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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.

Thermotoga maritima Arginine Binding Protein (TmArgBP) is a valuable candidate for arginine biosensing in diagnostics. This protein is endowed with unusual structural properties that include an extraordinary thermal/chemical stability, a domain swapped structure that undergoes large tertiary and quaternary structural transition, and the ability to form non-canonical oligomeric species. As the intrinsic stability of TmArgBP allows for extensive protein manipulations, we here dissected its structure in two parts: its main body deprived of the swapping fragment (TmArgBP(20-233)) and the C-terminal peptide corresponding to the helical swapping element. Both elements have been characterized independently or in combination using a repertoire of biophysical/structural techniques. Present investigations clearly indicate that TmArgBP(20-233) represents a better scaffold for arginine sensing compared to the wild-type protein. Moreover, our data demonstrate that the ligand-free and the ligand-bound forms respond very differently to this helix deletion. This drastic perturbation has an important impact on the ligand-bound form of TmArgBP(20-233) stability whereas it barely affects its ligand-free state. The crystallographic structures of these forms provide a rationale to this puzzling observation. Indeed, the arginine-bound state is very rigid and virtually unchanged upon protein truncation. On the other hand, the flexible ligand-free TmArgBP(20-233) is able to adopt a novel state as a consequence of the helix deletion. Therefore, the flexibility of the ligand-free form endows this state with a remarkable robustness upon severe perturbations. In this scenario, TmArgBP dissection highlights an intriguing connection between destabilizing/stabilizing effects and the overall flexibility that could operate also in other proteins.

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

We examined the effects of metallic silver island films on resonance energy transfer (RET) between a donor and acceptor bound to double helical DNA. The donor was 4′,6-diamidino-2-phenylindole (DAPI) and the acceptor was propidium iodide (PI). Proximity of the labeled DNA to the silver particles resulted in a dramatic increase in RET as seen from the emission spectra and the donor decay times. Proximity to silver particles results in an increase of the Förster distance from 35 Å to an apparent value of 166 Å(.) These results suggest a new type of DNA hybridization assays based on RET over distances much longer than the free-space Forster distance.

Damage-associated molecular patterns (DAMPs) are molecules that can be actively or passively released by injured tissues and that activate the immune system. Here we show that nicotinate phosphoribosyltransferase (NAPRT), detected by antibody-mediated assays and mass spectrometry, is an extracellular ligand for Toll-like receptor 4 (TLR4) and a critical mediator of inflammation, acting as a DAMP. Exposure of human and mouse macrophages to NAPRT activates the inflammasome and NF-κB for secretion of inflammatory cytokines. Furthermore, NAPRT enhances monocyte differentiation into macrophages by inducing macrophage colony-stimulating factor. These NAPRT-induced effects are independent of NAD-biosynthetic activity, but rely on NAPRT binding to TLR4. In line with our finding that NAPRT mediates endotoxin tolerance in vitro and in vivo, sera from patients with sepsis contain the highest levels of NAPRT, compared to patients with other chronic inflammatory conditions. Together, these data identify NAPRT as a endogenous ligand for TLR4 and a mediator of inflammation., The enzyme nicotinate phosphoribosyltransferase (NAPRT) mediates the rate-limiting step in NAD salvage pathway starting from nicotinic acid. Here the authors show that NAPRT can be detected extracellularly, binds to Toll like receptor 4, and activates NF-kB signaling and cytokine production in macrophage via NAD synthesis-independent pathways.

This paper presents the effects of the composition of different media (i.e., Tryptic soy broth (TSB), Brain heart infusion (BHI), Listeria enrichment broth (LEB), Fraser broth (FB) and University of Vermont medium (UVM)) on the detection of a short peptide fragment PepD specific to the p60 protein (p60) of L. monocytogenes by a monoclonal antibody (anti-PepD mAb). Expression of the p60 obtained was demonstrated to be proportional to the cellular growth of Listeria monocytogenes regardless of the tested growth medium. However, the early growth of L monocytogenes and the expression of the p60 were negatively affected by the presence of selective agents present in LEB, FB and UVM. Among those three selective enrichment media commonly used for L. monocytogenes, LEB allowed a better expression of L. monocytogenes p60 after an incubation period of 18 h. Optimization of the LEB revealed that the dextrose concentration was the critical factor for improving the expression of p60 and promotes the early expression of p60. Moreover, an optimal dextrose concentration of 0.5% (w/v) in LEB, coupled with anti-PepD mAb immobilized to solid support, reduced the detection of p60 from 18 h to 9 h for an initial concentration of L. monocytogenes of 10(8) CFU/ml. (C) 2019 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

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.

Even if cancer specific biomarkers are present in peripheral blood of cancer patients, it is very difficult to detect them with conventional technology because of their low concentration. A potential cancer biomarker is the HMGA1b protein, whose overexpression is a feature of several human malignant neoplasias. By taking advantage of the surface plasmon resonance (SPR) phenomenon, we realized a specific nano/technology-based assay for cancer detection. More in details, anti-HMGA1b monoclonal antibodies, whose affinity was previously defined by ELISA, were immobilized onto metallic surfaces to develop a direct SPR-based assay. After having analyzed blood samples from colorectal cancer patients and healthy people for the presence of HMGA1b, we observed a 2-fold increase of the HMGA1b levels in the blood of cancer patients with respect to the healthy control people. We conclude that the set-up technology might allow to detect a tumoral mass through the evaluation of HMGA1b protein blood levels.

In this paper we introduce a field diagnostic device based on the combination of advanced bio-sensing and photonics technologies, to tackle emerging and endemic viruses causing swine epidemics, and consequently significant economic damage in farms. The device is based on the use of microring resonators fabricated in silicon nitride with CMOS compatible techniques. In the paper, the designed and fabricated photonic integrated circuit (PIC) sensors are presented and characterized, showing an optimized performance in terms of optical losses (30 dB per ring) and extinction ration for ring resonances (15 dB). Furthermore, the results of an experiment for porcine circovirus 2 (PCV2) detection by using the developed biosensors are presented. Positive detection for different virus concentrations has been obtained. The device is currently under development in the framework of the EU Commission co-funded project SWINOSTICS.

ER–TGN contact sites (ERTGoCS) have been visualized by electron microscopy, but their location in the crowded perinuclear area has hampered their analysis via optical microscopy as well as their mechanistic study. To overcome these limits we developed a FRET-based approach and screened several candidates to search for molecular determinants of the ERTGoCS. These included the ER membrane proteins VAPA and VAPB and lipid transfer proteins possessing dual (ER and TGN) targeting motifs that have been hypothesized to contribute to the maintenance of ERTGoCS, such as the ceramide transfer protein CERT and several members of the oxysterol binding proteins. We found that VAP proteins, OSBP1, ORP9, and ORP10 are required, with OSBP1 playing a redundant role with ORP9, which does not involve its lipid transfer activity, and ORP10 being required due to its ability to transfer phosphatidylserine to the TGN. Our results indicate that both structural tethers and a proper lipid composition are needed for ERTGoCS integrity.

Authors present a very low-cost bio-sensing strategy, based on plastic optical fibers (POFs) and receptors, able to selective detection of substances in water solution. In this work several applications based on different kinds of receptors are reported, such as molecularly imprinted polymers (MIPs), chemical receptors (Deferoxamine and D,L-penicillamine) and bio-receptors (aptamers and antibodies). In particular, we have reported how, exploiting the same plasmonic platform in a D-shaped POF with these different kinds of receptors, interesting results have been achieved in medical diagnostics for cancer bio-markers detection, Celiac disease antigens monitoring, L-nicotine detection, and detection of Iron Fe(III) and copper Cu(II).

This paper presents the development of a support membrane based on chitosan, cellulose nanocrystals and glycerol (m-CCG) for the antibody immobilization by a covalent crosslinking using glutaraldehyde. The chemical characterization of the support by FTIR showed that m-CCG formation process was stabilized by the formation of hydrogen bonding between each component of m-CCG and the reactive amine groups allowing the antibody immobilization on m-CCG via glutaraldehyde. Moreover, this immobilization on m-CCG was optimized by mathematics modeling approaches, and it exhibited robustness and predictable detection in presence of 0.6% of cellulose nanocrystals (CNCs), 0.5 g of CCG solution per well, after 2 h of antibody immobilization. Results also showed that CNCs (0.6% w/v) was the most important factor of the optimization. At this concentration, CNCs improve the resistance of m-CCG during the crosslinking treatment by a modification of the surface topography and the reinforcement of the tensile strength of m-CCG at >30%.

Background Shp1, a tyrosine-phosphatase-1 containing the Src-homology 2 (SH2) domain, is involved in inflammatory and immune reactions, where it regulates diverse signalling pathways, usually by limiting cell responses through dephosphorylation of target molecules. Moreover, Shp1 regulates actin dynamics. One Shp1 target is Src, which controls many cellular functions including actin dynamics. Src has been previously shown to be activated by a signalling cascade initiated by the cytosolic-phospholipase A2 (cPLA2) metabolite glycerophosphoinositol 4-phosphate (GroPIns4P), which enhances actin polymerisation and motility. While the signalling cascade downstream Src has been fully defined, the mechanism by which GroPIns4P activates Src remains unknown. Methods Affinity chromatography, mass spectrometry and co-immunoprecipitation studies were employed to identify the GroPIns4P-interactors; among these Shp1 was selected for further analysis. The specific Shp1 residues interacting with GroPIns4P were revealed by NMR and validated by site-directed mutagenesis and biophysical methods such as circular dichroism, isothermal calorimetry, fluorescence spectroscopy, surface plasmon resonance and computational modelling. Morphological and motility assays were performed in NIH3T3 fibroblasts. Results We find that Shp1 is the direct cellular target of GroPIns4P. GroPIns4P directly binds to the Shp1-SH2 domain region (with the crucial residues being Ser 118, Arg 138 and Ser 140) and thereby promotes the association between Shp1 and Src, and the dephosphorylation of the Src-inhibitory phosphotyrosine in position 530, resulting in Src activation. As a consequence, fibroblast cells exposed to GroPIns4P show significantly enhanced wound healing capability, indicating that GroPIns4P has a stimulatory role to activate fibroblast migration. GroPIns4P is produced by cPLA2 upon stimulation by diverse receptors, including the EGF receptor. Indeed, endogenously-produced GroPIns4P was shown to mediate the EGF-induced cell motility. Conclusions This study identifies a so-far undescribed mechanism of Shp1/Src modulation that promotes cell motility and that is dependent on the cPLA2 metabolite GroPIns4P. We show that GroPIns4P is required for EGF-induced fibroblast migration and that it is part of a cPLA2/GroPIns4P/Shp1/Src cascade that might have broad implications for studies of immune-inflammatory response and cancer. Electronic supplementary material The online version of this article (10.1186/s12964-019-0329-3) contains supplementary material, which is available to authorized users.

In this paper, we present WaterSpy, a project developing an innovative, compact, cost-effective photonic device for pervasive water quality sensing, operating in the mid-IR spectral range. The approach combines the use of advanced Quantum Cascade Lasers (QCLs) employing the Vernier effect, used as light source, with novel, fibre-coupled, fast and sensitive Higher Operation Temperature (HOT) photodetectors, used as sensors. These will be complemented by optimised laser driving and detector electronics, laser modulation and signal conditioning technologies. The paper presents the WaterSpy concept, the requirements elicited, the preliminary architecture design of the device, the use cases in which it will be validated, while highlighting the innovative technologies that contribute to the advancement of the current state of the art.

The Arginine Binding Protein isolated from Thermotoga maritima (TmArgBP) is a protein endowed with several peculiar properties. We have previously shown that TmArgBP dimerization is a consequence of the swapping of the C-terminal helix. Here we explored the structural determinants of TmArgBP domain swapping and oligomerization. In particular, we report a mutational analysis of the residue Pro235, which is located in the hinge region of the swapping dimer. This residue was either replaced with a Gly-Lys dipeptide (TmArgBP P235GK ) or a Gly residue (TmArgBP P235G ). Different forms of these mutants were generated and extensively characterized using biophysical techniques. For both TmArgBP P235GK and TmArgBP P235G mutants, the occurrence of multiple oligomerization states (monomers, dimers and trimers) was detected. The formation of well-folded monomeric forms for these mutants indicates that the dimerization through C-terminal domain swapping observed in wild-type TmArgBP is driven by conformational restraints imposed by the presence of Pro235 in the hinge region. Molecular dynamics studies corroborate this observation by showing that Gly235 assumes conformational states forbidden for Pro residues in the TmArgBP P235G monomer. Unexpectedly, the trimeric forms present: (a) peculiar circular dichroism spectra, (b) a great susceptibility to heating, and (c) the ability to bind the Thioflavin T dye. The present findings clearly demonstrate that single-point mutations have an important impact on the TmArgBP oligomerization process. In a wider context, they also indicate that proteins endowed with an intrinsic propensity to swap have an easy access to states with altered structural and, possibly, functional properties.

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.

This work describes the development of membrane based on chitosan (CHI), cellulose nanocrystals (CNCs), and glycerol (GLY), and optimization of the formulation for immobilization of monoclonal anti-Shiga toxin 2B antibody (mAnti-stx2B-Ab) for E. coli O157:H7 detection. The effect of CHI deacetylation degree & viscosity, CNCs and GLY concentrations on Anti-stx2B-Ab immobilization efficiency was evaluated. Fractional factorial and Box-Behnken designs were applied to screen the effects of compounds interactions and optimize their concentrations for detection of Anti-stx2B-Ab. The results demonstrated that the use of 0.6 % (w/v) CNCs improved significantly the Anti-stx2B-Ab immobilization and the level of signal detection. The detection limit of Escherichia coli O157:H7 by developed optimized membrane is 1 log CFU/mL. The time needed for detection of E. coli O157:H7 was only 4 h of enrichment compared to 24 h with conventional methods. The developed immobilization support has potential for future pathogen detection in food and biomedical samples.

A simple approach to low-cost plasmonic sensing is obtained exploiting Plastic Optical Fibers (POFs). POFs are especially advantageous for their properties and, when receptors are used for bio/chemicals detection, are suitable for different application fields.

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.

We have applied a combined computational procedure based on inverse and direct docking in order to identify putative protein targets of a panel of mycotoxins and xenobiotic compounds that can contaminate food and that are known to have several detrimental effects on human health. This procedure allowed us to identify a panel of human proteins as possible targets for aflatoxins, gliotoxin, ochratoxin A and deoxynivalenol. Steady-state fluorescence and microscale thermophoresis experiments allowed us to confirm the binding of some of these mycotoxins to acetylcholinesterase and X-linked neuroligin 4, two proteins involved in synapse activity and, particularly for the second protein, neuronal plasticity and development. Considering the possible involvement of X-linked neuroligin 4 in the etiopathogenesis of autism spectrum syndrome, this finding opens up a new avenue to explore the hypothetical role of these xenobiotic compounds in the onset of this pathology.

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.

The Thermotoga maritima arginine-binding protein (TmArgBP) has been modified to create a reagentless fluorescent protein biosensor. Two design methods for biosensor construction are compared: 1) solvent accessibility of environmentally-sensitive probes and 2) fluorescence deactivation due to photo-induced electron transfer (PET). Nine single cysteine TmArgBP mutants were created and labeled with three different environmentally sensitive fluorescent probes. These mutants demonstrated limited changes in fluorescence emission upon the addition of arginine. In contrast, the PET-based biosensor provides significant enhancements over the traditional approach and provides a fluorescence quenching mechanism that was capable of providing quantitative detection of arginine. Site-directed mutagenesis of TmArgBP was used to create attachment points for the fluorescent probe (K145C) and for an internal aromatic residue (D18X) to serve as the PET quencher. Both tyrosine and tryptophan, but not phenylalanine, were able to quench the emission of the fluorescent probe by more than 80% upon the addition of arginine. The dissociation constant for arginine ranged from 0.87 to 1.5 mu M across the different sensors. This PET-based strategy provides a simple and broadly applicable approach for the analytical detection of small molecules that may be applied to any protein that exhibits conformational switching in a ligand dependent manner. (C) 2017 Elsevier Inc. All rights reserved.

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.

Glycine is involved in several physiological functions, e.g. as a neurotransmitter in the central nervous system, and sarcosine has been identified as a differential metabolite greatly enhanced during prostate cancer progression and metastasis. Glycine oxidase from Bacillus subtilis (GO) was engineered with the final aim of producing specific analytical systems to detect these small achiral amino acids. Based on in silico analysis, site-saturation mutagenesis was independently performed at 11 positions: a total of 16 single-point GO variants were analyzed. Significantly improved kinetic parameters were observed on glycine for the A54R, H244K-N-Q-R, Y246W and M261R variants. The introduction of multiple mutations then identified the H244K/M261R variant showing a 5.4-fold increase in maximal activity on glycine. With sarcosine as substrate, a number of single-point variants showed increased maximal activity and/or affinity: the kinetic efficiency was increased 6-fold for the M49L variant. Two GO variants with a high substrate specificity ratio for glycine (versus sarcosine, i.e. H244K GO) or for sarcosine (versus glycine, i.e. M49L GO) combined with high substrate affinity were used to set up a simple fluorescence-based biosensor. This optical sensing assay represents a novel, inexpensive and fast tool to assay glycine or sarcosine concentrations in biological samples (detection limit

Combining photonic integrated circuits with a biologically based sensing approach has the ability to provide a new generation of portable and low-cost sensor devices with a high specificity and sensitivity for a number of applications in environmental monitoring, defense, and homeland security. We report herein on the specific biosensing under continuous air flow of DMMP, which is commonly used as a simulant and a precursor for the synthesis of Sarin. The proposed technology is based on the selective recognition of the targeted DMMP molecule by specifically modified proteins immobilized on photonic structures. The response of the biophotonic structures shows a high stability and accuracy over 3 months, allowing for the detection in diluted air of DMMP at concentration as low as 35 μg/m(3) (6.8 ppb) in less than 15 min. The performance of the developed technology satisfies most current homeland and military security requirements.

This paper presents the application of a new support based on chitosan and reinforced with cellulose nanocrystals (m-CCG) for the detection of Listeria monocytogenes p60 protein in sandwich ELISA. This support consists of biopolymer membrane optimized for the covalent immobilization of the specific monoclonal antibodies against the unique short peptide of eleven amino acids (PepD, QQQTAPKAPTE) of L. monocytogenes p60 protein (anti-PepD mAb). Using the optimized immobilization method of anti-PepD mAb on m-CCG (m-CCG + anti-PepD mAb), the detection of L. monocytogenes culture was improved by 17% in Tryptic soy broth and by 24% in Listeria enrichment broth containing 0.5% of dextrose (modified LEB) on m-CCG compared to standard ELISA support. Moreover, the evaluation of the selectivity of detection of L. monocytogenes by anti-PepD mAb immobilized on m-CCG allowed the absence of any cross-reaction between the support and the nine other bacteria commonly found in food environment.

Damage-associated molecular patterns (DAMPs) are molecules that can be actively or passively released by injured tissues and that activate the immune system. Here we show for the first time that the NAD biosynthetic enzyme nicotinate phosphoribosyltransferase (NAPRT), the rate-limiting enzymes in the intracellular synthesis of NAD from nicotinic acid, is physiologically present in human sera, where it acts as a novel DAMP. We detected NAPRT in plasma/sera from human donors by antibody-mediated luminex assays and mass spectrometry. Exposure of human and mouse macrophages to NAPRT triggers activation of ERK1/2, phosphorylation of IKKα/β and nuclear translocation of the p65 subunit of the NF-kB complex, with synthesis and secretion of inflammatory cytokines, including IL-1β, IL-8, TNFα and CCL3. Furthermore, NAPRT enhances monocyte differentiation into macrophages, by inducing macrophage colony-stimulating factor. These effects are independent of the NAPRT catalytic activity, but rely on the protein’s binding to TLR4, as demonstrated by showing direct in vitro interaction and by the in vivo lack of NAPRT effects in TLR4−/− macrophages. In line with the finding that NAPRT mediates endotoxin tolerance, sera from patients with sepsis or septic shock contain the highest levels of circulating NAPRT, compared to other chronic inflammatory conditions, including cancer. Importantly, patients with serum NAPRT >15 ng/ml are characterized by a worse clinical outcome, compared to the counterparts. Together, these data identify NAPRT as a novel endogenous ligand for TLR4 and a critical mediator of inflammation.

In this work, we present integrated photonic sensor design to detect Chemical, Biological, Radiological and Nuclear (CBRN) threats, where high sensitivity and no false positive are strictly required. The sensor is formed by several parallel micro-ring resonators exposed to the environment and functionalized to recognized target analytes in a free-label way. Experimental demonstration of the working principle was performed using simulant compounds that permit tests of the features of the Photonic Integrated Circuit (PIC) in a safe environment. The results show the possibility of using PICs to detect as low as 20 ppb of Ovalbumin (simulant for Ricin A toxin) and 200 CFU/ml of Bacillus cereus spores (simulant for Bacillus anthracis). These results push forward the possibility of using PICs as biochemical sensors, also for critical objectives.

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.

A fast and reliable detection of biological agents in air is of a crucial importance to respond to terrorist attacks. With the aim to efficiently react to such hazards there is the need to develop highly sensitive and specific detection analytical devices for selective and quantitative detection of biological threats such as the presence of Bacillus anthracis spores and/or the presence of Ricin A toxins. In this study we explored how to achieve an oriented immobilization of antibody molecules on silicon nitride surfaces to improve their efficiency to bind to specific target molecules. In particular, we used two different methods to covalently immobilize antibody molecules on silicon nitride surfaces, and here we report the obtained results.

Ci pare interessante ricordare brevemente la genesi dell'idea di "Future for Food and Food for Future" (F4F)2, ovvero di come sia accaduto che un matematico esperto di ICT (Marco Padula, ITC-CNR), un fisico con esperienza diplomatica nella ricerca Italiana in ambito internazionale (Angelo Volpi, UREI-CNR) e un ingegnere esperto di controlli di processi industriali e di sistemi complessi (Elisabetta Punta, IEIIT-CNR) abbiano accettato con entusiasmo l'invito di Sabato D'Auria, direttore di ISA-CNR, biologo esperto di biosensori per la sicurezza alimentare e la qualità degli alimenti, e caro amico, a organizzare un evento sull'alimentazione. Inizialmente i punti fermi su come impostare l'evento non erano molti; volevamo riuscire a seguire il filo logico dettato dal tema centrale di EXPO 2015, "Nutrire il pianeta, energia per la vita" e volevamo ipotizzare gli scenari futuri dell'alimentazione investigando le problematiche relative alla sicurezza e alla disponibilità alimentare senza scollegarle da quelle relative a salute, foodomics, ambiente, normative, giurisprudenza, energia, trasporti e ICT. Un altro aspetto era però fondamentale: il concetto di "piazza" intesa come agorà, il termine con il quale nell'antica Grecia si indicava il centro della polis, il cuore pulsante di ogni attività, dove si creavano e mantenevano relazioni personali e istituzionali, dove le decisioni erano prese e dove tutti i cittadini possedevano gli stessi diritti e gli stessi doveri. Volevamo che questi temi fossero indagati, quindi, in una vera piazza, in un luogo aperto a tutti e dove tutti avessero, appunto, il diritto e il dovere di confrontarsi e scambiare idee.

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.

Expo Milano 2015, l'Esposizione Universale che si è tenuta a Milano, Italia, dal 1° maggio al 31 ottobre 2015, ha coinvolto più di 140 paesi partecipanti, così come prestigiose organizzazioni internazionali, ed ha accolto oltre 20 milioni di visitatori. Le nazioni espositrici hanno avuto l'opportunità di mostrare il meglio della loro tecnologia, che mira a garantire un'alimentazione sana, sicura e sufficiente per tutti, nel rispetto del pianeta e del suo equilibrio. L'Esposizione universale ha costituito un contesto eccezionale dove incontrarsi, scambiare idee e condividere soluzioni sul tema del cibo; è stato così raggiunto l'obiettivo principale di Expo 2015 che era quello di stimolare la creatività dei Paesi e promuovere innovazioni per un futuro sostenibile. "Nutrire il pianeta, energia per la vita" era il tema centrale di Expo 2015, il filo logico che ha attraversato tutti gli eventi organizzati sia all'interno che all'esterno del sito espositivo. EXPO 2015 è stata l'occasione per riflettere e discutere i diversi tentativi di trovare soluzioni alle contraddizioni del nostro mondo; scelte politiche consapevoli, sviluppo di stili di vita sostenibili e tecnologia avanzata sono necessari per raggiungere il giusto equilibrio tra la disponibilità ed il consumo di risorse. La partecipazione del Consiglio Nazionale delle Ricerche di Italia (CNR) a EXPO 2015 si è inserita nell'accordo di collaborazione che ha indicato il CNR quale consulente scientifico di Padiglione Italia (CNRxEXPO). Durante EXPO 2015 sono stati organizzati 24 eventi CNR con caratteristiche innovative, in termini di contenuti e di comunicazione. Ogni evento aveva lo scopo di comunicare un messaggio chiaro su ricerca e sviluppo nelle tre aree di ricerca individuate come fondamentali dal CNR in ambito EXPO 2015: il Cibo e l'uomo, Cibo e produttività, Cibo e tecnologie di trasformazione. Il ruolo cruciale svolto dal CNR come consulente scientifico ed organizzatore di eventi per EXPO 2015 è stato considerato da ISA-CNR, ITC-CNR, IEIIT-CNR e UREI-CNR un'occasione unica per organizzare l'evento CNR "Il futuro dell'alimentazione e l'alimentazione del futuro" (F4F)2, che si proponeva di indagare le problematiche relative alla sicurezza alimentare, ma anche alla salute, all'ambiente, all'energia, ai trasporti, all'ICT

The structural and stability properties of a novel zinc-dependent alcohol dehydrogenase from the hyperthermophilic archaeon Pyrobaculum aerophilum (PyAeADHII) were investigated by Fourier transformed infrared spectroscopy (FTIR). This enzyme is a thermostable homo-tetramer belonging to the GroES-fold motif proteins characterized by an irregular β-barrel conformation. Our results revealed a protein with a secondary structure rich in β-sheet (32% of the total secondary elements) and it showed a three-step thermal unfolding pathway. The complete enzyme denaturation was preceded by the formation of a relaxed tertiary/quaternary structure and previously by an excited native-like conformation. Two-dimensional correlation spectroscopy analysis (2D-COS) and differential scanning calorimetry (DSC) experiments supported these data and allowed us to determine the protein melting temperature at 96.9 °C as well as to suggest the sequence of the events that occurred during the protein denaturation process.

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.

The gene encoding a novel alcohol dehydrogenase (ADH) that belongs to the medium chain dehydrogenase/reductase (MDR) superfamily was identified in the hyperthermophilic archaeon, Pyrobaculum aerophilum . The P. aerophilum ADH gene ( Pae2687 ) was over-expressed in Escherichia coli , and the protein (PyAeADHII) was purified to homogeneity and characterized. The PyAeADHII belongs to a medium chain class because its monomer size is 330 residues and even if it is structurally similar to other enzymes belonging to MDR superfamily, it lacks key residues involved in the coordination of the catalytic Zn ion and in the binding of alcoholic substrates typical of other ADHs. Consistently, PyAeADHII does not show activity on a large number of alcohols, aldheydes or ketones. It is active only when α-tetralone is used as a substrate. The enzyme has a strict requirement for NADP(H) as the coenzyme and has remarkable thermophilicity, displaying activity at temperatures up to 95 °C. The study of the metabolic pathways of P. aerophilum can provide information on the evolution of genes and enzymes and may be crucial for understanding the evolution of eukaryotic cells.

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.

Fluorescence and circular dichroism in far-UV region were used to study the stability of trehalose/maltose binding protein (TMBP) from hyper thermophilic archaeonThermococcus litoralisand its complex with glucose (TMBP/Glc). The evaluation of difference between free energy of native and unfolded state for TMBP and TMBP/Glc showed that both of them are several times higher than that of proteins from mesophilic organisms. Due to the high stability and innate ability to bind glucose this protein is a good candidate as a sensitive element in biosensor systems for sugar control.

Andrea Scire, Anna Marabotti, Maria Staiano: These authors contributed equally to the work.; International audience; The effect of temperature on the structure of the rat odorant-binding protein was investigated by spectroscopic and in silico methodologies. In particular, in this work, we examined the structural features of the rat OBP-1F by Fourier-transform infrared spectroscopy and molecular dynamics investigations. The obtained spectroscopic results were analyzed using the following three different methods based on the unexchanged amide hydrogens of the protein sample: (1) the analysis of difference spectra; (2) the generalized 2D-IR correlation spectroscopy; (3) the phase diagram method. The three methods indicated that at high temperatures the rOBP-1F structure undergoes a relaxation process involving the protein tertiary organization before undergoing the denaturation and aggregation processes, suggesting the presence of an intermediate state such as a molten globule-like state. Importantly, the proposed analyses represent a general approach that could be applied to the study of protein stability.

Members of the periplasmic binding protein superfamily are involved in the selective passage of ligands through bacterial cell membranes. The hyperthermophilic eubacterium Thermotoga maritima was found to encode a highly stable and specific periplasmic arginine-binding protein (TM0593). Following signal sequence removal and overexpression in Escherichia coli, TM0593 was purified by thermoprecipitation and affinity chromatography. The ultra-stable protein with a monomeric molecular weight of 27.7 kDa was found to exist as both a homodimer and homotrimer at appreciable concentrations even under strongly denaturing conditions, with an estimated transition temperature of 116 degrees C. Its multimeric structure may provide further evidence of the importance of quaternary structure in the movement of nutrients across bacterial membranes. Purified and refolded TM0593 was further characterized by fluorescence spectroscopy, mass spectrometry, and circular dichroism to demonstrate the specificity of the protein for arginine and to elucidate structural changes associated with arginine binding. The protein binds arginine with a dissociation constant of 20 muM as determined by surface plasmon resonance measurements. Due to its high thermodynamic stability, TM0593 may serve as a scaffold for the creation of a robust fluorescent biosensor.

Odorant-binding proteins are biomolecules belonging to the lipocalin family. Among all the odorant-binding proteins, the porcine odorant-binding protein has been well characterized. This protein is a monomer that is characterized by the presence of the beta-barrel structure and of the disulphide bridge. The internal cavity of the beta-barrel is the binding site. In this study we have investigated the structural properties of the porcine odorant-binding protein by mass spectrometry experiments. Our data allow us to hypothesize that specific deamidation mechanisms of specific amino acid residues can be responsible for the binding properties of this class of proteins.

Glutamine-binding protein (GlnBP) from Escherichia coli is a monomer (26 kDa) that is responsible for the first step in the active transport of L-glutamine across the cytoplasmic membrane. GlnBP consists of two domains (termed large and small) linked by two antiparallel β-strands. The large domain is similar to the small domain but it contains two additional α-helices and three more short antiparallel β-strands. The deep cleft formed between the two domains contains the ligand-binding site. The binding of L-glutamine leads to cleft closing and a significant structural change with the formation of the so-called “closed form” structure. The protein contains two tryptophan residues (W32 and W220) and 10 tyrosine residues. We used phosphorescence spectroscopy measurements to characterize the role of the two tryptophan residues in the protein structure in the absence and the presence of glutamine. Our results pointed out that the phosphorescence of GlnBP is easily detected in fluid solutions where the emission of the two tryptophan residues is readily discriminated by the drastic difference in the phosphorescence lifetime allowing the assignments of the short lifetime to W220 and the long lifetime to W32. In addition, our results showed that the triplet lifetime of the superficial W220 is unusually short because of intramolecular quenching by the proximal Y163. On the contrary, the lifetime of W32 is several hundred milliseconds long, implicating a well-ordered, compact fold of the surrounding polypeptide. The spectroscopic data were analyzed and discussed together with a detailed inspection of the 3D structure of GlnBP. Proteins 2008. © 2007 Wiley-Liss, Inc.

Antibiotics, such as benzyl-penicillin (PenG) and cephalosporin, are the most common compounds used in animal therapy. Their massive and illegal use in animal therapy and prophylaxis inevitably causes the presence of traces in foods of animal origin (milk and meat), which creates several problems for human health. With the aim to prevent the negative impact of ?-lactam and, in particular, PenG residues present in the milk on customer health, many countries have established maximum residue limits (MRLs). To cope with this problem here, we propose an effective alternative, compared to the analytical methods actually employed, to quantify the presence of penicillin G using the surface plasmon resonance (SPR) method. In particular, the PenG molecule was conjugated to a protein carrier to immunize a rabbit and produce polyclonal antibodies (anti-PenG). The produced antibodies were used as molecular recognition elements for the design of a competitive immune-assay for the detection of PenG by SPR experiments. The detection limit of the developed assay was found to be 8.0 pM, a value much lower than the MRL of the EU regulation limit that is fixed at 12 nM. Thus, our results clearly show that this system could be successfully suitable for the accurate and easy determination of PenG.

An SPR sensor in POF, with possible implementation as bio/chemical sensor, is presented. This SPR-POF platform can be used in different application fields: industrial, medical diagnostics, environmental monitoring, food safety and security. © 2015 OSA.