A simple and selective electrochemical magneto-assay for sea lice eDNA detection developed with a Quality by Design approach.

Environmental DNA (eDNA) is a novel, non-invasive sampling procedure that allows the obtaining of genetic material directly from environmental samples without any evidence of biological sources. The eDNA methodology can greatly benefit from coupling it to reliable, portable and cost-effective tools able to perform decentralized measurements directly at the site of need and in resource-limited settings. Herein, we report a simple method for the selective analysis of eDNA using a magneto-assay with electrochemical detection. The proposed method involves the polymerase chain-reaction (PCR) amplification of mitochondrial eDNA of parasitic Salmon lice (Lepeophtheirus salmonis), extracted from seawater samples. The eDNA sequence was targeted via sandwich hybridization onto magnetic beads and enzymatic labeling was performed to obtain an electroactive product measured by differential pulse voltammetry. Quality by Design (QbD), a recent concept of science- and risk-oriented quality paradigm, was used for the optimization of the different parameters of the assay. Response surface methodology and Monte Carlo simulations were performed to define the method operable design region. The optimized electrochemical magneto-assay attained a limit of detection of 2.9 amol µL-1 of the short synthetic sea louse DNA analogue (43 bp). In addition, robustness testing using a further experimental design approach was performed for monitoring eDNA amplicons. Seawater samples spiked with individuals of free-swimming L. salmonis copepodite stages and seawater collected from tanks with sea lice-infested fish were analyzed.

Gold nanoparticles modified graphene platforms for highly sensitive electrochemical detection of vitamin C in infant food and formulae.

An easy and reliable method based on a novel electroanalytical nanostructured sensor has been developed to perform quantification of vitamin C in commercial and fortified cow-milk-based formulae and foods for infants and young children. The work is motivated by the need of a reliable analytical tool to be applied in quality control laboratories for the quantitative assessment of vitamin C where its rapid and cost-effective monitoring is essential. The ad hoc designed sensor, based on disposable screen-printed carbon electrodes modified with Au nanoparticles decorated reduced graphene oxide flakes, exhibits a LOD of 0.088 mg L-1. The low cost, easy sample preparation, fast response and high reproducibility (RSD ≈ 8%) of the proposed method highlight its suitability for usage in quality control laboratories for determining vitamin C in real complex food matrices, envisaging the application of the sensing platform in the determination of other compounds relevant in food chemistry and food manufacturing.

Nanotoxicity assessment: A challenging application for cutting edge electroanalytical tools.

In the recent years, the number of commercial products containing engineered nanomaterials (ENMs) has increased exponentially. Consequently, the toxicological profile of ENMs on the ecosystems as well as on human health has to be carefully evaluated. Nanotoxicology, an interdisciplinary research area devoted to assessing the hazards associated with ENMs, is expanding rapidly. Many physicochemical techniques and biochemical methodologies have been proposed and are currently used to characterize nanomaterials from a toxicological point of view. Electroanalytical and bioelectrochemical methods can be useful in expanding the repertoire of accessible nanotoxicity-assessment technologies and to accelerate the testing and screening of the toxicological effects of ENMs. These methods can be used for elucidating the toxicological behavior of ENMs at single cell, cell population and whole-organism levels, for in vitro and in vivo measurements, respectively. The aim of this review is to provide an overview on the bioelectrochemical approaches that have been proposed for ENMs toxicity assessment. Furthermore, an overview on cutting-edge electroanalytical devices with a potential impact to this peculiar application is provided.

Trends and Perspectives in Immunosensors for Determination of Currently-Used Pesticides: The Case of Glyphosate, Organophosphates, and Neonicotinoids.

Pesticides, due to their intensive use and their peculiar chemical features, can persist in the environment and enter the trophic chain, thus representing an environmental risk for the ecosystems and human health. Although there are several robust and reliable standard analytical techniques for their monitoring, the high frequency of contamination caused by pesticides requires methods for massive monitoring campaigns that are capable of rapidly detecting these compounds in many samples of different origin. Immunosensors represent a potential tool for simple, rapid, and sensitive monitoring of pesticides. Antibodies coupled to electrochemical or optical transducers have resulted in effective detection devices. In this review, the new trends in immunosensor development and the application of immunosensors for the detection of pesticides of environmental concern-such as glyphosate, organophosphates, and neonicotinoids-are described.

Polydopamine: surface coating, molecular imprinting, and electrochemistry-successful applications and future perspectives in (bio)analysis.

Dopamine oxidation and self-polymerization have recently attracted great interest arising from the versatile chemistry of this endogenous catecholamine. Particularly interesting are the applications of polydopamine for surface coating, molecular imprinting, and electrochemistry, which are reviewed here, covering the broad fields of medicine, materials science, and (bio)analytical chemistry. Nonetheless, the peculiar physicochemical properties of dopamine and polydopamine, due to the redox potential of the catechol moiety, are not fully exploited. We have confidence in increasing the applications of dopamine through a large variety of research approaches, including the use of naturally occurring or synthetic dopamine analogues and copolymers. Accordingly, our efforts in this direction are focused on proposing a role for polydopamine in quantitative applications, evaluating analytical performance, cost, reproducibility, and versatility of the methods developed, and also revisiting standard (bio)analytical platforms.

Au nanoparticle in situ decorated RGO nanocomposites for highly sensitive electrochemical genosensors.

A novel hybrid nanocomposite formed by RGO flakes, surface functionalized by 1-pyrene carboxylic acid (PCA), densely and uniformly in situ decorated by Au NPs, that are concomitantly coordinated by the PCA carboxylic group, and by an aromatic thiol used as the reducing agent in the synthesis, both ensuring, at the same time, a stable non-covalent NPs anchorage to the RGO flakes, and an efficient interparticle electron coupling along the NP network onto the RGO, is reported. The obtained solution processable hybrid material is used to modify Screen-Printed Carbon Electrodes (SPCEs). The hybrid modified SPCEs, functionalized with a thiolated DNA capture probe, are tested in a streptavidin-alkaline-phosphatase catalyzed assay, for the detection of the biotinylated miRNA-221, and for its determination in spiked human blood serum samples. The proposed genosensor demonstrates a high sensitivity (LOD of 0.7 pM), attesting for a performance comparable with the most effective reported sensors. The enhanced sensitivity is explained in terms of the very fast heterogeneous electron transfer kinetics, the concomitant decrease of the electron transfer resistance at the electrode/electrolyte interface, the high electroactivity and the high surface area of the nanostructured hybrid modified SPCEs that provide a convenient platform for nucleic acid biosensing.

Innovative Biocatalysts as Tools to Detect and Inactivate Nerve Agents.

Pesticides and warfare nerve agents are frequently organophosphates (OPs) or related compounds. Their acute toxicity highlighted more than ever the need to explore applicable strategies for the sensing, decontamination and/or detoxification of these compounds. Herein, we report the use of two different thermostable enzyme families capable to detect and inactivate OPs. In particular, mutants of carboxylesterase-2 from Alicyclobacillus acidocaldarius and of phosphotriesterase-like lactonases from Sulfolobus solfataricus and Sulfolobus acidocaldarius, have been selected and assembled in an optimized format for the development of an electrochemical biosensor and a decontamination formulation, respectively. The features of the developed tools have been tested in an ad-hoc fabricated chamber, to mimic an alarming situation of exposure to a nerve agent. Choosing ethyl-paraoxon as nerve agent simulant, a limit of detection (LOD) of 0.4 nM, after 5 s of exposure time was obtained. Furthermore, an optimized enzymatic formulation was used for a fast and efficient environmental detoxification (>99%) of the nebulized nerve agent simulants in the air and on surfaces. Crucial, large-scale experiments have been possible thanks to production of grams amounts of pure (>90%) enzymes.

Glyphosate Determination by Coupling an Immuno-Magnetic Assay with Electrochemical Sensors.

Glyphosate (N-(phosphonomethyl)glycine) is the most frequently used broad-spectrum herbicide worldwide. Its mechanism of action is based on the inhibition of an enzyme that is essential to plant growth. Its intensive use has caused global contamination to occur, which has not only affected the ecosystems, but even food and other objects of common use. Thus, there is a pronounced need for developing analytical methods for glyphosate determination in different matrices. Here, an electrochemical competitive immunoassay, based on the use of antibody-modified magnetic particles, has been developed. Tetramethylbenzidine (TMB) has been used as an enzymatic substrate. The extent of the affinity reaction has been achieved by monitoring the current value, due to the reduction of the enzymatic product. A disposable screen-printed electrochemical cell has been used. The calibration curve has been recorded in the 0⁻10,000 ng/L concentration range, with a detection limit of 5 ng/L and quantification limit of 30 ng/L. The electrochemical immunoassay has also been applied to the analysis of spiked beer samples.

Evaluation of a QuEChERS-like extraction approach for the determination of PBDEs in mussels by immuno-assay-based screening methods.

A sample preparation method was evaluated for the determination of polybrominated diphenyl ethers (PBDEs) in mussel samples, by using colorimetric and electrochemical immunoassay-based screening methods. Herein, a rapid procedure based on QuEChERS-like extraction approach followed by solid phase purification was optimized for PBDE extraction from mussel samples. The detection limits for colorimetric and electrochemical immunoassays, calculated as BDE-47 equivalent concentration, were 0.6ngg-1 and 1.1ngg-1, respectively. Real mussel samples, including a Certified Reference Material (CRM), were analyzed. The samples were measured by colorimetric and electrochemical immunoassays as well as by GC-MS. In comparison to GC-MS results, 106% and 102% relative accuracy were obtained for the colorimetric and electrochemical immunoassays, respectively. The proposed method could be useful for massive environmental campaigns, being able to rapidly detect possible polluted seafood samples.

Direct determination of small RNAs using a biotinylated polythiophene impedimetric genosensor.

Herein, direct determination of small RNAs is described using a functional-polymer modified genosensor. The analytical strategy adopted involves deposition by electropolymerization of biotinylated polythiophene films on the surface of miniaturized, disposable, gold screen-printed electrodes, followed by the layer-by-layer deposition of streptavidin, and then biotynilated capture probes. A small RNA (miR-221) target was determined via the impedimetric measurement of the hybridization event in a label-free and PCR-free approach. Under optimized conditions, the limit of detection (LOD) was 0.7 pM miR-221 (15% RSD). The genosensor was applied for determination of miR-221 in total RNA extracted from human lung and breast cancer cell lines, discriminating between the cancer-positive and -negative cells, without any amplification step, in less than 2h.

Imidazo[1,2-a]pyrazin-8-amine core for the design of new adenosine receptor antagonists: Structural exploration to target the A3 and A2A subtypes.

The imidazo[1,2-a]pyrazine ring system has been chosen as a new decorable core skeleton for the design of novel adenosine receptor (AR) antagonists targeting either the human (h) A3 or the hA2A receptor subtype. The N8-(hetero)arylcarboxyamido substituted compounds 4-14 and 21-30, bearing a 6-phenyl moiety or not, respectively, show good hA3 receptor affinity and selectivity versus the other ARs. In contrast, the 8-amino-6-(hetero)aryl substituted derivatives designed for targeting the hA2A receptor subtype (compounds 31-38) and also the 6-phenyl analogues 18-20 do not bind the hA2A AR, or show hA1 or balanced hA1/hA2A AR affinity in the micromolar range. Molecular docking of the new hA3 antagonists was carried out to depict their hypothetical binding mode to our refined model of the hA3 receptor. Some derivatives were evaluated for their fluorescent potentiality and showed some fluorescent emission properties. One of the most active hA3 antagonists herein reported, i.e. the 2,6-diphenyl-8-(3-pyridoylamino)imidazo[1,2-a]pyrazine 29, tested in a rat model of cerebral ischemia, delayed the occurrence of anoxic depolarization caused by oxygen and glucose deprivation in the hippocampus and allowed disrupted synaptic activity to recover.

Improving impedimetric nucleic acid detection by using enzyme-decorated liposomes and nanostructured screen-printed electrodes.

Sensitive impedimetric detection of miR-222, a miRNA sequence found in many lung tumors, was investigated by using gold-nanostructured disposable carbon electrodes and enzyme-decorated liposomes. The proposed method was based on the immobilization of thiolated DNA capture probes onto gold-nanostructured carbon surfaces. Afterwards, the capture probes were allowed to hybridize to the target miRNAs. Finally, enzyme-decorated liposomes were used as labels to amplify the miRNA sensing, by their association with the probe-miRNA hybrids generated on the nanostructured transducer. By using this amplification route a limit of detection of 0.400 pM, a limit of quantification of 1.70 pM, and an assay range spanning three orders of magnitude (1.70-900 pM) were obtained (RSD % = 13). This limit of quantification was 20 times lower than that obtained using a simple enzyme conjugate for the detection. A comparison was also made with gold screen-printed transducers. In this case, a limit of quantification approximately 70 times lower was found by using the nanostructured transducers. Application of the optimized assay in serum samples was also demonstrated. Graphical abstract Alkaline Phosphatase-decorated liposomes and Au nanostructured screen-printed electrodes have been used for the impedimetric detection of miRNAs, via the bio-catalyzed precipitation of an insulating product onto the electrode surface.

Strategies for the development of an electrochemical bioassay for TNF-alpha detection by using a non-immunoglobulin bioreceptor.

TNF-α is an inflammatory cytokine produced by the immune system. Serum TNF-α level is elevated in some pathological states such as septic shock, graft rejection, HIV infection, neurodegenerative diseases, rheumatoid arthritis and cancer. Detecting trace amount of TNF-α is, also, very important for the understanding of tumor biological processes. Detection of this key biomarker is commonly achieved by use of ELISA or cytofluorimetric based methods. In this study the traditional optical detection was replaced by differential pulse voltammetry (DPV) and an affinity molecule, produced by evolutionary approaches, has been tested as capture bioreceptor. This molecule, namely a combinatorial non-immunoglobulin protein (Affibody®) interacts with TNF-α selectively and was here tested in a sandwich assay format. Moreover magnetic beads were used as support for bioreceptor immobilization and screen printed carbon electrodes were used as transducers. TNF-α calibration curve was performed, obtaining the detection limit of 38pg/mL, the quantification range of 76-5000pg/mL and RSD%=7. Preliminary results of serum samples analysis were also reported.

Health and carcinogenic risk evaluation for cohorts exposed to PAHs in petrochemical workplaces in Rawalpindi city (Pakistan).

This study presents the analyses of urinary biomarkers (1-OHPyr, α- and ß-naphthols) of polycyclic aromatic hydrocarbons (PAHs) exposure and biomarkers of effect (i.e. blood parameters) in petroleum-refinery workers (RFs) and auto-repair workers (MCs). Exposed subjects had higher concentrations of white blood cell (WBC) count than control subjects (CN) subjects (5.31 × 10(3) µL(-1) in exposed vs. 5.15 × 10(3) µL(-1) in CN subjects), while the biomarker of oxidative DNA damage (8-OHdG) was significantly higher in MCs. The exposure among these two cohorts could be influenced by the ambience of the workplaces; in fact, MCs' shops are relatively damp and enclosed workplaces in comparison with the indoor environment of refineries. PAHs in the dust samples from mechanical workshops probably originated from mixed sources (traffic exhaust and petroleum spills), while the incremental lifetime cancer risk (ILCR) for MCs showed moderate-to-low cancer risk from exposure to dust-bound PAHs. The study shows that increasing PAH exposure can be traced in MC workstations and needs to be investigated for the safety of public health.

Electrochemical detection of miRNA-222 by use of a magnetic bead-based bioassay.

MicroRNAs (miRNAs, miRs) are naturally occurring small RNAs (approximately 22 nucleotides in length) that have critical functions in a variety of biological processes, including tumorigenesis. They are an important target for detection technology for future medical diagnostics. In this paper we report an electrochemical method for miRNA detection based on paramagnetic beads and enzyme amplification. In particular, miR 222 was chosen as model sequence, because of its involvement in brain, lung, and liver cancers. The proposed bioassay is based on biotinylated DNA capture probes immobilized on streptavidin-coated paramagnetic beads. Total RNA was extracted from the cell sample, enriched for small RNA, biotinylated, and then hybridized with the capture probe on the beads. The beads were then incubated with streptavidin-alkaline phosphatase and exposed to the appropriate enzymatic substrate. The product of the enzymatic reaction was electrochemically monitored. The assay was finally tested with a compact microfluidic device which enables multiplexed analysis of eight different samples with a detection limit of 7 pmol L(-1) and RSD = 15 %. RNA samples from non-small-cell lung cancer and glioblastoma cell lines were also analyzed.

Electrochemical bioassay for the detection of TNF-α using magnetic beads and disposable screen-printed array of electrodes.

BACKGROUND: In this study we have developed an electrochemical bioassay for the analysis of TNF-α, coupling magnetic beads with disposable electrochemical platforms. TNF-α is a pro inflammatory cytokine that participates in the regulation of immune defense against various pathogens and the recovery from injury. It plays a central role in the development of many inflammatory diseases. The bioassay was based on a sandwich format using alkaline phosphatase as an enzymatic label and an eight-sensor screen-printed array as an electrochemical transducer. RESULTS: The modified magnetic beads were captured by a magnet on the surface of each graphite working electrode of the array and the electrochemical detection was thus achieved through the addition of the alkaline phosphatase substrate (1-naphthylphosphate); 1-naphthol produced during the enzymatic reaction was detected using differential pulse voltammetry. The parameters influencing the different steps of the assay were optimized in order to reach the best sensitivity and specificity. CONCLUSION: The proposed strategy offers great promise for analysis of clinical diagnostics, considering also that arrays allow the simultaneous analysis of different samples.

Disposable electrochemical DNA-array for PCR amplified detection of hazelnut allergens in foodstuffs.

An electrochemical low-density DNA-array has been designed and implemented to be used in combination with polymerase chain reaction (PCR) in order to investigate the presence of hazelnut major allergens (Cor a 1.04, Cor a 1.03) in foodstuff. Unmodified PCR products were captured at the sensor interface via sandwich hybridization with surface-tethered probes and biotinylated signalling probes. The resulting biotinylated hybrids were coupled with a streptavidin-alkaline phosphatase conjugate and then exposed to a alpha-naphthyl phosphate solution. Differential pulse voltammetry was finally used to detect the alpha-naphthol signal. The detection limits for Cor a 1.03 and Cor a 1.04 were 0.3 and 0.1 nmol L(-1), respectively (R.S.D. 10%). The optimized conditions were used to test several commercially available foodstuffs, claiming to contain or not the targeted nuts. The results were compared with those obtained with classical ELISA tests.

One-shot screen-printed thylakoid membrane-based biosensor for the detection of photosynthetic inhibitors in discrete samples.

Screen-printing technology offers the possibility to produce a large number of sensors at low cost. Thus, due to their intrinsic characteristics and reproducibility, screen-printed electrodes can be used in the development of disposable electrochemical devices. In the present work, carbon-based screen-printed electrodes (SPCEs) have been used to develop a one-shot-measure biosensor for the detection of photosynthetic inhibitors in discrete samples. The measurement was based on the electrochemical evaluation of the activity of photosystem II (PSII), a protein complex present in photosynthetic organisms and involved in the photosynthesis. The biosensor was prepared by the modification of the working electrode of a SPCE, using thylakoid membranes extracted from spinach leaves. The modified sensors were then used as one-shot system to measure the presence of PSII activity inhibitors in discrete standard solutions. The coupling of the developed biosensor with a custom-made cell made it possible to perform tests using only 50 microL of total sample volume with a measurement time of 10 min. Inhibition curves were recorded for some photosynthetic inhibitors in a concentration range of 10(-6) to 10(-8) molL(-1). A reproducibility (relative standard deviation, R.S.D.%) of 10% was found and the calculated limit of detections (LODs) were in the nanomolar range. The effect of storage on sensitivity and reproducibility of a biosensor prepared by direct lyophilisation of thylakoid membranes on the electrode surface was also evaluated, confirming the possible use of the modified sensor up to one week after the preparation. Measurements on real samples were also reported, comparing the results with those obtained using a fluorescence-based commercial instrument for the analysis of photosynthetic inhibitors.