burp; and only 9 out of 32 (28%) patients could vomit. The cosmetic result was good in 30 out of 32 (94%) patients. The weight/height ratio was satisfactory in 28 out of 32 (87.5%) patients. The quality of life was good in 28 out of 32 (87.5%) patients. Conclusions: Our experience shows that the long-term follow-up after LF produces a good clinical result and a good quality of life. The modified ESPGHAN’s Roma III questionnaire seems an effective way to check the longterm results, because it avoids submitting patients to long and not well tolerated instrumental exams.

In this work we present an innovative optical sensing methodology based on the use of biomolecules as molecular gating nano-systems. Here, as an example, we report on the detection of analytes related to climate change. In particular, we focused our attention on the detection of nitric oxide (NO) and oxygen (O2). Our methodology builds on the possibility of modulating the excitation intensity of a fluorescent probe used as a transducer and a sensor molecule whose absorption is strongly affected by the binding of an analyte of interest used as a filter. The two simple conditions that have to be fulfilled for the method to work are: (a) the absorption spectrum of the sensor placed inside the cuvette, and acting as the recognition element for the analyte of interest, should strongly change upon the binding of the analyte and (b) the fluorescence dye transducer should exhibit an excitation band which overlaps with one or more absorption bands of the sensor. The absorption band of the sensor affected by the binding of the specific analyte should overlap with the excitation band of the transducer. The high sensitivity of fluorescence detection combined with the use of proteins as highly selective sensors makes this method a powerful basis for the development of a new generation of analytical assays. Proof-of-principle results showing that cytochrome c peroxidase (CcP) for NO detection and myoglobin (Mb) for O2 detection can be successfully used by exploiting our new methodology are reported. The proposed technology can be easily expanded to the determination of different target analytes.

In this work the thermophilic trehalose/maltose-binding protein from Thermococcus litoralis is presented as a probe for the design of a high stable fluorescence biosensor for glucose. In particular, we show the possibility of modulating the protein specificity by changing temperature. In addition to glucose sensing, we also report on the possibility of utilizing odorant-binding proteins as a probe for the development of optical sensors for analytes of environmental interests.