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.

Evaluation of a Voltametric E-Tongue Combined with Data Preprocessing for Fast and Effective Machine Learning-Based Classification of Tomato Purées by Cultivar.

The potential of a voltametric E-tongue coupled with a custom data pre-processing stage to improve the performance of machine learning techniques for rapid discrimination of tomato purées between cultivars of different economic value has been investigated. To this aim, a sensor array with screen-printed carbon electrodes modified with gold nanoparticles (GNP), copper nanoparticles (CNP) and bulk gold subsequently modified with poly(3,4-ethylenedioxythiophene) (PEDOT), was developed to acquire data to be transformed by a custom pre-processing pipeline and then processed by a set of commonly used classifiers. The GNP and CNP-modified electrodes, selected based on their sensitivity to soluble monosaccharides, demonstrated good ability in discriminating samples of different cultivars. Among the different data analysis methods tested, Linear Discriminant Analysis (LDA) proved to be particularly suitable, obtaining an average F1 score of 99.26%. The pre-processing stage was beneficial in reducing the number of input features, decreasing the computational cost, i.e., the number of computing operations to be performed, of the entire method and aiding future cost-efficient hardware implementation. These findings proved that coupling the multi-sensing platform featuring properly modified sensors with the custom pre-processing method developed and LDA provided an optimal tradeoff between analytical problem solving and reliable chemical information, as well as accuracy and computational complexity. These results can be preliminary to the design of hardware solutions that could be embedded into low-cost portable devices.

Supramolecular Materials as Solid-Phase Microextraction Coatings in Environmental Analysis.

Solid-phase microextraction (SPME) has been widely proposed for the extraction, clean-up, and preconcentration of analytes of environmental concern. Enrichment capabilities, preconcentration efficiency, sample throughput, and selectivity in extracting target compounds greatly depend on the materials used as SPME coatings. Supramolecular materials have emerged as promising porous coatings to be used for the extraction of target compounds due to their unique selectivity, three-dimensional framework, flexible design, and possibility to promote the interaction between the analytes and the coating by means of multiple oriented functional groups. The present review will cover the state of the art of the last 5 years related to SPME coatings based on metal organic frameworks (MOFs), covalent organic frameworks (COFs), and supramolecular macrocycles used for environmental applications.

The Combined ICP-MS, ESEM-EDX, and HAADF-STEM-EDX Approach for the Assessment of Metal Sub-Micro- and Nanoparticles in Wheat Grain.

Metal sub-microparticles (SMPs) and nanoparticles (NPs) presence in food is attributable to increasing pollution from the environment in raw materials and finished products. In the present study, a multifaceted analytical strategy based on Environmental Scanning Electron Microscopy and High-Angle Annular Dark-Field-Scanning Transmission Electron Microscopy coupled with Energy-Dispersive X-ray Spectroscopy (ESEM-EDX, HAADF-STEM-EDX) and Inductively Coupled Plasma Mass Spectrometry (ICP-MS) was proposed for the detection and characterization of metal and metal-containing SMPs and NPs in durum wheat samples, covering a size measurement range from 1 nm to multiple µm. ESEM-EDX and ICP-MS techniques were applied for the assessment of SMP and NP contamination on the surface of wheat grains collected from seven geographical areas characterized by different natural and anthropic conditions, namely Italy, the USA, Australia, Slovakia, Mexico, Austria, and Russia. ICP-MS showed significant differences among the mean concentration levels of metals, with the USA and Italy having the highest level. ESEM-EDX analysis confirmed ICP-MS concentration measurements and measured the highest presence of particles < 0.8 µm in size in samples from Italy, followed by the USA. Less marked differences were observed when particles < 0.15 µm were considered. HAADF-STEM-EDX was applied to a selected number of samples for a preliminary assessment of internal contamination by metal SMPs and NPs, and to expand the measurable particle size range. The multifaceted approach provided similar results for Fe-containing SMPs and NPs. ICP-MS and ESEM-EDX also highlighted the presence of a significant abundance of Ti- and Al-containing particles, while for STEM-EDX, sample preparation artifacts complicated the interpretation. Finally, HAADF-STEM-EDX results provided relevant information about particles in the low nm range, since, by applying this technique, no particles smaller than 50 nm were observed in accordance with ESEM-EDX.

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.

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.

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.

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.

Smart Immunosensors for Point-of-Care Serological Tests Aimed at Assessing Natural or Vaccine-Induced SARS-CoV-2 Immunity.

Innovative and highly performing smart voltammetric immunosensors for rapid and effective serological tests aimed at the determination of SARS-CoV-2 antibodies were developed and validated in human serum matrix. Two immunosensors were developed for the determination of immunoglobulins directed against either the nucleocapsid or the spike viral antigen proteins. The immunosensors were realized using disposable screen-printed electrodes modified with nanostructured materials for the immobilization of the antigens. Fast quantitative detection was achieved, with analysis duration being around 1 h. Signal readout was carried out through a smart, compact and battery-powered potentiostat, based on a Wi-Fi protocol and devised for the Internet of Things (IoT) paradigm. This device is used for the acquisition, storage and sharing of clinical data. Outstanding immunosensors' sensitivity, specificity and accuracy (100%) were assessed, according to the diagnostic guidelines for epidemiological data. The overall performance of the sensing devices, combined with the portability of the IoT-based device, enables their suitability as a high-throughput diagnostic tool. Both of the immunosensors were validated using clinical human serum specimens from SARS-CoV-2 infected patients, provided by IRCCS Ospedale San Raffaele.

An Efficient Solid-Phase Microextraction-Gas Chromatography-Mass Spectrometry Method for the Analysis of Methyl Farnesoate Released in Growth Medium by Daphnia pulex.

Methyl farnesoate (MF), a juvenile hormone, can influence phenotypic traits and stimulates male production in daphnids. MF is produced endogenously in response to stressful conditions, but it is not known whether this hormone can also be released into the environment to mediate stress signaling. In the present study, for the first time, a reliable solid-phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS) method was developed and validated for the ultra-trace analysis of MF released in growth medium by Daphnia pulex maintained in presence of crowding w/o MK801, a putative upstream inhibitor of MF endogenous production. Two different clonal lineages, I and S clones, which differ in the sensitivity to the stimuli leading to male production, were also compared. A detection limit of 1.3 ng/L was achieved, along with good precision and trueness, thus enabling the quantitation of MF at ultra-trace level. The achieved results demonstrated the release of MF by both clones at the 20 ng/L level in control conditions, whereas a significant decrease in the presence of crowding was assessed. As expected, a further reduction was obtained in the presence of MK801. These findings strengthen the link between environmental stimuli and the MF signaling pathway. Daphnia pulex, by releasing the juvenile hormone MF in the medium, could regulate population dynamics by means of an autoregulatory feedback loop that controls the intra- and extra-individual-level release of MF produced by endogenous biosynthesis.

Deciphering the Supramolecular Organization of Multiple Guests Inside a Microporous MOF to Understand their Release Profile.

Metal-organic frameworks (MOFs) give the opportunity of confining guest molecules into their pores even by a post-synthetic protocol. PUM168 is a Zn-based MOF characterized by microporous cavities that allows the encapsulation of a significant number of guest molecules. The pores engineered with different binding sites show a remarkable guest affinity towards a series of natural essential oils components, such as eugenol, thymol and carvacrol, relevant for environmental applications. Exploiting single crystal X-ray diffraction, it was possible to step-wisely monitor the rather complex three-components guest exchange process involving dimethylformamide (DMF, the pristine solvent) and binary mixtures of the flavoring agents. A picture of the structural evolution of the DMF-to-guest replacement occurring inside the MOF crystal was reached by a detailed single-crystal-to-single-crystal monitoring. The relation of the supramolecular arrangement in the pores with selective guests release was then investigated as a function of time and temperature by static headspace GC-MS analysis.

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.

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.

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.

Single-Walled Carbon Nanotubes as Enhancing Substrates for PNA-Based Amperometric Genosensors.

A new amperometric sandwich-format genosensor has been implemented on single-walled carbon nanotubes screen printed electrodes (SWCNT-SPEs) and compared in terms of performance with analogous genoassays developed using the same methodology on non-nanostructured glassy carbon platforms (GC-SPE). The working principle of the genosensors is based on the covalent immobilization of Peptide Nucleic Acid (PNA) capture probes (CP) on the electrode surface, carried out through the carboxylic functions present on SWCNT-SPEs (carboxylated SWCNT) or electrochemically induced on GC-SPEs. The sequence of the CP was complementary to a 20-mer portion of the target DNA; a second biotin-tagged PNA signalling probe (SP), with sequence complementary to a different contiguous portion of the target DNA, was used to obtain a sandwich hybrid with an Alkaline Phosphatase-streptavidin conjugate (ALP-Strp). Comparison of the responses obtained from the SWCNT-SPEs with those produced from the non-nanostructured substrates evidenced the remarkable enhancement effect given by the nanostructured electrode platforms, achieved both in terms of loading capability of PNA probes and amplification of the electron transfer phenomena exploited for the signal transduction, giving rise to more than four-fold higher sensitivity when using SWCNT-SPEs. The nanostructured substrate allowed to reach limit of detection (LOD) of 71 pM and limit of quantitation (LOQ) of 256 pM, while the corresponding values obtained with GC-SPEs were 430 pM and 1.43 nM, respectively.

A metaproteomic approach dissecting major bacterial functions in the rhizosphere of plants living in serpentine soil.

A metaproteomic approach, based on liquid chromatography-high resolution mass spectrometry (LC-HRMS) analysis, was followed to map the major bacterial metabolic functions associated with the rhizosphere of metal-tolerant and metal hyperaccumulator plants, growing in a serpentine soil naturally contaminated by heavy metals such as Ni, Co and Cr. In particular, an "in-house" bacterial protein database was built based on the genera recognised by 16S rDNA profiling, then it was used for protein identification from LC-MS data. The combination of the information arising from three different extraction protocols, applied to each soil sample, permitted the identification of almost 800 proteins, corresponding to functions assigned to proper Gene Ontology categories. Mainly proteins involved in response to stimulus or in transport of metals and nutrients revealed variability of bacteria responses to microenvironment conditions. As for taxonomy, Phyllobacterium, Microbacterium oxidans, Pseudomonas oryzihabitans, Stenotrophomonas rhizophila and Bacillus methylotrophicus bacterial species were more represented in the rhizosphere samples of the metal-tolerant Biscutella laevigata and of the Ni hyperaccumulator Noccaea caerulescens with respect to bulk soil. Combining 16S rRNA gene-based sequencing and metaproteomic analysis, we get insights into microbial community functions and their interaction with plants colonising the stressful environment of serpentine soils.

Competitive immunosensor based on gliadin immobilization on disposable carbon-nanogold screen-printed electrodes for rapid determination of celiotoxic prolamins.

The first competitive disposable amperometric immunosensor based on gliadin-functionalized carbon/nanogold screen-printed electrodes was developed for rapid determination of celiotoxic prolamins. To date, no competitive spectrophotometric or electrochemical immunoassays have yet been successfully applied to gluten detection in processed food samples, which require the use of complex prolamin extraction solutions containing additives with denaturing, reducing and disaggregating functions. Thus, in this work, great effort was put into the optimization and performance evaluation of the immunosensor in terms of suitability as a screening tool for analysis of cereal-based food samples. For this purpose, aqueous ethanol or complex extraction mixtures, as the patented Cocktail Solution®, were proved effective in the extraction of gliadin. Good sensitivity was achieved after optimization of the immunocompetitive assay, giving limit of detection and limit of quantitation of 8 and 22 ng/ml of gliadin, respectively, for ethanol extracts. The immunosensor was proved to be suitable also for samples extracted with Cocktail Solution® after a proper dilution. Analysis of real samples of different flours proved the suitability of the immunosensing device as a powerful tool for safety assessment of raw materials used for the formulation of dietary products for celiac disease patients. This immunosensor combines good analytical performance using a very simplified set-up protocol with suitability for rapid screening analysis performed using inexpensive and portable instrumentation. Graphical abstract Depiction of the development and working principle of the competitive immunosensor.

Micro-solid-phase extraction coupled to desorption electrospray ionization-high-resolution mass spectrometry for the analysis of explosives in soil.

Home-made micro-solid-phase extraction (SPE) cartridges using different adsorbent materials were tested for the desorption electrospray ionization-high-resolution mass spectrometry (DESI-HRMS) determination of explosives like 2,4,6-trinitrotoluene, cyclotrimethylene-trinitramine, cyclotetramethylene-tetranitramine, pentaerythritol tetranitrate, and trinitrophenylmethylnitramine in soil samples. Quantitation limits in the low nanogram per kilogram range proved the reliability of the method for the detection of explosives at ultra-trace levels. The reduced sample preparation allowed for low costs and high-throughput analyses. Finally, the superior extraction capability of the method was proved by obtaining DESI-HRMS responses at least five times higher than those achieved by performing DESI-HRMS analyses of solid-liquid extracts spotted onto commercial polytetrafluoroethylene slides.

Cavitand-based solid-phase microextraction coating for the selective detection of nitroaromatic explosives in air and soil.

A selective cavitand-based solid-phase microextraction coating was synthesized for the determination of nitroaromatic explosives and explosive taggants at trace levels in air and soil. A quinoxaline cavitand functionalized with a carboxylic group at the upper rim was used to enhance selectivity toward analytes containing nitro groups. The fibers were characterized in terms of film thickness, morphology, thermal stability, and pH resistance. An average coating thickness of 50 (±4) µm, a thermal stability until 400 °C, and an excellent fiber-to-fiber and batch to batch repeatability with RSD lower than 4% were obtained. The capabilities of the developed coating for the selective sampling of nitroaromatic explosives were proved achieving LOD values in the low ppbv and ng kg(-1) range, respectively, for air and soil samples.

A novel magnetic ligand-based assay for the electrochemical determination of BRD4.

The first magnetic ligand-based electrochemical assay aimed at the determination of BRD4 was developed and validated. BRD4 is an epigenetic regulator of great interest in oncology in relation to its overexpression observed in the pathogenesis of several cancer diseases. BRD4 also represents a major target for the development of innovative treatments aimed at protein inhibition or degradation. Despite the relevance of BRD4 both for diagnostics and therapeutic purposes, current methodologies for its determination are limited to commercial ELISA kits. We present a novel magnetic ligand-based assay for the electrochemical determination of BRD4. The developed assay is based on the use of a small synthetic fragment of the natural protein ligand for BRD4 as receptor, thus exploiting the intrinsic biological protein-protein recognition mechanism. In addition, the assay features the use of magnetic beads as immobilization platforms and peroxidase-conjugated monoclonal anti-BRD4 antibody for the generation of the electrochemical signal. The ligand-based assay shows outstanding performance in terms of rapidity, with results achievable in less than 20 min, no matrix effect when applied to human plasma or cell lysate samples, and excellent specificity. The proposed method exhibits a limit of detection of 2.66 nM and a response range tunable as a function of the amount of immobilized receptor. The developed ligand-based assay was successfully applied to the accurate determination of BRD4 in untreated cell lysates, as proven by the ELISA reference method. The good performance of the proposed bioassay for determination of BRD4 showed potential application of this strategy in convenient point-of-care testing.