Plasmonic Metasurfaces for Specific SERS Detection of Shiga Toxins.

The interest in the development of nanoscale plasmonic technologies has dramatically increased in recent years. The photonic properties of plasmonic nanopatterns can be controlled and tuned via their size, shape, or the arrangement of their constituents. In this work, we propose a 2D hybrid metallic polymeric nanostructure based on the octupolar framework with enhanced sensing property. We analyze its plasmonic features both numerically and experimentally, demonstrating the higher values of their relevant figures of merit: we estimated a surface-enhanced Raman spectroscopy (SERS) enhancement factor of 9 × 107 and a SPR bulk sensitivity of 430 nm/RIU. In addition, our nanostructure exhibits a dual resonance in the visible and near-infrared region, enabling our system toward multispectral plasmonic analysis. Finally, we illustrate our design engineering strategy as enabled by electron beam lithography by the outstanding performance of a SERS-based biosensor that targets the Shiga toxin 2a, a clinically relevant bacterial toxin. To the best of our knowledge, this is the first time that a SERS fingerprint of this toxin has been evidenced.

Active thermography for real time monitoring of UV-B plant interactions.

Although Ultraviolet-B (UV-B)-plant interactions have been extensively analysed in the past years, many physiological aspects of the complex plant response mechanisms still need to be elucidated. Depending on the energy dose, this part of the electromagnetic spectrum can induce detrimental or beneficial effects in plant and fruit. In the present work, active thermography is used to analyse in real time the response of plants under different doses of artificial UV-B. In particular, we investigated the temporal variations of the leaf surface temperature (LST) to UV-B exposure by Long Pulse and Lock-in thermography in Epipremnum aureum and in Arabidopsis plants overexpressing or knockout mutants of UVR8, the known UV-B photoreceptor. In both cases, UV-B irradiation triggers a cooling effect, namely a thermal response characterised by a LST lower respect to the initial value. Lock-in thermography demonstrated that the cooling effect is associated with an immediate mobilization and accumulation of water in the leaves. Also, we demonstrated that thermographic responses change according to the different capability of plants to tolerate high UV-B radiation. Our study highlights new physiological and physical aspects of the plants response to UV-B radiation and, more in general, it opens new opportunities for the use of the thermography as smart tool for real-time monitoring of plant environmental interactions.

Dodecagonal plasmonic quasicrystals for phage-based biosensing.

In this work, we fabricate and characterize a novel sensitive two-dimensional surface enhanced Raman spectroscopy (SERS) substrate made of plasmonic nanocavities in a photonic quasicrystal arrangement characterized by a 12-fold rotational symmetry. Our SERS device is capable of detecting chemisorbed bacteriophages at a femtomolar range. Most importantly, the paper presents for the first time a study on the procedure to functionalize the plasmonic quasicrystal with bacteriophages of the Podoviridae family. The immobilization of the phages on the plasmonic substrate has been studied and verified through SERS measurements. A new stable peak, visible in the SERS spectra at 1326 cm-1 at a greater than 60 times amplification, confirms the immobilization of the phages on the substrate. This functionalization approach can be used also for other types of phages or plasmonic sensors and hence, our achievements could allow the development of novel systems for the specific detection of different species of bacteria.

Engineered nanopatterned substrates for high-sensitive localized surface plasmon resonance: an assay on biomacromolecules.

In this paper, we report on novel iso-Y-shaped-nanopillar based photonic crystals (PCs) engineered for plasmonic lab-on-a-chip advanced diagnostics. The iso-Y shaped units are selected on the basis of their plasmonic properties, analyzed numerically and experimentally. We show that by accurately choosing the nanopillar shape, dimensions and their geometrical disposal it is possible to obtain efficient optical 2D structures for biomolecule detection by high-sensitive localized surface plasmonic resonance (LSPR). In particular, an assay is realized by using bovine serum albumin (BSA), a widely recognized model for biosystem studies. BSA was simply deposited on a self-assembled monolayer (SAM) of 4-mercaptobenzoic acid (4-MBA) previously grown-up on the plasmonic substrate. We demonstrate that the geometries considered allow the design of LSPR nano-assays working in the visible-NIR region based on both intensity interrogation and the resonance peak shift permitting the sensing of BSA with a limit of detection in the order of picomoles (LOD = 233 pM).

A novel hybrid organic/inorganic photonic crystal slab showing a resonance action at the band edge.

Here we propose and experimentally demonstrate a hybrid photonic crystal (PC) slab consisting of air rods in a nanocomposite prepared by incorporating CdSe/CdS core/shell NRs (NR) in a polymer. Since the styrene methyl acrylate based polymer (ZEP) is transparent in the visible spectral range and is an electron-sensitive material, it was chosen as the embedding matrix for the NRs. Scanning electron microscopy and luminance measurements were used to characterize the experimental structure. The vertical extraction of the light, by the coupling of the modes guided by the PC slab to the free radiation via Bragg scattering, consists of a narrow orange emission band at 592 nm with a full width at half-maximum (FWHM) of 17 nm. The original characteristics of hybrid materials based on polymers and colloidal NRs, able to combine the unique optical properties of the inorganic moiety with the processability of the host matrix, are extremely appealing in view of their technological impact on the development of new high performing optical devices such as organic light-emitting diodes, ultra-low threshold lasers and non-linear devices.

Spectroscopic ellipsometry study of liquid crystal and polymeric thin films in visible and near infrared.

In this work, we propose spectroscopic ellipsometry as a suitable method for measuring optical properties in soft materials, polymers and liquid crystals, specially selected for use in photonics applications. We show the results of our measurements on some multilayered samples, in the range from visible to the near-IR region, of interest for telecom applications. We point out potentialities and limits of the technique and compare the obtained results with another experimental method, the m-lines spectroscopy, and/or with existing data in the literature. The results about the optical parameters for the analysed materials (the nematic liquid crystal 5CB, one commercial and one lab made optical polymer, and an Indium Tin Oxide film) are useful and interesting by themselves. In fact, as a paradigmatic example, we briefly discuss how an incomplete knowledge of this kind of data can lead to a wrong design of a Bragg grating device. However, more than in the provided data, we put the interest of the present analysis in the warnings about spectroscopic ellipsometry utilization and eventually the necessity of getting complementary information.

6-Phosphogluconate dehydrogenase: the mechanism of action investigated by a comparison of the enzyme from different species.

The mechanism of action of 6-phosphogluconate dehydrogenase with the alternative substrate 2-deoxy 6-phosphogluconate was investigated using enzymes from sheep liver, human erythrocytes and Trypanosoma brucei. The three enzymes oxidize 2-deoxy 6-phosphogluconate, but only the sheep liver enzyme releases the intermediate 2-deoxy,3-keto 6-phosphogluconate. Kinetic comparison showed that an increase in the rate of NADP+ reduction at high pH is due to increased release of the intermediate, rather than an increase in the overall reaction rate. 2-Deoxy,3-keto 6-phosphogluconate is decarboxylated by the erythrocyte and trypanosome enzymes but not the liver one in the absence of either NADPH or 6-phosphogluconate, which act as activators. The pH dependence of decarboxylation and the degree of activation suggest that 6-phosphogluconate is the activator which operates under normal assay conditions, while NADPH acts mainly by increasing the binding of the intermediate. The data suggest that the activity of 6PGDH is subjected to a two-way regulation: NADPH, which regulates the pentose phosphate pathway, inhibits the enzyme, while 6-phosphogluconate, levels of which rise when NADPH inhibition is removed, acts as an activator ensuring that 6-phosphogluconate is rapidly removed.

The cross-linking by o-phthalaldehyde of two amino acid residues at the active site of 6-phosphogluconate dehydrogenase.

o-phthalaldehyde inactivates homodimeric, NADP+ dependent, 6-phosphogluconate dehydrogenase from sheep liver, upon formation of a single isoindole derivative per enzyme subunit. This indicates that the thiol group of a cysteine residue or the epsilon-amino group of a lysine residue located within 3 A and crosslinked by the reagent is essential for catalysis. Fluorescence analyses of the modified enzyme suggest that the isoindole derivative forms at the binding site of the nicotinamide moiety of NADP+. The enzymes from Trypanosoma brucei and Lactococcus lactis are also inactivated suggesting a similar three-dimensional structure in this domain. The isoindole derivative does not form with two mutants of the T. brucei enzyme (Lys185His and Lys185Leu), this allowing to identify not only the lysine but also the cysteine involved in the cross-linking. The formation of the isoindole derivative inactivates not only the oxidative decarboxylation, but also two partial reactions catalysed by the enzyme.

6-Phosphogluconate dehydrogenase from Trypanosoma brucei. Kinetic analysis and inhibition by trypanocidal drugs.

The kinetics of 6-phosphogluconate dehydrogenase from Trypanosoma brucei was examined and compared to those of the same enzyme from lamb's liver. Variation of kinetic parameters as a function of pH suggests a chemical mechanism similar to other 6-phosphogluconate dehydrogenases. The comparison extended to a detailed analysis of the effect on enzyme activity by several inhibitors including the trypanocidal drugs suramin, melarsoprol and analogues of these compounds. The T. brucei enzyme differs significantly from its mammalian counterpart with respect to several inhibitors, particularly the substrate analogue 6-phospho-2-deoxygluconate and the coenzyme analogue adenosine 2',5'-bisphosphate which have respectively 170-fold and 40-fold higher affinity for the parasite enzyme.

Properties of diacetyl (acetoin) reductase from Bacillus stearothermophilus.

The cells of Bacillus stearothermophilus contain an NADH-dependent diacetyl (acetoin) reductase. The enzyme was easily purified to homogeneity, partially characterised, and found to be composed of two subunits with the same molecular weight. In the presence of NADH, it catalyses the stereospecific reduction of diacetyl first to (3S)-acetoin and then to (2S,3S)-butanediol; in the presence of NAD+, it catalyses the oxidation of (2S,3S)- and meso-butanediol, respectively, to (3S)-acetoin and to (3R)-acetoin, but is unable to oxidise these compounds to diacetyl. The enzyme is also able to catalyse redox reactions involving some endo-bicyclic octen- and heptenols and the related ketones, and its use is suggested also for the recycling of NAD+ and NADH in enzymatic redox reactions useful in organic syntheses.

Bromopyruvate for the affinity labelling of a single cysteine residue near the carboxylate binding site of lamb liver 6-phosphogluconate dehydrogenase.

Bromopyruvate inactivates 6-phosphogluconate dehydrogenase by affinity labeling. Kinetic analyses, stoichiometry and isolation of a single labelled tryptic peptide of the modified protein indicate that inactivation is due to the affinity labeling of a single cysteine residue, identified as cysteine 401. It thus appears that this cysteine is within a short distance from the protein site involved in the binding of the carboxylate group of the substrate. These results suggest that the carboxylate binding site of proteins could be used as an anchorage point for affinity labeling, and that bromopyruvate can be used to individuate an amino acid residue within few A from this site.

Inactivation and cleavage of liver 6-P-gluconate dehydrogenase during irradiation in the presence of vanadate.

Lamb liver phosphogluconate dehydrogenase is inactivated and selectively cleaved during irradiation in the presence of vanadate. Under our experimental conditions, the correlation between the species of vanadate in solution and rates of enzyme inactivation and cleavage indicates tetravanadate as the most likely photosensitizing agent, in agreement with previous data on other proteins. The enzyme is inactivated more rapidly at acidic pH and is partially protected by the coenzyme NADP, but not by the substrate phosphogluconate. Complete inactivation is obtained when only half of the protein is cleaved into smaller peptides. Differences in the pattern of the peptides produced are observed when irradiation is carried out in phosphate rather than in Hepes buffer: in the former instance cleavage results into formation of a main peptide of 47 kDa, while in latter case two additional peptides of 31 and 25 kDa are produced.

Oxidized ATP. An irreversible inhibitor of the macrophage purinergic P2Z receptor.

The effects of oxidized ATP (oATP) on responses triggered by extracellular adenosine 5'-triphosphate (ATPe) were investigated in the mouse macrophage-like cell line J774. ATPe induced in this cell line two kinds of responses mediated by two different P2 purinergic receptors: 1) an early permeabilization of the plasma membrane to extracellular hydrophilic markers of M(r) up to 900 mediated by P2Z receptors; and 2) a fast mobilization of Ca2+ from intracellular stores mediated by P2Y receptors. Low oATP concentrations (100 microM) completely blocked the first response without affecting the second. ATPe-dependent cell swelling, vacuolization, and lysis were also inhibited. Antagonism developed slowly, as an incubation at 37 degrees C for at least 2 h in the presence of oATP was needed and was irreversible, thus suggesting that the inhibitory action was due to covalent modification of the receptor. The rate of hydrolysis of exogenous ATP was slightly decreased by oATP, indicating a minor blocking effect of this compound on plasma membrane ecto-ATPases in the concentration range tested. These observations suggest that oATP may be a potentially very useful tool for isolation and characterization of the P2Z purinergic receptor.

Use of trinitrobenzensulfonate for affinity labeling of lysine residues at phosphate binding sites of some enzymes.

Trinitrobenzensulfonate, a reagent for lysine residues, inactivates lamb liver 6-phosphogluconate dehydrogenase through affinity labeling. Complete inactivation is due to the binding of only one residue of reagent per enzyme subunit. Other enzymes with a phosphate binding site are also inactivated by affinity labeling. It appears that trinitrobenzensulfonate, when used at low concentrations, first binds to a phosphate binding site, then reacts with a nearby lysine residue. This reagent presents some advantages over pyridoxal phosphate, which has similar characteristics.

Subunits asymmetry in the ternary complex of lamb liver 6-phosphogluconate dehydrogenase detected by a NADP analogue.

Incubation of lamb liver 6-phosphogluconate dehydrogenase, a dimeric enzyme with periodate-oxidized NADP causes the inactivation of the enzyme due to the covalent binding of 2 mol of inhibitor/mol of dimer. In the presence of substrate, the inactivation is faster and is almost complete after the labelling of only one subunit. These results not only confirm the hypothesis of a 'half-of-the-sites' mechanism of action of the enzyme, but also suggest that the formation of the ternary complex (enzyme-substrate-coenzyme) in one subunit causes a conformational change that makes the other subunit unable to bind the coenzyme (and even the adenylic part of it) and, thus, this subunit becomes inactive. It appears that while one subunit catalyses the oxidation of 6-phosphogluconate the other is inactive in this reaction.

NADPH activates a decarboxylation reaction catalysed by lamb liver 6-phosphogluconate dehydrogenase.

NADP-dependent lamb liver 6-phosphogluconate dehydrogenase catalyses the oxidative decarboxylation of 2-deoxy-6-phosphogluconate, an analogue of the natural substrate. The first products of the reaction are NADPH and 3-keto-2-deoxy-6-phosphogluconate. The NADPH, released from the enzyme, binds to the coenzyme site of the same or the other subunit, activating the decarboxylation reaction in which has not a redox role, since it can be substituted by an analogue devoid of enzymatic redox power. These findings are compared to those obtained with other NADP-dependent decarboxylating dehydrogenases.

Identification of the lysine residue involved in the inactivation of lamb liver 6-phosphogluconate dehydrogenase by fluorescein 5'-isothiocyanate.

Fluorescein 5'-isothiocyanate binds almost selectively at the active site of lamb liver NADP-dependent 6-phosphogluconate dehydrogenase causing the inactivation of the enzyme. The substrate and the coenzyme protect against the loss of catalytic activity. The enzyme derivative was digested with trypsin, the labelled peptide was isolated by h.p.l.c. and its amino acid analysis allowed to establish that the inactivator binds to lysine 166 at the active site of the protein.

A rapid method for purification of myelin basic protein.

A rapid procedure for purification of myelin basic protein has been developed. White matter is delipidated with 2-butanol, and the residue is extracted at pH 7.5 and 8.5. Myelin basic protein is solubilized by extraction in acetate buffer, pH 4.5. The entire procedure requires less than 4 h, and gives homogeneous protein essentially free of protease activity. This procedure can be scaled down to process milligram amounts of white matter; thus it can be very useful for purification of myelin basic protein from very limited amounts of human white matter obtained during surgery.