Electrochemical Immunosensor for the Determination of Antibodies against Prostate-Specific Antigen Based on ZnO Nanostructures.

In this study, ZnO nanostructures with different types of morphologies and particle sizes were evaluated and applied for the development of an immunosensor. The first material was composed of spherical, polydisperse nanostructures with a particle size in the range of 10-160 nm. The second was made up of more compact rod-like spherical nanostructures with the diameter of these rods in the range of 50-400 nm, and approximately 98% of the particles were in the range of 20-70 nm. The last sample of ZnO was made up of rod-shaped particles with a diameter of 10-80 nm. These ZnO nanostructures were mixed with Nafion solution and drop-casted onto screen-printed carbon electrodes (SPCE), followed by a further immobilization of the prostate-specific antigen (PSA). The affinity interaction of PSA with monoclonal antibodies against PSA (anti-PSA) was evaluated using the differential pulse voltammetry technique. The limit of detection and limit of quantification of anti-PSA were determined as 1.35 nM and 4.08 nM for compact rod-shaped spherical ZnO nanostructures, and 2.36 nM and 7.15 nM for rod-shaped ZnO nanostructures, respectively.

Last Trends in Point-of-Care (POC) Diagnostics for the Management of Hematological Indices in Home Care Patients.

Today, complete blood count (CBC) analyses are highly automated and allow for high throughput and accurate and reliable results. However, new analytical tools are in great demand to provide simple, rapid and cost-effective management of hematological indices in home care patients. Chronic disease monitoring at home has become a benefit for patients who are finding cost savings in programs designed to monitor/treat patients in offsite locations. This review reports the latest trends in point-of-care (POC) diagnostics useful for home testing of key hematological counts that may be affected during home therapy treatment.

Investigation of Photosystem II Functional Size in Higher Plants under Physiological and Stress Conditions Using Radiation Target Analysis and Sucrose Gradient Ultracentrifugation.

The photosystem II (PSII) reaction centre is the critical supramolecular pigment-protein complex in the chloroplast which catalyses the light-induced transfer of electrons from water to plastoquinone. Structural studies have demonstrated the existence of an oligomeric PSII. We carried out radiation inactivation target analysis (RTA), together with sucrose gradient ultracentrifugation (SGU) of PSII, to study the functional size of PSII in diverse plant species under physiological and stress conditions. Two PSII populations, made of dimeric and monomeric core particles, were revealed in Pisum sativum, Spinacea oleracea, Phaseulus vulgaris, Medicago sativa, Zea mais and Triticum durum. However, this core pattern was not ubiquitous in the higher plants since we found one monomeric core population in Vicia faba and a dimeric core in the Triticum durum yellow-green strain, respectively. The PSII functional sizes measured in the plant seedlings in vivo, as a decay of the maximum quantum yield of PSII for primary photochemistry, were in the range of 75-101 ± 18 kDa, 2 to 3 times lower than those determined in vitro. Two abiotic stresses, heat and drought, imposed individually on Pisum sativum, increased the content of the dimeric core in SGU and the minimum functional size determined by RTA in vivo. These data suggest that PSII can also function as a monomer in vivo, while under heat and drought stress conditions, the dimeric PSII structure is predominant.

A Proof-of-Concept Electrochemical Cytosensor Based on Chlamydomonas reinhardtii Functionalized Carbon Black Screen-Printed Electrodes: Detection of Escherichia coli in Wastewater as a Case Study.

Herein, we report a proof-of-concept algal cytosensor for the electrochemical quantification of bacteria in wastewater, exploiting the green photosynthetic alga Chlamydomonas reinhardtii immobilized on carbon black (CB) nanomodified screen-printed electrodes. The CB nanoparticles are used as nanomodifiers, as they are able to sense the oxygen produced by the algae and thus the current increases when algae are exposed to increasing concentrations of bacteria. The sensor was tested on both standard solutions and real wastewater samples for the detection Escherichia coli in a linear range of response from 100 to 2000 CFU/100 mL, showing a limit of detection of 92 CFU/100 mL, in agreement with the maximum E. coli concentration established by the Italian law for wastewater (less than 5000 CFU/100 mL). This bacterium was exploited as a case study target of the algal cytosensor to demonstrate its ability as an early warning analytical system to signal heavy loads of pathogens in waters leaving the wastewater treatment plants. Indeed, the cytosensor is not selective towards E. coli but it is capable of sensing all the bacteria that induce the algae oxygen evolution by exploiting the effect of their interaction. Other known toxicants, commonly present in wastewater, were also analyzed to test the cytosensor selectivity, with any significant effect, apart from atrazine, which is a specific target of the D1 protein of the Chlamydomonas photosystem II. However, the latter can also be detected by chlorophyll fluorescence simultaneously to the amperometric measurements. The matrix effect was evaluated, and the recovery values were calculated as 105 ± 8, 83 ± 7, and 88 ± 7% for 1000 CFU/100 mL of E. coli in Lignano, San Giorgio, and Pescara wastewater samples, respectively.

A dual electro-optical biosensor based on Chlamydomonas reinhardtii immobilised on paper-based nanomodified screen-printed electrodes for herbicide monitoring.

The indiscriminate use of herbicides in agriculture contributes to soil and water pollution, with important endangering consequences on the ecosystems. Among the available analytical systems, algal biosensors have demonstrated to be valid tools thanks to their high sensitivity, cost-effectiveness, and eco-design. Herein, we report the development of a dual electro-optical biosensor for herbicide monitoring, based on Chlamydomonas reinhardtii whole cells immobilised on paper-based screen-printed electrodes modified with carbon black nanomaterials. To this aim, a systematic study was performed for the selection and characterisation of a collection among 28 different genetic variants of the alga with difference response behaviour towards diverse herbicide classes. Thus, CC125 strain was exploited as case study for the study of the analytical parameters. The biosensor was tested in standard solutions and real samples, providing high sensitivity (detection limit in the pico/nanomolar), high repeatability (RSD of 5% with n = 100), long lasting working (10 h) and storage stability (3 weeks), any interference in the presence of heavy metals and insecticides, and low matrix effect in drinking water and moderate effect in surface one.

Quantum dots functionalised artificial peptides bioinspired to the D1 protein from the Photosystem II of Chlamydomonas reinhardtii for endocrine disruptor optosensing.

Herein we describe the design and synthesis of novel artificial peptides mimicking the plastoquinone binding niche of the D1 protein from the green photosynthetic alga Chlamydomonas reinhardtii, also able to bind herbicides. In particular, molecular dynamics (MD) simulations were performed to model in silico the behaviour of three peptides, D1Pep70-H, D1Pep70-S264K and D1Pep70-S268C, as genetic variants with different affinity towards the photosynthetic herbicide atrazine. Then the photosynthetic peptides were functionalised with quantum dots for the development of a hybrid optosensor for the detection of atrazine, one of the most employed herbicides for weed control in agriculture as well as considered as a putative endocrine disruptor case study. The excellent agreement between computational and experimental results self consistently shows resistance or super-sensitivity toward the atrazine target, with detection limits in the µg/L concentration range, meeting the requirements of E.U. legislation.

High-Tech and Nature-Made Nanocomposites and Their Applications in the Field of Sensors and Biosensors for Gas Detection.

Gas sensors have been object of increasing attention by the scientific community in recent years. For the development of the sensing element, two major trends seem to have appeared. On one hand, the possibility of creating complex structures at the nanoscale level has given rise to ever more sensitive sensors based on metal oxides and metal-polymer combinations. On the other hand, gas biosensors have started to be developed, thanks to their intrinsic ability to be selective for the target analyte. In this review, we analyze the recent progress in both areas and underline their strength, current problems, and future perspectives.

Novel atrazine-binding biomimetics inspired to the D1 protein from the photosystem II of Chlamydomonas reinhardtii.

Biomimetic design represents an emerging field for improving knowledge of natural molecules, as well as to project novel artificial tools with specific functions for biosensing. Effective strategies have been exploited to design artificial bioreceptors, taking inspiration from complex supramolecular assemblies. Among them, size-minimization strategy sounds promising to provide bioreceptors with tuned sensitivity, stability, and selectivity, through the ad hoc manipulation of chemical species at the molecular scale. Herein, a novel biomimetic peptide enabling herbicide binding was designed bioinspired to the D1 protein of the Photosystem II of the green alga Chlamydomonas reinhardtii. The D1 protein portion corresponding to the QB plastoquinone binding niche is capable of interacting with photosynthetic herbicides. A 50-mer peptide in the region of D1 protein from the residue 211 to 280 was designed in silico, and molecular dynamic simulations were performed alone and in complex with atrazine. An equilibrated structure was obtained with a stable pocked for atrazine binding by three H-bonds with SER222, ASN247, and HIS272 residues. Computational data were confirmed by fluorescence spectroscopy and circular dichroism on the peptide obtained by automated synthesis. Atrazine binding at nanomolar concentrations was followed by fluorescence spectroscopy, highlighting peptide suitability for optical sensing of herbicides at safety limits.

Electrospray deposition as a smart technique for laccase immobilisation on carbon black-nanomodified screen-printed electrodes.

Enzymes immobilisation represents a critical issue in the design of biosensors to achieve standardization as well as suitable analytical performances in terms of sensitivity, selectivity, and stability. In this work electrospray deposition (ESD) has been exploited as a novel technique for the immobilisation of laccase enzyme on carbon black modified screen-printed electrodes. The aim is to fabricate an amperometric biosensor for phenolic compound detection. The electrodes produced by ESD have been analysed by scanning electron microscopy and characterised electrochemically to prove that this immobilisation technique is suited to manufacture high performance biosensors. The results show that the laccase enzyme maintains its activity after undergoing the electrospray ionisation process and deposition and the fabricated biosensor has improved performances in terms of storage (up to 3 months at room temperature) and working (up to 25 measurements on the same electrode) stability. The laccase-based biosensor has been tested for phenolic compound detection, with catechol as target analyte, in the linear range 2.5-50 µM, with 2.0 µM limit of detection, without interference from lead, cadmium, atrazine, and paraoxon, and without matrix effect in drinking, surface, and wastewater.

Preventive or potential therapeutic value of nutraceuticals against ionizing radiation-induced oxidative stress in exposed subjects and frequent fliers.

Humans are constantly exposed to ionizing radiation deriving from outer space sources or activities related to medical care. Absorption of ionizing radiation doses over a prolonged period of time can result in oxidative damage and cellular dysfunction inducing several diseases, especially in ageing subjects. In this report, we analyze the effects of ionizing radiation, particularly at low doses, in relation to a variety of human pathologies, including cancer, and cardiovascular and retinal diseases. We discuss scientific data in support of protection strategies by safe antioxidant formulations that can provide preventive or potential therapeutic value in response to long-term diseases that may develop following exposure.

Design and biophysical characterization of atrazine-sensing peptides mimicking the Chlamydomonas reinhardtii plastoquinone binding niche.

The plastoquinone (Q(B)) binding niche of the Photosystem II (PSII) D1 protein is the subject of intense research due to its capability to bind also anthropogenic pollutants. In this work, the Chlamydomonas reinhardtii D1 primary structure was used as a template to computationally design novel peptides enabling the binding of the herbicide atrazine. Three biomimetic molecules, containing the Q(B)-binding site in a loop shaped by two α-helices, were reconstituted by automated protein synthesis, and their structural and functional features deeply analysed by biophysical techniques. Standing out among the others, the biomimetic mutant peptide, D1pepMut, showed high ability to mimic the D1 protein in binding both Q(B) and atrazine. Circular dichroism spectra suggested a typical properly-folded α-helical structure, while isothermal titration calorimetry (ITC) provided a complete thermodynamic characterization of the molecular interaction. Atrazine binds to the D1pepMut with a high affinity (Kd = 2.84 µM), and a favourable enthalpic contribution (ΔH = -11.9 kcal mol(-1)) driving the interaction. Fluorescence spectroscopy assays, in parallel to ITC data, provided hyperbolic titration curves indicating the occurrence of a single atrazine binding site. The binding resulted in structural stabilisation of the D1pepMut molecule, as suggested by atrazine-induced cooperative profiles for the fold-unfold transition. The interaction dynamics and the structural stability of the peptides in response to the ligand were particularly considered as mandatory parameters for biosensor/biochip development. These studies paved the way to the set-up of an array of synthetic mutant peptides with a wide range of affinity towards different classes of target analytes, for the development of optical nanosensing platforms for herbicide detection.

Directed evolution and in silico analysis of reaction centre proteins reveal molecular signatures of photosynthesis adaptation to radiation pressure.

Evolutionary mechanisms adopted by the photosynthetic apparatus to modifications in the Earth's atmosphere on a geological time-scale remain a focus of intense research. The photosynthetic machinery has had to cope with continuously changing environmental conditions and particularly with the complex ionizing radiation emitted by solar flares. The photosynthetic D1 protein, being the site of electron tunneling-mediated charge separation and solar energy transduction, is a hot spot for the generation of radiation-induced radical injuries. We explored the possibility to produce D1 variants tolerant to ionizing radiation in Chlamydomonas reinhardtii and clarified the effect of radiation-induced oxidative damage on the photosynthetic proteins evolution. In vitro directed evolution strategies targeted at the D1 protein were adopted to create libraries of chlamydomonas random mutants, subsequently selected by exposures to radical-generating proton or neutron sources. The common trend observed in the D1 aminoacidic substitutions was the replacement of less polar by more polar amino acids. The applied selection pressure forced replacement of residues more sensitive to oxidative damage with less sensitive ones, suggesting that ionizing radiation may have been one of the driving forces in the evolution of the eukaryotic photosynthetic apparatus. A set of the identified aminoacidic substitutions, close to the secondary plastoquinone binding niche and oxygen evolving complex, were introduced by site-directed mutagenesis in un-transformed strains, and their sensitivity to free radicals attack analyzed. Mutants displayed reduced electron transport efficiency in physiological conditions, and increased photosynthetic performance stability and oxygen evolution capacity in stressful high-light conditions. Finally, comparative in silico analyses of D1 aminoacidic sequences of organisms differently located in the evolution chain, revealed a higher ratio of residues more sensitive to oxidative damage in the eukaryotic/cyanobacterial proteins compared to their bacterial orthologs. These results led us to hypothesize an archaean atmosphere less challenging in terms of ionizing radiation than the present one.

The NUTRA-SNACKS project: basic research and biotechnological programs on nutraceutics.

The Nutra-Snacks project aims at creating novel high quality ready-to-eat foods with functional activity, useful for promoting public health. The team is composed of seven research institutes and three SMEs from different countries whose activities span from basic to applied research providing the right technological transfer to small and medium industries involved in the novel food production chain. Strategic objectives include the application of plant cell and in vitro culture systems to create very large amounts of high-value plant secondary metabolites with recognized anticancer, antilipidemic, anticholesterol, antimicrobial, antiviral, antihypertensive and anti-inflammatory properties and to include them in specific food products. To this end, the screening of a vast number of working organisms capable of accumulating the desired compounds and the characterization of their expression profiles represent fundamental steps in the research program. The information allows the identification of plant species hyper-producing metabolites and selection of those metabolites capable of specifically counteracting the oxidative stress that underlies the development of important pathologies and diseases. In addition, devising safe metabolite extraction procedures is also crucial in order to provide nutraceutical-enriched extracts compatible with human health. New biotechnological approaches are also undertaken including the exploitation of photosynthetic algal strains in bio-farms to enhance the synthesis ofantioxidant compounds and the design of novel bioreactors for small and large scale biomass production. Further outstanding objectives include the development of (i) safety and quality control protocols (ii) biosensor techniques for the analysis of the emerging ready-to-eat food and (iii) a contribution to define a standard for new regulations on nutraceutics.