Enzymatic Synthesis and Structural Modeling of Bio-Based Oligoesters as an Approach for the Fast Screening of Marine Biodegradation and Ecotoxicity.

Given the widespread use of esters and polyesters in products like cosmetics, fishing nets, lubricants and adhesives, whose specific application(s) may cause their dispersion in open environments, there is a critical need for stringent eco-design criteria based on biodegradability and ecotoxicity evidence. Our approach integrates experimental and computational methods based on short oligomers, offering a screening tool for the rapid identification of sustainable monomers and oligomers, with a special focus on bio-based alternates. We provide insights into the relationships between the chemical structure and properties of bio-based oligomers in terms of biodegradability in marine environments and toxicity in benchmark organisms. The experimental results reveal that the considered aromatic monomers (terephthalic acid and 2,5-furandicarboxylic acid) accumulate under the tested conditions (OECD 306), although some slight biodegradation is observable when the inoculum derives from sites affected by industrial and urban pollution, which suggests that ecosystems adapt to non-natural chemical pollutants. While clean seas are more susceptible to toxic chemical buildup, biotic catalytic activities offer promise for plastic pollution mitigation. Without prejudice to the fact that biodegradability inherently signifies a desirable trait in plastic products, nor that it automatically grants them a sustainable "license", this study is intended to facilitate the rational design of new polymers and materials on the basis of specific uses and applications.

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.

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.

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.

Rapid Quantification of SARS-Cov-2 Spike Protein Enhanced with a Machine Learning Technique Integrated in a Smart and Portable Immunosensor.

An IoT-WiFi smart and portable electrochemical immunosensor for the quantification of SARS-CoV-2 spike protein was developed with integrated machine learning features. The immunoenzymatic sensor is based on the immobilization of monoclonal antibodies directed at the SARS-CoV-2 S1 subunit on Screen-Printed Electrodes functionalized with gold nanoparticles. The analytical protocol involves a single-step sample incubation. Immunosensor performance was validated in a viral transfer medium which is commonly used for the desorption of nasopharyngeal swabs. Remarkable specificity of the response was demonstrated by testing H1N1 Hemagglutinin from swine-origin influenza A virus and Spike Protein S1 from Middle East respiratory syndrome coronavirus. Machine learning was successfully used for data processing and analysis. Different support vector machine classifiers were evaluated, proving that algorithms affect the classifier accuracy. The test accuracy of the best classification model in terms of true positive/true negative sample classification was 97.3%. In addition, the ML algorithm can be easily integrated into cloud-based portable Wi-Fi devices. Finally, the immunosensor was successfully tested using a third generation replicating incompetent lentiviral vector pseudotyped with SARS-CoV-2 spike glycoprotein, thus proving the applicability of the immunosensor to whole virus detection.

A Folding-Based Electrochemical Aptasensor for the Single-Step Detection of the SARS-CoV-2 Spike Protein.

Efficient and timely testing has taken center stage in the management, control, and monitoring of the current COVID-19 pandemic. Simple, rapid, cost-effective diagnostics are needed that can complement current polymerase chain reaction-based methods and lateral flow immunoassays. Here, we report the development of an electrochemical sensing platform based on single-walled carbon nanotube screen-printed electrodes (SWCNT-SPEs) functionalized with a redox-tagged DNA aptamer that specifically binds to the receptor binding domain of the SARS-CoV-2 spike protein S1 subunit. Single-step, reagentless detection of the S1 protein is achieved through a binding-induced, concentration-dependent folding of the DNA aptamer that reduces the efficiency of the electron transfer process between the redox tag and the electrode surface and causes a suppression of the resulting amperometric signal. This aptasensor is specific for the target S1 protein with a dissociation constant (KD) value of 43 ± 4 nM and a limit of detection of 7 nM. We demonstrate that the target S1 protein can be detected both in a buffer solution and in an artificial viral transport medium widely used for the collection of nasopharyngeal swabs, and that no cross-reactivity is observed in the presence of different, non-target viral proteins. We expect that this SWCNT-SPE-based format of electrochemical aptasensor will prove useful for the detection of other protein targets for which nucleic acid aptamer ligands are made available.

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.

PNA-functionalized magnetic microbeads as substrates for enzyme-labelled voltammetric genoassay for DNA sensing applied to identification of GMO in food.

A novel enzyme-labelled voltammetric magnetogenoassay for DNA sensing based on the use of carboxyl-surface coated magnetic microbeads functionalized with PNA probes and subsequent read-out on screen-printed electrode (SPE) substrates was developed. The assay was validated for determination of non-amplified genomic DNA from genetically modified Roundup Ready soy. Outstanding performance with respect to other genoassays requiring preliminary amplification of target DNA via PCR was demonstrated. The analytical performance was also improved compared to previous methods based on the immobilization of the same PNA probes on SPE substrates, since the method was found capable of achieving LOD and LOQ of 415 fM and 995 fM, respectively. The ability of the magnetogenoassay to detect the presence of Roundup Ready soy DNA sequence was tested on genomic DNA extract from European Reference Material soy flours, demonstrating the capability of the method to match the European Union regulation for labelling of food containing a percentage of GM products greater than 0,9%.

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.

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.

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.

Novel amperometric genosensor based on peptide nucleic acid (PNA) probes immobilized on carbon nanotubes-screen printed electrodes for the determination of trace levels of non-amplified DNA in genetically modified (GM) soy.

A novel amperometric genosensor based on PNA probes covalently bound on the surface of Single Walled Carbon Nanotubes - Screen Printed Electrodes (SWCNT-SPEs) was developed and validated in samples of non-amplified genomic DNA extracted from genetically modified (GM)-Soy. The sandwich assay is based on a first recognition of a 20-mer portion of the target DNA by a complementary PNA Capture Probe (CP) and a second hybridization with a PNA Signalling Probe (SP), with a complementary sequence to a different portion of the target DNA. The SP was labelled with biotin to measure current signal by means of a final incubation of an Alkaline Phosphatase-streptavidin conjugate (ALP-Strp). The electrochemical detection was carried out using hydroquinone diphosphate (HQDP) as enzymatic substrate. The genoassay provided a linear range from 250 pM to 2.5 nM, LOD of 64 pM and LOQ of 215 pM Excellent selectivity towards one base mismatch (1-MM) or scrambled (SCR) sequences was obtained. A simple protocol for extraction and analysis of non-amplified soybean genomic DNA without sample treatment was developed and validated. Our study provides insight into how the outstanding recognition efficiency of PNAs can be combined with the unique properties of CNTs in terms of signal response enhancement for direct detection of genomic DNA samples at the level of interest without previous amplification.

Competitive amperometric immunosensor for determination of p53 protein in urine with carbon nanotubes/gold nanoparticles screen-printed electrodes: A potential rapid and noninvasive screening tool for early diagnosis of urinary tract carcinoma.

Since p53 protein has become recognized biomarker for both diagnostic and therapeutic purposes in oncological diseases with particular relevance for bladder cancer, it is highly desirable to search for a novel sensing tool for detecting the patient's p53 level at the early stage. Here we report the first study on the development and validation of a novel disposable competitive amperometric immunosensor for determination of p53 protein at subnanomolar levels, based on p53 immobilization on gold nanoparticles/carbon nanotubes modified screen-printed carbon electrodes. The assay protocol requires the use of single anti-p53 mouse monoclonal antibody (DO-7 clone), able to recognize both wild-type and mutant p53. The developed immunosensor as well as the protocol of the electrochemical immunoassay were optimized by means of an experimental design procedure to assess the suitability of the device to be validated and applied for the determination of p53 in untreated and undiluted urine samples. It was found that the developed competitive immunodevice was able to achieve wide linear range detection of wild-type p53 from 20 pM to 10 nM with a low detection limit of 14 pM in synthetic urine samples, suggesting the sensor's capability of working in a complex sample matrix. The excellent performance results also in terms of selectivity, trueness and precision, coupled with the advantages of an easy preparation and low-cost assay in contrast to other methods which require very complex, time-consuming and costly nanostructured architectures, makes the developed competitive immunosensor an analytically robust diagnostic tool, valuable for implementation of screening and follow-up programs in patients with urologic malignancies.

Acartia tonsa eggs as a biomonitor to evaluate bioavailability/toxicity of persistent contaminants in anoxic/sulfidic conditions: The case of cadmium and nickel.

The evaluation of toxicity due to persistent pollutants in anoxic aquatic environments has met with various problems, as most test organisms can not withstand oxygen lack and exposure to free sulfide. We evaluated the suitability of the eggs of the brackish copepod Acartia tonsa for bioassays in anoxic/sulfidic conditions: when exposed to deep hypoxia and free sulfide, the eggs become quiescent and are able to resume hatching after restoring normoxic conditions. Tests with cadmium and nickel were performed in normoxic and deeply hypoxic conditions and in anoxic water containing H2S or H2S+FeSO4 on an equimolar basis. Active and quiescent eggs showed equivalent sensitivity to the metals, both suffering significant reductions in hatching success at 89µM Cd and 17µM Ni. As expected on the basis of the SEM/AVS model, Cd toxicity was almost completely suppressed in presence of sulfides. Dissolved Cd concentration drastically dropped and hatching success was generally >80%, as against values <6% observed in sulfide-free water, indicating that the applied experimental procedure can simulate metal-sulfide interaction. Ni toxicity was only slightly reduced by the presence of sulfides. High dissolved Ni concentrations were detected and mean hatching percentages were ≤32%, suggesting that Ni bioavailability/toxicity was only partially controlled by excess reactive sulfides. The results suggest that A. tonsa eggs could be a useful biomonitor to evaluate toxicity due persistent contaminants in anoxic conditions and the role of sulfides in reducing metal bioavailability/toxicity.

Electrochemical decompatibilisation leads to morphology rearrangements in host-guest polymer blend films.

Controlled phase separation in a polymer film, with subsequent morphology rearrangement on the micro-scale, provides novel perspectives in smart materials. Based on our experience on supramolecularly compatibilised polymer blends consisting of polystyrene and poly(butyl methacrylate), we demonstrate here physical segregation of the blend in the solid state by the application of an electrochemical stimulus. The thereby occurring changes in film morphology, namely the appearance of voids and grains, have been characterised by atomic force microscopy in spin coated and in Langmuir-Schaefer deposited films.

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.

Multiplex liquid chromatography-tandem mass spectrometry for the detection of wheat, oat, barley and rye prolamins towards the assessment of gluten-free product safety.

Celiac patients should feel confident in the safety of foods labelled or expected to be gluten-free. In this context, a targeted proteomic approach based on liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) technique was proposed to assess the presence of celiotoxic cereals, namely wheat, oats, barley and rye, in raw and processed food products. To this aim, unique marker peptides were properly selected in order to distinguish between the different cereal types. A revised cocktail solution based on reducing and denaturing agents was exploited for prolamin extraction from raw and processed food; in addition, defatting with hexane was carried out for sample clean-up, allowing to largely reduce problems related to matrix effect. Method validation on fortified rice flour showed good analytical performance in terms of sensitivity (limits of detection in the 2-18 mg kg(-1) range). However, poor trueness was calculated for self-made incurred bread (between 3 and 30% depending on the peptide), probably due to baking processes, which reduce gluten extractability. Thus, it is evident that in the case of processed foods further insights into sample treatment efficiency and reference materials for protein calibration are required to obtain accurate gluten determination. Finally, the developed method was applied for the analysis of market food products, offering the possibility to discriminate among cereals, with good agreement with labelled ingredients for gluten-containing foodstuffs.

Reliability of the TTC approach: learning from inclusion of pesticide active substances in the supporting database.

Data on pesticide active substances were used to assess the reliability of the Threshold of Toxicological Concern (TTC) approach. Pesticides were chosen as a robust test because of their potential for toxicity. 328 pesticide substances were classified on the basis of their chemical structure, according to the generic scheme proposed by the European Food Safety Authority. 43 carbamates and organophosphates were allocated to the group for neurotoxicity alerts, and 279 substances to Cramer structural Class III. For Class III, the 5th percentile value as calculated from the cumulative distribution curve of the no-observed-effect levels (0.20 mg/kg bw per day), was slightly higher than that determined by Munro (0.15 mg/kg bw per day) from his original database. The difference is explained by the inclusion of carbamates and organophosphates in Munro's Class III. Consideration of the acceptable daily intakes and their underlying toxicity data showed that the TTC approach is conservative for 96.2% of the substances. Overall, this analysis gives added support to the utility of the generic scheme of application of the TTC approach for hazard assessment of substances for which few or no experimental toxicity data are available. A convenient alternative to the Cramer decision tree is proposed.