Combined endophytic inoculants enhance nickel phytoextraction from serpentine soil in the hyperaccumulator Noccaea caerulescens

This study assesses the effects of specific bacterial endophytes on the phytoextraction capacity of the Ni-hyperaccumulator Noccaea caerulescens, spontaneously growing in a serpentine soil environment. Five metal-tolerant endophytes had already been selected for their high Ni tolerance (6 mM) and plant growth promoting ability. Here we demonstrate that individual bacterial inoculation is ineffective in enhancing Ni translocation and growth of N. caerulescens in serpentine soil, except for specific strains Ncr-1 and Ncr-8, belonging to the Arthrobacter and Microbacterium genera, which showed the highest indole acetic acid production and 1-aminocyclopropane-1-carboxylic acid-deaminase activity. Ncr-1 and Ncr-8 co-inoculation was even more efficient in promoting plant growth, soil Ni removal, and translocation of Ni, together with that of Fe, Co, and Cu. Bacteria of both strains densely colonized the root surfaces and intercellular spaces of leaf epidermal tissue. These two bacterial strains also turned out to stimulate root length, shoot biomass, and Ni uptake in Arabidopsis thaliana grown in MS agar medium supplemented with Ni. It is concluded that adaptation of N. caerulescens in highly Ni-contaminated serpentine soil can be enhanced by an integrated community of bacterial endophytes rather than by single strains; of the former, Arthrobacter and Microbacterium may be useful candidates for future phytoremediation trials in multiple metal-contaminated sites, with possible extension to non-hyperaccumulator plants

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 point-of-care accurate screening of disease biomarkers, such as human epididymis protein 4 (HE4), a recently discovered serum markers 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 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, 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 required for diagnostic purposes, with high potential for applications as portable and smart diagnostic tool for point-of-care testing

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

Optimization of a rapid QuEChERS sample treatment method for HILIC-MS2 analysis of paralytic shellfish poisoning (PSP) toxins in mussels

A rapid and simple QuEChERS sample treatment was proposed for the development of a selective hydrophilic interaction liquid chromatography-ESI-MS2-based method for the determination of saxitoxins (STXs) in mussel samples. Among different sorbents, ABS Elut-NEXUS phase, composed of polystyrene cross-linked with 50 % divinyl benzene and poly(methyl methacrylate), provided the best results. The effects of experimental parameters, including sorbent amount, vortexing time and centrifugation time were investigated and optimized by experimental design. In particular, regression models and desirability functions were applied to find the experimental conditions providing the highest global extraction response. The method was validated under the optimized conditions; detection and quantification limits in the 3-159 μg/kg and 7-436 μg/kg ranges respectively were obtained, except for C2 for which highest values were calculated due to its low ESI ionization efficiency. Finally, the analysis of twenty-eight mussel samples permitted to detect and quantify some of the investigated STXs, proving the applicability of the devised method

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

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

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

Liquid Chromatography/Mass Spectrometry in Environmental Analysis

Liquid chromatography/mass spectrometry (LC-MS) technique enables analysis of emergent polar compounds without the need for derivatization reaction, while the introduction of high-resolution mass analyzers in hybrid configurations makes possible to elucidate chemical structure, also without reference standards. Over the past 10 years, besides target analysis, the coupling of LC to high-resolution mass spectrometry (HRMS) has allowed screening of compounds expected to be in the environment, as well as nontarget screening of unknown compounds, such as transformation products (TPs) formed in the environment or during technical wastewater and sewage purification treatments. Nowadays, analytical chemists are trying to achieve real nontarget analysis of numerous unknowns in environmental samples for a comprehensive view of the true overall contamination present in samples, but it still remains a very challenging issue. The development of new software tools to extract relevant data, spectra libraries, and databases will be of great help to solve this issue, enhancing the success rate. This article represents the second edition of a previous manuscript by Careri,2000 which was updated to take into account the huge progress in MS instrumentation and related methodologies and also advances in environmental analytical chemistry. In this overview, among the environmental applications of LC-MS, current and innovative approaches for the analysis of selected classes of emerging pollutants, such as novel brominated flame retardants, nanomaterials, hormones and other endocrine disruptors, and pharmaceutical and personal care products in environmental samples are discussed

Innovative gold-free carbon nanotube/chitosan-based competitive immunosensor for determination of HIV-related p24 capsid protein in serum

In the past decade, the need for simple, rapid, sensitive, specific and inexpensive screening methods for diagnosis of HIV infection has led to a focus on the HIV1-related capsid protein p24. In this work, the first competitive electrochemical immunosensor for the detection of p24 in untreated human serum was developed as a simple, easy-to-use and promising tool for serum screening for early diagnosis of HIV infection. The immunodevice was implemented on disposable gold-free single-walled carbon nanotube-functionalized screen-printed electrodes. The competitive sensor is based on the immobilization of the target protein on the electrode surface using a chitosan/glutaraldehyde crosslinking system, able to ensure, under mild conditions, a robust immobilization and a proper exposition of p24 for interaction with a mouse anti-p24 IgG1. The immunosensor setup as well as the assay's experimental conditions were then optimized, achieving a wide linear detection range of 10 pM to 1 nM, with a low detection limit of 2 pM in human serum. The good performance, also in terms of selectivity, trueness and precision, coupled with the advantages of an easy preparation compared to other methods requiring very complex conjugation procedures between bioreceptors and expensive nanostructures, makes the immunosensor a powerful diagnostic tool, valuable for implementation of large-scale screening programs for early diagnosis of seropositivity