Development of a flow system for decentralized electrochemical analysis of heavy metals using screen-printed electrodes: the importance of sensor stability.

Year after year, the need for decentralized tools to tackle the monitoring of heavy metal levels in the environment gradually increases. In this context, suitable electrochemical methodologies are widely established and particularly attractive for the production of low-cost miniaturized field-deployable analytical platforms. This work focused on the development of an automatable portable system based on square-wave anodic stripping voltammetry (SWASV) for the on-line detection of heavy metals. The surface of the sensors is appropriately modified and coupled with a fluidic system equipped with an ad-hoc designed flow cell. A custom software tool was introduced to handle the remote-controlled potentiostat and automate the various steps of the procedure, including stirring operations, cleaning phases, SWASV measurements, and data collection. After studying technical and analytical challenges, the final system developed was applied to the simultaneous detection of Cd(II), Pb(II), and Cu(II) in solution, achieving sub-ppb detection limits. Additionally, the practical applicability of the method was successfully applied to river water samples collected from the Loire basin in France.

Bioactive Compounds from Agrifood Byproducts: Their Use in Medicine and Biology.

Agrifood produces a high amount of waste, millions of tons per year worldwide, the disposal of which is a significant environmental, organizational, logistical, economic and ethic problem and in the last decades the scientific interest about this argument has increased significantly [...].

A microfluidic card-based electrochemical assay for the detection of sulfonamide resistance genes.

Most electroanalytical detection schemes for DNA markers require considerable time and effort from expert personnel to thoroughly follow the analysis and obtain reliable outcomes. This work aims to present an electrochemical assay performed inside a small card-based platform powered by microfluidic manipulation, requiring minimal human intervention and consumables. The assay couples a sample/signal dual amplification and DNA-modified magnetic particles for the detection of DNA amplification products. Particularly, the sul1 and sul4 genes involved in the resistance against sulfonamide antibiotics were analyzed. As recognized by the World Health Organization, antimicrobial resistance threatens global public health by hampering medication efficacy against infections. Consequently, analytical methods for the determination of such genes in environmental and clinical matrices are imperative. Herein, the resistance genes were extracted from E. coli cells and amplified using an enzyme-assisted isothermal amplification at 37 °C. The amplification products were analyzed in an easily-produced, low-cost, card-based set-up implementing a microfluidic system, demanding limited manual work and small sample volumes. The target amplicon was thus captured and isolated using versatile DNA-modified magnetic beads injected into the microchannel and exposed to the various reagents in a continuously controlled microfluidic flow. After the optimization of the efficiency of each phase of the assay, the platform achieved limits of detections of 44.2 pmol L-1 for sul1 and 48.5 pmol L-1 for sul4, and was able to detect down to ≥500-fold diluted amplification products of sul1 extracted from E. coli living cells in around 1 h, thus enabling numerous end-point analyses with a single amplification reaction.

Progesterone and ß-hCG Determination Using an Electrochemical Combo-Strip for Pregnancy Monitoring.

The development of analytical devices that can allow an easy, rapid and cost-effective measurement of multiple markers, such as progesterone and ß-hCG, could have a role in decreasing the burden associated with pregnancy-related complications, such as ectopic pregnancies. Indeed, ectopic pregnancies are a significant contributor to maternal morbidity and mortality in both high-income and low-income countries. In this work, an effective and highly performing electrochemical strip for a combo determination of progesterone and ß-hCG was developed. Two immunosensing approaches were optimized for the determination of these two hormones on the same strip. The immunosensors were realized using cost-effective disposable electrode arrays and reagent-saving procedures. Each working electrode of the array was modified with both the IgG anti-ß-hCG and anti-progesterone, respectively. By adding the specific reagents, progesterone or ß-hCG can then be determined. Fast quantitative detection was achieved, with the analysis duration being around 1 h. Sensitivity and selectivity were assessed with a limit of detection of 1.5 × 10-2 ng/mL and 2.45 IU/L for progesterone and ß-hCG, respectively. The proposed electrochemical combo-strip offers great promise for rapid, simple, cost-effective, and on-site analysis of these hormones and, thus, for the development of a point-of-care diagnostic tool for early detection of pregnancy-related complications.

Characterization of a Ruthenium(II) Complex in Singlet Oxygen-Mediated Photoelectrochemical Sensing.

A water-soluble ruthenium(II) complex (L), capable of producing singlet oxygen (1O2) when irradiated with visible light, was used to modify the surface of an indium-tin oxide (ITO) electrode decorated with a nanostructured layer of TiO2 (TiO2/ITO). Singlet oxygen triggers the appearance of a cathodic photocurrent when the electrode is illuminated and biased at a proper reduction potential value. The L/TiO2/ITO electrode was first characterized with cyclic voltammetry, impedance spectroscopy, NMR, and Raman spectroscopy. The rate constant of singlet oxygen production was evaluated by spectrophotometric measurements. Taking advantage of the oxidative process initiated by 1O2, the analysis of phenolic compounds was accomplished. Particularly, the 1O2-driven oxidation of hydroquinone (HQ) produced quinone moieties, which could be reduced back at the electrode surface, biased at -0.3 V vs Ag/AgCl. Such a light-actuated redox cycle produced a photocurrent dependent on the concentration of HQ in solution, exhibiting a limit of detection (LOD) of 0.3 µmol dm-3. The L/TiO2/ITO platform was also evaluated for the analysis of p-aminophenol, a commonly used reagent in affinity sensing based on alkaline phosphatase.

Electrochemical sensors based on sewage sludge-derived biochar for the analysis of anthocyanins in berry fruits.

The reutilization of waste and the reduction of the general environmental impact of every production are fundamental goals that must be achieved in the framework of a circular economy. Recycled carbon-rich materials may represent a promising alternative to other less-sustainable carbonaceous materials used in the production of electrochemical sensing platforms. Herein, we propose an innovative carbon paste electrode (CPE) composed of biochar derived from biological sludge obtained from municipal and industrial wastewater treatment plants. The physicochemical properties of the biochar after a chemical treatment with an acidic solution obtained from industrial by-products were investigated. The electrode surface characterization was carried out by analyzing common redox probes and multiple phenols bearing varying numbers of -OH and -OCH3 groups in their structure. Furthermore, the CPE was also tested on the evaluation of the phenolic fingerprints of Vaccinium myrtillus, Vaccinium uliginosum subsp. gaultherioides, and Fragaria × ananassa. Standard anthocyanin mixtures and extracts of the aforementioned fruits were analyzed to provide a phenolic characterization of real samples. The obtained results show that the sewage sludge-derived biochar can be a promising material for the development of electroanalytical sensors.

Paper-based genetic assays with bioconjugated gold nanorods and an automated readout pipeline.

Paper-based biosensors featuring immunoconjugated gold nanoparticles have gained extraordinary momentum in recent times as the platform of choice in key cases of field applications, including the so-called rapid antigen tests for SARS-CoV-2. Here, we propose a revision of this format, one that may leverage on the most recent advances in materials science and data processing. In particular, we target an amplifiable DNA rather than a protein analyte, and we replace gold nanospheres with anisotropic nanorods, which are intrinsically brighter by a factor of ~ 10, and multiplexable. By comparison with a gold-standard method for dot-blot readout with digoxigenin, we show that gold nanorods entail much faster and easier processing, at the cost of a higher limit of detection (from below 1 to 10 ppm in the case of plasmid DNA containing a target transgene, in our current setup). In addition, we test a complete workflow to acquire and process photographs of dot-blot membranes with custom-made hardware and regression tools, as a strategy to gain more analytical sensitivity and potential for quantification. A leave-one-out approach for training and validation with as few as 36 sample instances already improves the limit of detection reached by the naked eye by a factor around 2. Taken together, we conjecture that the synergistic combination of new materials and innovative tools for data processing may bring the analytical sensitivity of paper-based biosensors to approach the level of lab-grade molecular tests.

Innovative Eco-Friendly Conductive Ink Based on Carbonized Lignin for the Production of Flexible and Stretchable Bio-Sensors.

In this study, we report a novel way to produce carbon-based conductive inks for electronic and sensor technology applications. Carbonized lignin, obtained from the waste products of the Eucalyptus globulus tree paper industry, was used to produce a stable conductive ink. To this end, liquid-phase compositions were tested with different amounts of carbonized lignin powder to obtain an ink with optimal conductivity and rheological properties for different possible uses. The combination that showed the best properties, both regarding electrochemical properties and green compatibility of the materials employed, was cyclohexanone/cellulose acetate/carbonized lignin 5% (w/w), which was used to produce screen-printed electrodes. The electrodes were characterized from a structural and electrochemical point of view, resulting in an electrochemically active area of 0.1813 cm2, compared to the electrochemically active area of 0.1420 cm2 obtained by employing geometrically similar petroleum-based screen-printed electrodes and, finally, their performance was demonstrated for the quantification of uric acid, with a limit of detection of 0.3 µM, and their biocompatibility was assessed by testing it with the laccase enzyme and achieving a limit of detection of 2.01 µM for catechol as the substrate. The results suggest that the developed ink could be of great use in both sensor and electronic industries, reducing the overall ecological impact of traditionally used petroleum-based inks.

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.

Sustainable Printed Electrochemical Platforms for Greener Analytics.

The development of miniaturized electrochemical platforms holds considerable importance for the in situ analytical monitoring of clinical, environmental, food, and forensic samples. However, it is crucial to pay attention to the sustainability of materials chosen to fabricate these devices, in order to decrease the amount and the impact of waste coming from their production and use. In the framework of a circular economy and an environmental footprint reduction, the electrochemical sensor production technology must discover the potentiality of innovative approaches based on techniques and materials that can satisfy the needs of environmental-friendly and greener analytics. The aim of this review is to describe some of the printing technologies most used for sensor production, including screen-printing, inkjet-printing, and 3D-printing, and the low-impact materials that are recently proposed for these techniques, such as polylactic acid, cellulose, silk proteins, biochar.

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.

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.

PBDEs in Italian sewage sludge and environmental risk of using sewage sludge for land application.

Polybrominated diphenyl ethers (PBDEs) were determined in sewage sludge samples collected from eight Italian wastewater treatment plants (WWTPs) between June 2009 and March 2010. Total PBDE concentrations ranged from 158.3 to 9427 ng g(-1) dw, while deca-BDE (BDE-209) (concentrations ranging from 130.6 to 9411 ng g(-1) dw) dominated the congener profile in all the samples, contributing between 77% and 99.8% of total PBDE. The suitability of using a magnetic particle enzyme-linked immunoassay (ELISA) to analyse PBDEs in sewage sludge was also tested. The ELISA results, expressed as BDE-47 equivalents, were well correlated with those obtained by GC-NCI-MS, with correlation coefficients (r(2)) of 0.899 and 0.959, depending on the extraction procedure adopted. The risk assessment of PBDEs in sewage sludge addressed to land application was calculated. PEC(soil) values compared to the relative PNEC(soil) for penta and deca-BDE suggests that there is a low risk to the soil environment.

A new gravity-driven microfluidic-based electrochemical assay coupled to magnetic beads for nucleic acid detection.

In this work, the characterisation and the optimisation of hybridisation assays based on a novel, rapid and sensitive micro-analytical, gravity-driven, flow device is reported. This device combines a special chip containing eight polymer microchannels, with a portable, computer-controlled instrument. The device is used as a platform for affinity experiments using oligonucleotide-modified paramagnetic particles. In our approach, both hybridisation and labelling events are performed on streptavidin-coated paramagnetic microparticles functionalized with a biotinylated capture probe. Modified particles, introduced in the microchannel inlet of the chip, accumulate near the electrode surface by virtue of a magnetic holder. After hybridisation with the complementary sequence, the hybrid is labelled with an alkaline phosphatase conjugate. The electrochemical substrate for alkaline phosphatase revelation is p-aminophenyl phosphate. Solutions and reagents are sequentially passed through the microchannels, until enzyme substrate is added for in situ signal detection. Upon readout, the magnet array is flipped away, beads are removed by addition of regeneration buffer, and the so-regenerated chip is ready for further analysis. This protocol has been applied to the analytical detection of specific DNA sequences of Legionella pneumophila, with an RSD=8.5% and a detection limit of 0.33 nM.

Enzyme-amplified electrochemical hybridization assay based on PNA, LNA and DNA probe-modified micro-magnetic beads.

In the present study, we investigated the properties of PNA and LNA capture probes in the development of an electrochemical hybridization assay. Streptavidin-coated paramagnetic micro-beads were used as a solid phase to immobilize biotinylated DNA, PNA and LNA capture probes, respectively. The target sequence was then recognized via hybridization with the capture probe. After labeling the biotinylated hybrid with a streptavidin-enzyme conjugate, the electrochemical detection of the enzymatic product was performed onto the surface of a disposable electrode. The assay was applied to the analytical detection of biotinylated DNA as well as RNA sequences. Detection limits, calculated considering the slope of the linear portion of the calibration curve in the range 0-2 nM were found to be 152, 118 and 91 pM, coupled with a reproducibility of the analysis equal to 5, 9 and 6%, calculated as RSD%, for DNA, PNA and LNA probes respectively, using the DNA target. In the case of the RNA target, the detection limits were found to be 51, 60 and 78 pM for DNA, PNA and LNA probes respectively.

Electrochemical biosensor technology: application to pesticide detection.

In recent years, electrochemical sensors and biosensors are becoming an accepted part of analytical chemistry since they satisfy the expanding need for rapid and reliable measurements. An area in which electrochemical biosensors perhaps show the greatest diversity and potential for development involves the measurement of environmentally significant parameters. The increasing number of pollutants in the environment calls for fast and cost-effective analytical requirements. In this context, biosensors appear as suitable alternative or complementary analytical tools. The aim of this chapter is to review some basic concept concerning the electrochemical biosensors and to illustrate a protocol for the detection of environmental organic pollutants on the basis of electrochemical biosensors. In particular, a method based on the inhibition of the enzyme acetylcholinesterase (AChE) for the detection of organophosphorus and carbamate pesticides will be described in detail.

Strategies for electrochemical detection in immunochemistry.

In this review, the current status of research in electrochemical immunosensors is considered. We primarily focus on label-free and enzyme-labeled immunosensors, and the analytical capabilities of these devices are discussed. Moreover, the use of magnetic beads as new materials for immunosensors coupled with electrochemical sensing is also described, together with the application of new molecules such as aptamers as specific biorecognition elements. Examples of the applicability of these devices in solving various analytical problems in clinical, environmental and food fields are reported. Finally, the prospects for the further development of immunosensor technologies are shown.

Microfluidic-based electrochemical genosensor coupled to magnetic beads for hybridization detection.

This paper describes the development of a rapid and sensitive enzyme-linked electrochemical genosensor using a novel microfluidic-based platform. In this work, hybridization was performed on streptavidin-coated paramagnetic micro-beads functionalized with a biotinylated capture probe. The complementary sequence was then recognized via sandwich hybridization with a capture probe and a biotinylated signaling probe. After labeling the biotinylated hybrid with a streptavidin-alkaline phosphatase conjugate, the beads were introduced in a disposable cartridge composed of eight parallel microchannels etched in a polyimide substrate. The modified beads were trapped with a magnet addressing each microchannel individually. The presence of microelectrodes in each channel allowed direct electrochemical detection of the enzymatic product within the microchannel. Detection was performed in parallel within the eight microchannels, giving rise to the possibility of performing a multiparameter assay. Quantitative determinations of the analyte concentrations were obtained by following the kinetics of the enzymatic reaction in each channel. The chip was regenerated after each assay by removing the magnet and thus releasing the magnetic beads. The system was applied to the analytical detection of PCR amplified samples with a RSD%=6. A detection limit of 0.2 nM was evaluated.

Electrochemical behavior of colchicine using graphite-based screen-printed electrodes.

Miniaturized electrochemical graphite based sensor for the analysis of colchicine, along with its detailed construction was described. The electrochemical behavior of colchicine, both by cyclic and differential pulse voltammetry was investigated, showing an irreversible reduction and oxidation mechanism. The effect of several different electrochemical mediators was studied in order to decrease the oxidation potential of colchicine, but none of them showed a favorable electron transfer between the alkaloid and the electrode's surface. The influences of different biomolecules (calf thymus DNA, bovine serum albumin and beta-tubulin) over colchicine's electrochemical behavior were also presented. For quantitative determinations the anodic differential pulse voltammetric method in H(3)PO(4)/HClO(4) 0.01 M (pH 2.05) was selected, evaluating one of the oxidation peaks of colchicine at +970 mV versus Ag pseudo-reference. The electrochemical scanning parameters were optimized by an experimental design using response surface modelling. The method was validated according to ICH guidelines in the concentration range 85-1200 ng mL(-1) (R(2)=0.9966, n=7) colchicine in terms of linearity, accuracy, limits of detection (LOD) (41 ng mL(-1)), limits of quantification (LOQ) and fidelity and it was successfully applied to the determination of colchicine in tablets, without the interference of the excipients. The studied method showed the same sensitivity as the more complex HPLC procedure and micellar electrokinetic chromatography with spectrophotometric detection.

Tuning the charge distribution and photoswitchable properties of cobalt-dioxolene complexes by using molecular techniques.

A series of cobalt complexes [Co(Me(n)tpa)(diox)]PF(6)sol (diox=3,5-di-tert-butyl-1,2-dioxolene; sol=ethanol, toluene; tpa=tris(2-pyridylmethyl)amine) were prepared by using tripod-like Me(n)tpa (n=0, 1, 2, 3), derived from tpa by successive introduction of methyl groups into the 6-position of the pyridine moieties, as an ancillary ligand. The steric hindrance induced by this substitution modulates the redox properties of the metal acceptor, thus determining the charge distribution of the metal-dioxolene moiety at room temperature. All of these complexes were characterised by using diffractometric studies, electronic spectroscopic analysis, and magnetic susceptibility measurements. In the solid state, the [Co(Me(n)tpa)(diox)](+) ions (n=0, 1) can be described as diamagnetic cobalt(III)-catecholato derivatives, whereas a cobalt(II)-semiquinonato description seems appropriate for the paramagnetic [Co(Me(3)tpa)(diox)](+) complex. The complex [Co(Me(2)tpa)(diox)]PF(6)C(2)H(5)OH undergoes entropy-driven valence tautomeric interconversion at room temperature. Optically induced valence tautomerism was observed by irradiation of [Co(Me(n)tpa)(diox)]PF(6) complexes (n=0, 1, 2) at cryogenic temperatures. The different relaxation kinetics of the photoinduced metastable phases are related to the respective free-energy changes of the interconversion, as estimated by cyclic voltammetric experiments at room temperature, and to the different lattice interactions, as supported by structural data. These results show the importance of molecular techniques for controlling the relaxation properties of photoinduced metastable species. At the same time, this behaviour strongly suggests that this paradigm exhibits intrinsic limits because of the less controllable factors that affect the process.