Detection of canine and equine procalcitonin for sepsis diagnosis in veterinary clinic by the development of novel MIP-based SPR biosensors.

Procalcitonin (PCT) has emerged as a promising biomarker for the rapid identification of sepsis both in human and veterinary medicine. Nevertheless, the only analytical method currently available for the detection of PCT in veterinary species, is represented by immunoassays, useful only for research purposes. In this work, we report the development of two biosensors which utilize molecularly imprinted polymers (MIPs) for the detection of canine and equine PCT. Dopamine (DA) and norepinephrine (NE) were used as monomers for the synthesis of the MIP films on surface plasmon resonance (SPR) gold chips and the imprinting efficiency of canine and equine PCT in terms of binding affinity toward the analyte, selectivity, and sensitivity were compared. After optimization in buffer conditions, PCTs calibration was successfully achieved also in animal plasma, with good specificity and reproducibility. More effective protein binding and imprinting was obtained with polynorepinephrine (PNE) for both PCTs, and the SPR biosensors were able to detect the biomarkers in plasma with a LOD of 15 ng mL-1 and 30 ng mL-1 respectively for equine and canine PCT.

Norepinephrine as new functional monomer for molecular imprinting: An applicative study for the optical sensing of cardiac biomarkers.

The continuous research for alternatives to antibody-based detection drove, in the last decades, the development of numerous strategies. Molecularly imprinted polymers (MIPs) emerged thanks to the low-cost and long-term stability features, where the choice of natural functional monomer(s) represents the key step for efficient imprinting of biomolecules. The chemical structure of dopamine (DA), one of the most used natural functional monomers, provided the inspiration for this work. We wondered why norepinephrine (NE) that differs from dopamine only for an additional hydroxyl group was not investigated at all in biosensing applications. In fact, there is only one paper exploiting polynorepinephrine (PNE) in molecular recognition applications, taking advantage of molecular imprinting, but not for biosensing purposes. In contrast, hundreds of papers describe polydopamine-based sensors. Therefore, we firstly investigated how the additional hydroxyl group of NE could affect the properties of the resulting polymer, and how these properties could be exploited for biosensing applications. The results highlighted the reduced non-specific adsorption of proteins onto PNE with respect to dopamine polymer. Furthermore, as a case study, we successfully developed a PNE-based imprinted biosensor for the early detection of Troponin I, a crucial biomarker for heart failure, by coupling the MIP biosensor with surface plasmon resonance (SPR) detection. The results indicate the feasible use of imprinted PNE as synthetic receptor for biomolecules, opening new perspectives for this biopolymer, so far not considered, and encouraging further investigations on its potential application in biosensing.

High purity single photons entangled with an atomic qubit.

Trapped atomic ions are an ideal candidate for quantum network nodes, with long-lived identical qubit memories that can be locally entangled through their Coulomb interaction and remotely entangled through photonic channels. The integrity of this photonic interface is generally reliant on the purity of single photons produced by the quantum memory. Here, we demonstrate a single-photon source for quantum networking based on a trapped 138Ba+ ion with a single photon purity of g (2)(0)=(8.1±2.3)×10-5 without background subtraction. We further optimize the tradeoff between the photonic generation rate and the memory-photon entanglement fidelity for the case of polarization photonic qubits by tailoring the spatial mode of the collected light.

Determination of fermentable sugars in beer wort by gold nanoparticles@polydopamine: A layer-by-layer approach for Localized Surface Plasmon Resonance measurements at fixed wavelength.

Polydopamine decorated in-situ with Localized Surface Plasmon Resonance (LSPR)-active gold nanoparticles (AuNPs) may extend the applicability of nanoplasmonic materials to original and innovative applications in several fields. Here we report the modification of disposable UV-Vis polystyrene cuvettes with AuNPs@PDA for refractive index LSPR-based measurements. An original layer-by-layer deposition method of PDA followed by AuNPs growth is here developed, showing linear correlation between PDA thickness and optical properties. In particular, the modulation from wavelength sensitivity toward absorbance sensitivity is obtained, allowing measurements at fixed wavelength (578 nm). As applicative example of the photonic cuvettes, the measurement of fermentable sugars in beer wort is here reported. The analytical performance of our approach has been directly compared to portable refractometer of reference, displaying excellent results in terms of the precise estimation of sugars in beer wort (expressed in degrees Brix), reproducibility and sensitivity. The approach may be extended to other materials of interest in LSPR based optical sensors, e.g. optical fibers.

Cardiac Troponin T capture and detection in real-time via epitope-imprinted polymer and optical biosensing.

Millions of premature deaths per year from cardiovascular diseases represent a global threat urging governments to increase global initiatives, as advised by World Health Organization. In particular, together with prevention and management of risk factors, the development of portable platforms for early diagnosis of cardiovascular disorders appears a fundamental task to carry out. Contemporary assays demonstrated very good accuracy for diagnosis of acute myocardial infarction (AMI), but they are based on expensive and fragile capture antibodies. Accordingly, also considering the massive demand from developing countries, we have devoted our study to an affinity-based biosensor for detection of troponin T (TnT), a preferred biomarker of AMI. This combines a stable and inexpensive molecularly imprinted polymer (MIP) based on polydopamine (PDA) with surface plasmon resonance (SPR) transduction. Herein we report the fast and specific answer upon TnT binding onto an epitope-imprinted surface that strongly encourages the further development toward antibody-free point-of-care testing for cardiac injury.

The early nucleation stage of gold nanoparticles formation in solution as powerful tool for the colorimetric determination of reducing agents: The case of xylitol and total polyols in oral fluid.

Herein we report a simple non-enzymatic assay for xylitol and total polyols in water and oral fluid based on the time resolved formation of gold NPs in solution, and their colorimetric detection at fixed wavelength (520 nm). The key novelty of the proposed approach relies on the exploitation of information given by the early nucleation step of NPs formation instead of those related to final products at the end point of AuNPs growth, as generally reported in literature. We demonstrate that the nucleation stage is linearly correlated to the concentration of the reducing agent in solution. On the contrary, the optical reading carried out the end point of the reaction shows non-linear correlation and several undesired features. As case study, we applied the proposed method to xylitol and polyols determination, first tested in water and spiked oral fluid samples. The detection limits obtained on xylitol resulted 180 mg L-1 (CV% = 6.9) and 44 mg L-1 (CV% = 6.5) in water and oral fluid, respectively. Afterward, we successfully performed the monitoring of total polyols in oral fluid over time during xylitol-containing gums consumption. Data here reported show high correspondence with available data in literature. The proposed approach is fast, cheap, highly reproducible, and can be extended to other reducing substances of interest for analytical purposes.

Toward sensitive immuno-based detection of tau protein by surface plasmon resonance coupled to carbon nanostructures as signal amplifiers.

Interest on Tau protein is fast increasing in Alzheimer's disease (AD) diagnosis. There is the urgent need of highly sensitive and specific diagnostic platforms for its quantification, also in combination with the other AD hallmarks. Up to now, SPR has been poorly exploited for tau detection by immunosensing, due to sensitivity limits at nanomolar level, whereas the clinical requirement is in the picomolar range. Molecular architectures built in a layer-by-layer fashion, biomolecules and nanostructures (metallic or not) may amplify the SPR signal and improve the limit of detection to the desired sensitivity. Mostly gold nanostructures are widely employed to this aim, but great interest is also emerging in Multi Walled Carbon Nanotubes (MWCNTs). Here MWCNTs are modified and then decorated with the secondary antibody for tau protein. Eventually we took advantage from MWCNTs-antibody conjugate to obtain a sandwich-based bioassay with the capability to increase the SPR signal of about 102 folds compared to direct detection and conventional unconjugated sandwich. With respect to these results, we hope to give a strong impulse for further investigation on studying possible roles of carbon nanotubes in optical-based biosensing.

Coupling non invasive and fast sampling of proteins from work of art surfaces to surface plasmon resonance biosensing: Differential and simultaneous detection of egg components for cultural heritage diagnosis and conservation.

Despite the wide application of surface plasmon resonance (SPR) to a broad area of interests, from environment to food analysis, from drug discovery to diagnostics, its exploitation in cultural heritage conservation is still unexplored. Water-based highly viscous polymeric dispersions (HVPD) composed by partially hydrolyzed polyvinyl acetate (PVA), borax, and water, were recently developed and successfully applied for the selective removal of surface degradation patinas (i.e. protein materials, natural resins etc.) from paintings of historical and artistic interest. This approach is here coupled for the first time to a SPR biosensor to simultaneously recognize albumen, yolk, or their mixtures in HVPD extracts. Ovalbumin and immunoglobulin Y are selected as analytes for egg white and yolk recognition, respectively. The biosensor was first characterized on standard analytes within the range 0-400mgL(-1) and then on fresh and dried egg albumen and yolk down to 2·10(^4) and 1·10(^5) dilution factors, respectively. Once optimized, the biosensor was combined to the HVPD application on simulated and real art samples for the evaluation of hen egg presence in the extract, i.e. albumen, yolk, or their co-presence in the matrix. For a contemporary 'sacred icon', realized by the traditional egg tempera procedure described by Cennino Cennini, the biosensor successfully distinguished different uses of egg components for the realization of painted and gilded areas, i.e. yolk and albumen, respectively. Finally, a XVIII century italian painting whose the realization technique is unknown, was tested confirming its egg tempera-based realization technique.

Design of a dual aptamer-based recognition strategy for human matrix metalloproteinase 9 protein by piezoelectric biosensors.

MMP-9, human matrix metalloproteinase 9, belongs to the family of zinc-dependent peptide-bond hydrolases and is involved in the degradation of the extracellular matrix (ECM). In clinics, it is well known that elevated MMP-9 serum levels are associated with cardiovascular dysfunctions, several aspects of the physiology and pathology of the central nervous system, neuropsychiatric disorders and degenerative diseases related to brain tumors, and excitotoxic/neuroinflammatory processes. Due to the large interest of diagnostics in this protein, efforts to set up sensitive methods to detect MMP-9 for early diagnosis of a number of metabolic alterations are rapidly increasing. In this panorama, biosensors could play a key role; therefore we explored for the first time the development of an aptamer-based piezoelectric biosensor for a sensitive, label free, and real time detection of MMP-9. The detecting strategy involved two different aptamers in a sandwich-like approach able to detect down to 100 pg mL(-1) (1.2 pM) of MMP-9 as detection limit in standard solution. As proof of principle, commercial serum was investigated in terms of possible interferents, their identification and role in MMP-9 detection. The estimated detection limit for MMP-9 is about 560 pg mL(-1) (6.8 pM) in untreated serum.

Self-powered microneedle-based biosensors for pain-free high-accuracy measurement of glycaemia in interstitial fluid.

In this work a novel self-powered microneedle-based transdermal biosensor for pain-free high-accuracy real-time measurement of glycaemia in interstitial fluid (ISF) is reported. The proposed transdermal biosensor makes use of an array of silicon-dioxide hollow microneedles that are about one order of magnitude both smaller (borehole down to 4µm) and more densely-packed (up to 1×10(6)needles/cm(2)) than state-of-the-art microneedles used for biosensing so far. This allows self-powered (i.e. pump-free) uptake of ISF to be carried out with high efficacy and reliability in a few seconds (uptake rate up to 1µl/s) by exploiting capillarity in the microneedles. By coupling the microneedles operating under capillary-action with an enzymatic glucose biosensor integrated on the back-side of the needle-chip, glucose measurements are performed with high accuracy (±20% of the actual glucose level for 96% of measures) and reproducibility (coefficient of variation 8.56%) in real-time (30s) over the range 0-630mg/dl, thus significantly improving microneedle-based biosensor performance with respect to the state-of-the-art.

Single nucleotide polymorphism detection by optical DNA-based sensing coupled with whole genomic amplification.

The work presented here deals with the optimization of a strategy for detection of single nucleotide polymorphisms based on surface plasmon resonance imaging. First, a sandwich-like assay was designed, and oligonucleotide sequences were computationally selected in order to study optimized conditions for the detection of the rs1045642 single nucleotide polymorphism in the gene ABCB1. Then the strategy was optimized on a surface plasmon resonance imaging biosensor using synthetic DNA sequences in order to evaluate the best conditions for the detection of a single mismatching base. Finally, the assay was tested on DNA extracted from human blood which was subsequently amplified using a whole genome amplification kit. The direct detection of the polymorphism was successfully achieved. The biochip was highly regenerable and reusable for up to 20 measurements. Furthermore, coupling these promising results with the multiarray assay, we can foresee applying this biosensor in clinical research extended to concurrent analysis of different polymorphisms.

Direct detection of genomic DNA by surface plasmon resonance imaging: an optimized approach.

The direct detection of specific sequences in genomic DNA samples is very challenging in the biosensor-based approach. In this work we developed an optimized strategy for the direct detection of DNA sequences in human genomic samples by a surface plasmon resonance imaging technology. As model study, the target analyte was identified in a DNA sequence mapping the human ABCB1 gene. The computed-assisted approach was here applied for probe design. After a preliminary evaluation of the probe functioning by the complementary synthetic target, the system was applied to the direct detection of the target sequence in human genomic DNA extracted from lymphocytes. To achieve this result, several steps aimed to improve the analytical performances of the biosensor were studied and optimized. The immobilization chemistry, based on thiolated probes, was adapted here to non-amplified sequence detection. DNA sample pre-treatments, i.e. genomic fragmentation by ultrasounds and dsDNA denaturation by thermal treatment were also investigated. A sandwich-like strategy, by using a secondary probe, was also applied to understand and confirm the selectivity of the developed biosensor in detecting ABCB1 gene in genomic samples. Finally, a reliable calibration curve of ABCB1 was obtained with an experimental detection limit of 140 aM. Furthermore, the biosensor was well regenerable, assuring up to thirty cycles of effective measurements.

SPR detection of human hepcidin-25: a critical approach by immuno- and biomimetic-based biosensing.

The human hepcidin-25 hormone has a key role in iron regulation in blood. The clinical relevance of this hepatic ~2.8 kDa cysteine-rich peptide is rapidly increasing, since altered levels can be associated with inflammatory events and iron dysfunctions, such as hereditary hemochromatosis and iron overload. Moreover, hepcidin has also attracted the anti-doping field for its possible role as indirect marker of erythropoietin blood doping. Methods currently reported are based on immunoassays (ELISA and RIA), or various types of mass spectroscopy (MS)-based protocols, semi-quantitative or quantitative. Despite the great effort in optimizing robust and simple assays measuring hepcidin in real matrices, at present this challenge remains still an open issue. To explore the possibility to face hepcidin detection through the development of affinity-based biosensors, we set up a comparative study by surface plasmon resonance (SPR) technology. An immuno-based, on anti-hepcidin-25 IgG, and a biomimetic-based, on a synthetic peptide corresponding to the hepcidin-binding site on ferroportin (HBD), biosensors were developed. Here we report behaviors and analytical performances of the two systems, discussing limits and potentialities.

Surface Plasmon Resonance imaging-based sensing for anti-bovine immunoglobulins detection in human milk and serum.

Only few papers deal with Surface Plasmon Resonance imaging (SPRi) direct detection on complex matrices, limiting the biosensor application to real analytical problems. In this work a SPRi biosensor for anti-bovine IgG detection in untreated human bodily fluids, i.e. diluted human serum and milk, was developed. Enhanced levels of cow's milk antibodies in children's serum are suspected for their possible correlation with Type 1 diabetes during childhood and their detection in real samples was up to now performed by classical immunoassays based on indirect detection. The biosensor was optimised in standard samples and then in untreated human milk for anti-bovine IgG direct detection. The key novelty of the work is the evaluation of matrix effect by applying to real samples an experimental and ex ante method previously developed for SPRi signal sampling in standard solutions, called "Data Analyzer"; it punctually visualises and analyses the behaviour of receptor spots of the array, to select only spot areas with the best specific vs. unspecific signal values. In this way, benefits provide by SPRi image analysis are exploited here to quantify and minimise drawbacks due to the matrix effect, allowing to by-pass every matrix pre-treatment except dilution.

A rational approach in probe design for nucleic acid-based biosensing.

Development of nucleic acid-based sensing attracts the interest of many researchers in the field of both basic and applied research in chemistry. Major factors for the fabrication of a successful nucleic acid sensor include the design of probes for target sequence hybridization and their immobilization on the chip surface. Here we demonstrate that a rational choice of bioprobes has important impact on the sensor's analytical performances. Computational evaluations, by a simple and freely available program, successfully led to the design of the best probes for a given target, with direct application to nucleic acid-based sensing. We developed here an optimized and reproducible strategy for in silico probe design supported by optical transduction experiments. In particular Surface Plasmon Resonance imaging (SPRi), at the forefront of optical sensing, was used here as proof of principle. Five probes were selected, immobilized on gold chip surfaces by widely consolidated thiol chemistry and tested to validate the computational model. Using SPRi as the transducting component, real-time and label free analysis was performed, taking the Homo sapiens actin beta (ACTB) gene fragment as model system in nucleic acid detection. The experimental sensor behavior was further studied by evaluating the strength of the secondary structure of probes using melting experiments. Dedicated software was also used to evaluate probes' folding, to support our criteria. The SPRi experimental results fully validate the computational evaluations, revealing this approach highly promising as a useful tool to design biosensor probes with optimized performances.

Genetic and phenotypic diversity of autochthonous Saccharomyces spp. strains associated to natural fermentation of 'Malvasia delle Lipari'.

AIMS: Characterize from both genetic and phenotypic standpoints the indigenous strains of Saccharomyces spp. associated with natural fermentation of 'Malvasia delle Lipari'. METHODS AND RESULTS: A total of 192 yeast isolates were obtained from completed fermentation of a mix of 'Malvasia delle Lipari' (92%) and 'Corinto nero' (8%) grapes in two wineries in Salina Island (Sicily, Italy). Fifty-one Saccharomyces spp. isolates were characterized using ITS-PCR, random amplified polymorphic DNA-PCR and mitochondrial DNA restriction fragment length polymorphism and 12 biotypes were identified. Representative strains of each biotype, tested for their physiological traits, exhibit different killer activity, fermentation vigour, production of hydrogen sulphide and show similar beta-glucosidase and proteolytic activity. CONCLUSIONS: It is possible to cluster in different groups naturally occurring indigenous biotypes of Saccharomyces cerevisiae from 'Malvasia delle Lipari' on the basis of molecular profiles. SIGNIFICANCE AND IMPACT OF THE STUDY: Deeper insight on indigenous wine yeast of a conserved environment. The knowledge gained might offer a contribution to the selection of autochthonous wine yeast as starters for controlled fermentations.

Pharyngolaryngeal reflux in outpatient clinical practice: personal experience.

Proximal pharyngolaryngeal reflux now appears to be strictly related to symptoms and clinical patterns frequently encountered in clinical ORL practice and which are, more often than not, differentiated from classical gastro-oesophageal reflux disease. The aim of the study was to evaluate the incidence of the symptoms and clinical signs of gastro-oesophageal reflux disease, together with the symptomatologic response to a cycle of acid suppression treatment with proton pump inhibitors in a non-selected population comprising consecutive patients coming to the outpatient unit of the Laryngology Clinic of Università Cattolica of Rome between June and December 2001, all of whom had been examined by the same practitioner. Of the 1300 patients evaluated, 52 presented a clinical history and chronic pharyngolaryngeal symptoms strongly indicative of gastro-oesophageal reflux disease. All selected patients were prescribed a two-month regimen of acid suppression therapy (20 mg Omeprazole twice daily, sufficient to confirm the clinical suspicion of pharyngolaryngeal reflux ex adiuvantibus (Omeprazole test), at the same time obtaining a satisfactory response in the symptomatology, as laid out in the guidelines emerging from the 1997 Consensus Conference Report on pharyngolaryngeal reflux. The same patients were later interviewed by telephone to evaluate the effectiveness of treatment, exclusively in terms of improvement in the symptoms. Analysis of the data from the 33 who answered the telephone questionnaire revealed a complete symptomatological response in 24 (72.7%), partial response in 4 (12.1%) and no response in 5. The Authors stress that, despite the known limits of this treatment, linked to proton pump inhibitors resistance, the "Omeprazole test", on account of excellent tolerability, lack of adverse effects and, above all, non-invasive nature, is feasible in the preliminary evaluation to confirm clinical suspicion of gastro-oesophageal reflux disease in outpatients, especially when a large-scale pH-metric multielectrode investigation is not possible. Furthermore, despite the many epidemiological, aetiopathogenetic, clinico-diagnostic and therapeutic aspects which remain to be clarified, there is no doubt that gastro-oesophageal reflux disease on account of the particular clinical features, directly involves the ORL specialist not only in the diagnostic, but also in the therapeutic phase.

Multicentre study of the in vitro evaluation of moxifloxacin and other quinolones against community acquired respiratory pathogens.

The in vitro activity of moxifloxacin was compared with that of ciprofloxacin, levofloxacin, ofloxacin and trovafloxacin against 710 strains (180 Streptococcus pneumoniae, 180 Haemophilus influenzae, 160 Moraxella catarrhalis and 190 Streptococcus pyogenes) isolated from patients with community-acquired respiratory tract infections. MIC values for moxifloxacin, trovafloxacin were 0.25/0.25, 0.03/0.03, 0.06/0.03 and 0.125/0.0125 mg/l for S. pneumoniae, H. influenzae, M. catharralis and S. pyogenes. Based upon the MIC(90) values and the MIC distributions, moxifloxacin and trovafloxacin were the most active of the quinolones tested. They showed enhanced activity against Gram-positive organisms including penicillin non susceptible S. pneumoniae strains. Moxifloxacin was also highly active against ciprofloxacin-resistant S. pneumoniae strains.

Comparative in vitro activity of gemifloxacin against gram-positive and gram-negative clinical isolates in Argentina.

The in vitro activity of gemifloxacin against 1,000 clinical isolates of 147 Streptococcus pneumoniae (115, penicilin susceptible; 26, intermediate penicillin-resistant and 6, penicillin-resistant), 127 Hemophilus influenzae (109, beta lactamasa non-producer; 18, beta lactamase producers), 95 Streptococcus pyogenes (6, azytromycin-resistant), 84 Moraxella catarrhalis (79, beta lactamase producers), 110 Staphilococcus aureus (89, methicillin-susceptible; 21, methicilin-resistant), 98 Eenterococcus faecalis and 339 Enterobacteriacea, (recovered from patients with respiratory tract infection; skin and soft tissue infection and urinary tract infection), was compared with the activities of four fluorquinolones and five other antimicrobial agents. Of the quinolones tested, gemifloxacin was the most potent against Streptococcus pneumoniae, including penicillin intermediate and resistant strains. Mic(90) values obtained for gemifloxacin, ciprofloxacin, ofloxacin, levofloxacin and trvafloxacin were 0.03, 2, 2, 1 and 0.25 mg/L respectively. Gemifloxacin was 16 fold more potent than ciprofloxacin against methicillin-susceptible Staphylococcus aureus and 32 fold more potent than ciprofloxacin against Streptococcus pyogenes. When tested against Hemophilus influenzae, Moraxella catarrhalis and Enterobacteriaceae, all the quinolones showed similar activity. Our results demonstrate that gemifloxacin has similar activity than the other quinolones tested against Gram-negative organisms and is considerably more potent against Gram-positive organisms.