Are Aptamers Really Promising as Receptors for Analytical Purposes? Insights into Anti-Lysozyme DNA Aptamers through a Multitechnique Study.

Aptamers are recognition elements increasingly used for the development of biosensing strategies, especially in the detection of proteins or small molecule targets. Lysozyme, which is recognized as an important biomarker for various diseases and a major allergenic protein found in egg whites, is one of the main analytical targets of aptamer-based biosensors. However, since aptamer-based strategies can be prone to artifacts and data misinterpretation, rigorous strategies for multifaceted characterization of the aptamer-target interaction are needed. In this work, a multitechnique approach has been devised to get further insights into the binding performance of the anti-lysozyme DNA aptamers commonly used in the literature. To study molecular interactions between lysozyme and different anti-lysozyme DNA aptamers, measurements based on a magneto-electrochemical apta-assay, circular dichroism spectroscopy, fluorescence spectroscopy, and asymmetrical flow field-flow fractionation were performed. The reliability and versatility of the approach were proved by investigating a SELEX-selected RNA aptamer reported in the literature, that acts as a positive control. The results confirmed that an interaction in the low micromolar range is present in the investigated binding buffers, and the binding is not associated with a conformational change of either the protein or the DNA aptamer. The similar behavior of the anti-lysozyme DNA aptamers compared to that of randomized sequences and polythymine, used as negative controls, showed nonsequence-specific interactions. This study demonstrates that severe testing of aptamers resulting from SELEX selection is the unique way to push these biorecognition elements toward reliable and reproducible results in the analytical field.

Microbial consortia and biochar as sustainable biofertilisers: Analysis of their impact on wheat growth and production.

The European Union is among the top wheat producers in the world, but its productivity relies on adequate soil fertilisation. Biofertilisers, either alone or in combination with biochar, can be a preferable alternative to chemical fertilisers. However, the addition of biofertilisers, specifically plant growth promoting microbes (PGPM), could modify grain composition, and/or deteriorate the soil composition. In this study, the two wheat cultivars Triticum aestivum (Bramante) and T. durum (Svevo) were cultivated in open fields for two consecutive years in the presence of a commercial PGPM mix supplied alone or in combination with biochar. An in-depth analysis was conducted by collecting physiological and agronomic data throughout the growth period. The effects of PGPM and biochar were investigated in detail; specifically, soil chemistry and rhizosphere microbial composition were characterized, along with the treatment effects on seed storage proteins. The results demonstrated that the addition of commercial microbial consortia and biochar, alone or in combination, did not modify the rhizospheric microbial community; however, it increased grain yield, especially in the cultivar Svevo (increase of 6.8 %-13.6 %), even though the factors driving the most variations were associated with both climate and cultivar. The total gluten content of the flours was not affected, whereas the main effect of the treatments was a variation in gliadins and low-molecular-weight-glutenin subunits in both cultivars when treated with PGPM and biochar. This suggested improved grain quality, especially regarding the viscoelastic properties of the dough, when the filling period occurred in a dry climate. The results indicate that the application of biofertilisers and biochar may aid the effective management of sustainable wheat cultivation, to support environmental health without altering the biodiversity of the resident microbiome.

A novel PGPR strain homologous to Beijerinckia fluminensis induces biochemical and molecular changes involved in Arabidopsis thaliana salt tolerance.

The use of PGPR is widely accepted as a promising tool for a more sustainable agricultural production and improved plant abiotic stress resistance. This study tested the ability of PVr_9, a novel bacterial strain, homologous to Beijerinckia fluminensis, to increase salt stress tolerance in A. thaliana. In vitro plantlets inoculated with PVr_9 and treated with 150 mM NaCl showed a reduction in primary root growth inhibition compared to uninoculated ones, and a leaf area significantly less affected by salt. Furthermore, salt-stressed PVr_9-inoculated plants had low ROS and 8-oxo-dG, osmolytes, and ABA content along with a modulation in antioxidant enzymatic activities. A significant decrease in Na+ in the leaves and a corresponding increase in the roots were also observed in salt-stressed inoculated plants. SOS1, NHX1 genes involved in plant salt tolerance, were up-regulated in PVr_9-inoculated plants, while different MYB genes involved in salt stress signal response were down-regulated in both roots and shoots. Thus, PVr_9 was able to increase salt tolerance in A. thaliana, thereby suggesting a role in ion homeostasis by reducing salt stress rather than inhibiting total Na+ uptake. These results showed a possible molecular mechanism of crosstalk between PVr_9 and plant roots to enhance salt tolerance, and highlighted this bacterium as a promising PGPR for field applications on agronomical crops.

Ultra-high Performance Liquid Chromatography-Ion Mobility-High-Resolution Mass Spectrometry to Evaluate the Metabolomic Response of Durum Wheat to Sustainable Treatments.

Sustainable agriculture aims at achieving a healthy food production while reducing the use of fertilizers and greenhouse gas emissions using biostimulants and soil amendments. Untargeted metabolomics by ultra-high performance liquid chromatography-ion mobility-high-resolution mass spectrometry, operating in a high-definition MSE mode, was applied to investigate the metabolome of durum wheat in response to sustainable treatments, i.e., the addition of biochar, commercial plant growth promoting microbes, and their combination. Partial least squares-discriminant analysis provided a good discrimination among treatments with sensitivity, specificity, and a non-error rate close to 1. A total of 88 and 45 discriminant compounds having biological, nutritional, and technological implications were tentatively identified in samples grown in 2020 and 2021. The addition of biochar-biostimulants produced the highest up-regulation of lipids and flavonoids, with the glycolipid desaturation being the most impacted pathway, whereas carbohydrates were mostly down-regulated. The findings achieved suggest the safe use of the combined biochar-biostimulant treatment for sustainable wheat cultivation.

Ultra-high performance liquid chromatography high-resolution mass spectrometry for metabolomic analysis of dental calculus from Duke Alessandro Farnese and Maria D'Aviz.

Dental calculus is a valuable resource for the reconstruction of dietary habits and oral microbiome of past populations. In 2020 the remains of Duke Alessandro Farnese and his wife Maria D'Aviz were exhumed to get novel insights into the causes of death. This study aimed to investigate the dental calculus metabolome of the noble couple by untargeted metabolomics. The pulverized samples were decalcified in a water-formic acid mixture, extracted using methanol/acetonitrile and analyzed by ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS) using a reversed-phase separation followed by electrospray ionization and full scan in positive and negative ion mode. Waters Synapt-G2-Si High-Definition hybrid quadrupole time-of-flight mass spectrometer was used. Significant features were then identified using MSE acquisition mode, recording information on exact mass precursor and fragment ions within the same run. This approach, together with data pre-treatment and multivariate statistical analysis allowed for the identification of compounds able to differentiate between the investigated samples. More than 200 metabolites were identified, being fatty acids, alcohols, aldehydes, phosphatidylcholines, phosphatidylglycerols, ceramides and phosphatidylserines the most abundant classes. Metabolites deriving from food, bacteria and fungi were also determined, providing information on the habits and oral health status of the couple.

Mass spectrometry-based proteomic strategy for ecchymotic skin examination in forensic pathology.

Mass spectrometry (MS)-based proteomics has recently attracted the attention from forensic pathologists. This work is the first report of the development of a shotgun bottom-up proteomic approach based on rapid protein extraction and nano-liquid chromatography/high-resolution mass spectrometry applied to full-thickness human skin for the differential analysis of normal and ecchymotic tissues to identify new biomarkers for bruise characterization and dating. We identified around 2000 proteins from each pooled extract. The method showed excellent precision on independent replicates, with Pearson correlation coefficients always higher than 95%. Glycophorin A, a known biomarker of vital wounds from immunochemical studies, was identified only in ecchymotic tissues, as confirmed by Western blotting analysis. This finding suggests that this protein can be used as a MS-detectable biomarker of wound vitality. By focusing on skin samples from individuals with known wound dating, besides Glycophorin A, other proteins differentially expressed in ecchymotic samples and dependant on wound age were identified, although further analysis on larger datasets are needed to validate these findings. This study paves the way for an in-depth investigation of the potential of MS-based techniques for wound examination in forensic pathology, overcoming the limitations of immunochemical assays.

Metrological traceability in process analytical technologies and point-of-need technologies for food safety and quality control: not a straightforward issue.

Traditional techniques for food analysis are based on off-line laboratory methods that are expensive and time-consuming and often require qualified personnel. Despite the high standards of accuracy and metrological traceability, these well-established methods do not facilitate real-time process monitoring and timely on-site decision-making as required for food safety and quality control. The future of food testing includes rapid, cost-effective, portable, and simple methods for both qualitative screening and quantification of food contaminants, as well as continuous, real-time measurement in production lines. Process automatization through process analytical technologies (PAT) is an increasing trend in the food industry as a way to achieve improved product quality, safety, and consistency, reduced production cycle times, minimal product waste or reworks, and the possibility for real-time product release. Novel methods of analysis for point-of-need (PON) screening could greatly improve food testing by allowing non-experts, such as consumers, to test in situ food products using portable instruments, smartphones, or even visual naked-eye inspections, or farmers and small producers to monitor products in the field. This requires the attention of the research community and devices manufacturers to ensure reliability of measurement results from PAT strategy and PON tests through the demonstration and critical evaluation of performance characteristics. The fitness for purpose of methods in real-life conditions is a priority that should not be overlooked in order to maintain an effective and harmonized food safety policy.

A spotlight on analytical prospects in food allergens: From emerging allergens and novel foods to bioplastics and plant-based sustainable food contact materials.

The present review throws a spotlight on new and emerging food safety concerns in view of a well-established food allergen risk arising from global socio-economic changes, international trade, circular economy, environmental sustainability, and upcycling. Food culture globalization needs harmonization of regulations, technical specifications, and reference materials towards mutually recognised results. In parallel, routine laboratories require high-throughput reliable analytical strategies, even in-situ testing devices, to test both food products and food contact surfaces for residual allergens. Finally, the currently neglected safety issues associated to possible allergen exposure due to the newly proposed bio- and plant-based sustainable food contact materials require an in-depth investigation.

Controlling Dynamic DNA Reactions at the Surface of Single-Walled Carbon Nanotube Electrodes to Design Hybridization Platforms with a Specific Amperometric Readout.

Carbon nanotube (CNT)-based electrodes are cheap, highly performing, and robust platforms for the fabrication of electrochemical sensors. Engineering programmable DNA nanotechnologies on the CNT surface can support the construction of new electrochemical DNA sensors providing an amperometric output in response to biomolecular recognition. This is a significant challenge, since it requires gaining control of specific hybridization processes and functional DNA systems at the interface, while limiting DNA physisorption on the electrode surface, which contributes to nonspecific signal. In this study, we provide design rules to program dynamic DNA structures at the surface of single-walled carbon nanotubes electrodes, showing that specific DNA interactions can be monitored through measurement of the current signal provided by redox-tagged DNA strands. We propose the use of pyrene as a backfilling agent to reduce nonspecific adsorption of reporter DNA strands and demonstrate the controlled formation of DNA duplexes on the electrode surface, which we then apply in the design and conduction of programmable DNA strand displacement reactions. Expanding on this aspect, we report the development of novel amperometric hybridization platforms based on artificial DNA structures templated by the small molecule melamine. These platforms enable dynamic strand exchange reactions orthogonal to conventional toehold-mediated strand displacement and may support new strategies in electrochemical sensing of biomolecular targets, combining the physicochemical properties of nanostructured carbon-based materials with programmable nucleic acid hybridization.

FFF-based high-throughput sequence shortlisting to support the development of aptamer-based analytical strategies.

Aptamers are biomimetic receptors that are increasingly exploited for the development of optical and electrochemical aptasensors. They are selected in vitro by the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) procedure, but although they are promising recognition elements, for their reliable applicability for analytical purposes, one cannot ignore sample components that cause matrix effects. This particularly applies when different SELEX-selected aptamers and related truncated sequences are available for a certain target, and the choice of the aptamer should be driven by the specific downstream application. In this context, the present work aimed at investigating the potentialities of asymmetrical flow field-flow fractionation (AF4) with UV detection for the development of a screening method of a large number of anti-lysozyme aptamers towards lysozyme, including randomized sequences and an interfering agent (serum albumin). The possibility to work in native conditions and selectively monitor the evolution of untagged aptamer signal as a result of aptamer-protein binding makes the devised method effective as a strategy for shortlisting the most promising aptamers both in terms of affinity and in terms of selectivity, to support subsequent development of aptamer-based analytical devices.

Aptamer-based assays: strategies in the use of aptamers conjugated to magnetic micro- and nanobeads as recognition elements in food control.

An outlook on the current status of different strategies for magnetic micro- and nanosized bead functionalization with aptamers as prominent bioreceptors is given with a focus on electrochemical and optical apta-assays, as well as on aptamer-modified magnetic bead-based miniaturized extraction techniques in food control. Critical aspects that affect interaction of aptamers with target molecules, as well as the possible side effects caused by aptamer interaction with other molecules due to non-specific binding, are discussed. Challenges concerning the real potential and limitations of aptamers as bioreceptors when facing analytical problems in food control are addressed.

Reversed-phase and weak anion-exchange mixed-mode stationary phase for fast separation of medium-, long- and very long chain free fatty acids by ultra-high-performance liquid chromatography-high resolution mass spectrometry.

Two commercial stationary phases allowing both reversed phase mechanism and anion-exchange with different selectivity, i.e. CSH C18 and Atlantis PREMIER BEH C18 AX, were tested for the separation of a complex mixture of 21 fatty acids (FAs) encompassing saturated medium-, long- and very long chain FAs, unsaturated long and very long chain FAs, cis/trans isomers, and isomers of odd- and branched-chain FAs. For this purpose, the role of surface area of stationary phase and the effect of pH of the mobile phase on the retention of the analytes were investigated. Separation was performed by ultra-high-performance liquid chromatography coupled with high resolution mass spectrometry (UHPLC-HRMS). BEH C18 AX was shown to be more versatile and to offer superior retention of these analytes to CSH C18 owing to a higher surface area and anion-exchange capacity up to pH 8.5. The UHPLC system allows shortening analysis time, the chromatographic analysis being accomplished in about 5 min, affording a high throughput of samples without the need for derivatization or ion-pairing reagents compared to techniques based upon gas chromatography approaches or LC. Finally, the application of the BEH C18 AX column using UHPLC-HRMS was demonstrated for the separation and unambiguous identification of FAs of nutritional interest in a dietary supplement sample.

Sprouting of Sorghum (Sorghum bicolor [L.] Moench): Effect of Drying Treatment on Protein and Starch Features.

The nutritional and physicochemical properties of sorghum proteins and starch make the use of this cereal for food production challenging. Sprouting is a cost-effective technology to improve the nutritional and functional profile of grains. Two drying treatments were used after sorghum sprouting to investigate whether the drying phase could improve the protein and starch functionalities. Results showed that the drying treatment at lower temperature/longer time (40 °C for 12 h) extended the enzymatic activity that started during sprouting compared to the one performed at higher temperature/shorter time (50 °C for 6 h). An increased protein hydrolysis and water- and oil-holding capacity were found in the flour obtained by the former treatment. Higher protein matrix hydrolysis caused high exposure of starch to enzymes, thus increasing its digestibility, while worsening the technological functionality. Overall, modulating drying conditions could represent a further way, in addition to sprouting, to improve sorghum flour's nutritional profile.

Electrochemical immunomagnetic assay as biosensing strategy for determination of ovarian cancer antigen HE4 in human serum.

Prompt cancer diagnosis and treatment represent fundamental aspects to significantly improve patient survival rate. Human epididymis protein 4 (HE4) has recently been identified as promising single serum biomarker of epithelial ovarian cancer with improved diagnostic performances respect to current reference biomarkers. In this study we present the first competitive immunosensing strategy for HE4 determination implemented on magnetic microbeads functionalized with HE4 antigen. A full factorial design and multiple linear regression allowed to find the optimal experimental conditions providing the maximum inhibition rate within the explored domain. Method validation was performed in serum to ensure reliable data to support decision in clinical practice. This method allowed matching the clinically relevant concentration values for the serum biomarker, limits of detection and quantification being 2.8 and 23.0 pM, respectively. Also recovery rate in the 89 ± 7-103 ± 5% range resulted suitable for method applicability for diagnostic purposes.

The role of surface in desorption electrospray ionization-mass spectrometry: advances and future trends.

An outlook on the current status and trends in desorption electrospray ionization-mass spectrometry (DESI-MS), one of the most common spray-based techniques for ambient ionization, is given with a focus on the main advances recently achieved or still in progress regarding studies of surface properties affecting the signal stability and efficiency of the DESI process. Future directions that the field may take in the years to come are discussed, with particular focus on bioanalytical research.

3D-printed chitosan scaffolds modified with D-(+) raffinose and enriched with type IV collagen to improve epithelial cell colonization.

Tissue regeneration often requires the use of biocompatible resorbable scaffolds to support the ingrowth of cells from neighboring tissues into a localized tissue defect. Such scaffolds must possess surface molecular cues that stimulate cells to populate the device, the first necessary condition for the formation of a healthy tissue. Chitosan is a natural polymer that has long been tested in biomedical applications because of its high biocompatibility, which can be further increased by modifying its formulation, e.g. adding D-(+) raffinose. We used this formulation in an ad hoc designed 3D printer to create regularly ordered scaffolds, which we then enriched with type IV collagen, an isoform of collagen that is exclusively found in basement membranes. Human epithelial A549 cells were then seeded on control scaffolds or on scaffolds coated with collagen, which was precipitated, or on scaffolds first collagenized and then exposed to either UVB or UVC radiation. Observations by the transmission light microscope, confocal microscope after staining with calcein-AM/propidium iodide, and by environmental scanning electron microscope revealed that collagen-enriched UV-treated scaffolds promoted the attachment of a higher number of cells, which covered a more extensive area of the scaffold, as also confirmed by alamar blue viability assay. Together these data confirm that coating 3D-printed scaffolds made of D-(+) raffinose-modified chitosan with type IV collagen and exposing them to UV light sensibly increases the cell compatibility of scaffolds, making them a better candidate to serve as a tool for the regeneration of epithelia.

A Self-Calibrating IoT Portable Electrochemical Immunosensor for Serum Human Epididymis Protein 4 as a Tumor Biomarker for Ovarian Cancer.

Nowadays, analytical techniques are moving towards the development of smart biosensing strategies for the point-of-care accurate screening of disease biomarkers, such as human epididymis protein 4 (HE4), a recently discovered serum marker for early ovarian cancer diagnosis. In this context, the present work represents the first implementation of a competitive enzyme-labelled magneto-immunoassay exploiting a homemade IoT Wi-Fi cloud-based portable potentiostat for differential pulse voltammetry readout. The electrochemical device was specifically designed to be capable of autonomous calibration and data processing, switching between calibration, and measurement modes: in particular, firstly, a baseline estimation algorithm is applied for correct peak computation, then calibration function is built by interpolating data with a four-parameter logistic function. The calibration function parameters are stored on the cloud for inverse prediction to determine the concentration of unknown samples. Interpolation function calibration and concentration evaluation are performed directly on-board, thus reducing the power consumption. The analytical device was validated in human serum, demonstrating good sensing performance for analysis of HE4 with detection and quantitation limits in human serum of 3.5 and 29.2 pM, respectively, reaching the sensitivity that is required for diagnostic purposes, with high potential for applications as portable and smart diagnostic tool for point-of-care testing.

Analytical screening of marine algal toxins for seafood safety assessment in a protected Mediterranean shallow water environment.

Microalgal species growing in marine and aquaculture environments can be responsible for harmful events because of their ability to produce potent natural toxins that can accumulate in edible mollusc species. Their consumption can cause severe illness and even be lethal. The European Union provides comprehensive regulations covering various general food safety aspects to manage the risk of contamination in shellfish farms. Many analytical methods have been proposed to evaluate algal toxins presence in the environment and in food products, for conducting surveillance studies of the main molluscs production sites and, where necessary, immediate monitoring of possible contamination of shellfish. In this work, a one-year analytical surveillance study was carried out to verify the possible presence of algal biotoxins in molluscs from a Mediterranean breeding area. Water and molluscs were sampled from a district of the North-East coast of Sicily, consisting of a unique brackish ecosystem of two lakes connected to each other and to the sea by narrow canals. Water samples were collected to investigate phytoplankton i by microscope analysis to assess the presence of potentially toxin-producing species, such as Pseudo-nitzschia spp, Alexandrium spp and Gonyaulax spinifera, although the presence of toxic phytoplankton has never reached alert levels. Mussels and clams samples were submitted to analysis of paralytic shellfish poisoning toxins, amnesic shellfish poisoning toxins and lipophilic toxins by liquid chromatography-based methods Only a few yessotoxins were detected, having concentrations always below the regulation limits. An existing liquid chromatography-tandem mass spectrometry-based multiresidue method for lipophilic biotoxins was adopted and extended to cover emerging biotoxins such as cyclic imines. The performance of the analytical method for Gymnodimine A and Spirolide 13-desMeC was assessed, obtaining respective quantitation limits of 20 and 10 µg kg-1, a precision always lower than 13% and trueness in the 81-120% range. Method applicability was confirmed using certified materials and a naturally contaminated sample.

Vertebrate odorant binding proteins as antimicrobial humoral components of innate immunity for pathogenic microorganisms.

The bacterium Pseudomonas aeruginosa (PA) and the yeast Candida albicans (CA) are pathogens that cohabit the mucosa of the respiratory tracts of animals and humans. Their virulence is largely determined by chemical communication driven by quorum sensing systems (QS), and the cross perception of their quorum sensing molecules (QSM) can modulate the prevalence of one microorganism over the other. Aiming to investigate whether some of the protein components dissolved in the mucus layering the respiratory mucosa might interfere with virulence and cross-communication of these, and eventually other microorganisms, ligand binding assays were carried out to test the scavenging potential of the bovine and porcine forms of the Lipocalin odorant binding protein (OBP) for several QSMs (farnesol, and acylhomoserine lactones), and for pyocyanin, a toxin produced by PA. In addition, the direct antimicrobial activity of the OBPs was tested by time kill assay (TKA) against CA, PA and other bacteria and yeasts. The positivity of all the ligand binding assays and the antimicrobial activity determined for CA, and for some of the other microorganisms tested, let hypothesize that vertebrate OBPs might behave as humoral components of innate immunity, active against pathogenic bacteria and fungi. In addition, TKAs with mutants of bovine OBP with structural properties different from those of the native form, and with OBP forms tagged with histidines at the amino terminal, provided information about the mechanisms responsible of their antimicrobial activity and suggested possible applications of the OBPs as alternative or co-adjuvants to antibiotic therapeutic treatments.

The role of incurred materials in method development and validation to account for food processing effects in food allergen analysis.

The issue of undeclared allergens represents a matter of great concern, being the subject of many alert notifications by the Rapid Alert System for Food and Feed portal of the European Commission, often leading to food recalls. The availability of reliable analytical approaches able to detect and quantify hidden allergens in processed foods is increasingly requested by the food industry, food safety authorities and regulatory bodies to protect sensitive consumers' health. The present review discusses the fundamental role of incurred materials for method development and analytical performance assessment in a metrology perspective on testing for undeclared allergens in processed foodstuffs. Due to the nature of the analytes and their susceptibility to various processing effects, reliability and comparability of results have posed a great challenge. In this context, the use of incurred samples as reference materials permits simulation of the effects of food processing on target analyte structure affecting analyte extractability and detectability. Graphical abstract ᅟ.