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.

Ruthenium(II) Polypyridyl Complexes with Benzoxazole Derivatives and Non-Innocent Ligands as Effective Antioxidants in Human Neuroblasts.

Ruthenium(II) polypyridyl complexes continue to raise increasing interest for the encouraging results in several biomedical areas. Considering their vast chemical-physical repertoire, in particular the possibility to switch from the sensitization of reactive oxygen species (ROS) to ROS-scavenging abilities by tuning the nature of their ligands, it is therefore surprising that their potential as antioxidants has not been largely investigated so far. Herein, we explored the antioxidant behaviour of the novel ruthenium compound [Ru(dbpy)(2,3-DAN)Cl]PF6 (Ru1), featuring a benzoxazole derivative (dpby=2,6-bis(4-methyl-2-benzoxazolyl)pyridine) and the non-innocent 2,3-diamminonaftalene (2,3-DAN) ligand, along with the reference tpy-containing analogue [Ru(tpy)(2,3-DAN)Cl]PF6 (Ru2) (tpy=2,2':6',2''-terpyridine). Following the synthesis and the electrochemical characterization, chemical antioxidant assays highlighted the beneficial role of dpby for the ROS-scavenging properties of Ru1. These data have been corroborated by the highest protective effect of Ru1 against the oxidative stress induced in SH-SY5Y human neuroblastoma, which exerts pro-survival and anti-inflammatory actions. The results herein reported highlight the potential of Ru1 as pharmacological tool in neurodegenerative diseases and specially prove that the antioxidant properties of such compounds are likely the result of a non-trivial synergetic action involving the bioactive ligands in their chemical architectures.

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.

Practical tips and new trends in electrochemical biosensing of cancer-related extracellular vesicles.

To tackle cancer and provide prompt diagnoses and prognoses, the constantly evolving biosensing field is continuously on the lookout for novel markers that can be non-invasively analysed. Extracellular vesicles (EVs) may represent a promising biomarker that also works as a source of biomarkers. The augmented cellular activity of cancerous cells leads to the production of higher numbers of EVs, which can give direct information on the disease due to the presence of general and cancer-specific surface-tethered molecules. Moreover, the intravesicular space is enriched with other molecules that can considerably help in the early detection of neoplasia. Even though EV-targeted research has indubitably received broad attention lately, there still is a wide lack of practical and effective quantitative procedures due to difficulties in pre-analytical and analytical phases. This review aims at providing an exhaustive outline of the recent progress in EV detection using electrochemical and photoelectrochemical biosensors, with a focus on handling approaches and trends in the selection of bioreceptors and molecular targets related to EVs that might guide researchers that are approaching such an unstandardised field.

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.

Highly Charged Ru(II) Polypyridyl Complexes as Photosensitizer Agents in Photodynamic Therapy of Epithelial Ovarian Cancer Cells.

Ovarian cancer recurrence is frequent and associated with chemoresistance, leading to extremely poor prognosis. Herein, we explored the potential anti-cancer effect of a series of highly charged Ru(II)-polypyridyl complexes as photosensitizers in photodynamic therapy (PDT), which were able to efficiently sensitize the formation of singlet oxygen upon irradiation (Ru12+ and Ru22+) and to produce reactive oxygen species (ROS) in their corresponding dinuclear metal complexes with the Fenton active Cu(II) ion/s ([CuRu1]4+ and [Cu2Ru2]6+). Their cytotoxic and anti-tumor effects were evaluated on human ovarian cancer A2780 cells both in the absence or presence of photoirradiation, respectively. All the compounds tested were well tolerated under dark conditions, whereas they switched to exert anti-tumor activity following photoirradiation. The specific effect was mediated by the onset of programed cell death, but only in the case of Ru12+ and Ru22+ was preceded by the loss of mitochondrial membrane potential soon after photoactivation and ROS production, thus supporting the occurrence of apoptosis via type II photochemical reactions. Thus, Ru(II)-polypyridyl-based photosensitizers represent challenging tools to be further investigated in the identification of new therapeutic approaches to overcome the innate chemoresistance to platinum derivatives of some ovarian epithelial cancers and to find innovative drugs for recurrent ovarian cancer.

Alteration of the Nucleotide Excision Repair (NER) Pathway in Soft Tissue Sarcoma.

Clinical responses to anticancer therapies in advanced soft tissue sarcoma (STS) are unluckily restricted to a small subgroup of patients. Much of the inter-individual variability in treatment efficacy is as result of polymorphisms in genes encoding proteins involved in drug pharmacokinetics and pharmacodynamics. The nucleotide excision repair (NER) system is the main defense mechanism for repairing DNA damage caused by carcinogens and chemotherapy drugs. Single nucleotide polymorphisms (SNPs) of NER pathway key genes, altering mRNA expression or protein activity, can be significantly associated with response to chemotherapy, toxicities, tumor relapse or risk of developing cancer. In the present study, in a cohort of STS patients, we performed DNA extraction and genotyping by SNP assay, RNA extraction and quantitative real-time reverse transcription PCR (qPCR), a molecular dynamics simulation in order to characterize the NER pathway in STS. We observed a severe deregulation of the NER pathway and we describe for the first time the effect of SNP rs1047768 in the ERCC5 structure, suggesting a role in modulating single-stranded DNA (ssDNA) binding. Our results evidenced, for the first time, the correlation between a specific genotype profile of ERCC genes and proficiency of the NER pathway in STS.

An improved NMR approach for metabolomics of intact serum samples.

NMR metabolomics has inherent capabilities for studying biofluids, such as reproducibility, minimal sample preparation, non-destructiveness, and molecular structure elucidation; however, reliable quantitation of metabolites is still a challenge because of the complex matrix of the samples. The serum is one of the most common samples in clinical studies but possibly the most difficult for NMR analysis because of the high content of proteins, which hampers the detection and quantification of metabolites. Different processes for protein removal, such as ultrafiltration and precipitation, have been proposed, but require sample manipulation, increase time and cost, and possibly lead to loss of information in the metabolic profile. Alternative methods that rely on filtering protein signals by NMR pulse sequencing are commonly used, but standardisation of acquisition parameters and spectra calibration is far from being reached. The present technical note is a critical assessment of the sparsely suggested calibrants, pulse sequences and acquisition parameters toward an optimised combination of the three for accurate and reproducible quantification of metabolites in intact serum.

Bicyclic peptide-based assay for uPA cancer biomarker.

The use of synthetic bioreceptors to develop biosensing platforms has been recently gaining momentum. This case study compares the performance of a biosensing platform for the human biomarker urokinase-type plasminogen activator (h-uPA) when using two bicyclic peptides (P1 and P2) with different affinities for the target protein. The bioreceptors P1 and P2 were immobilized on magnetic microbeads and tested within a sandwich-type affinity electrochemical assay. Apart from enabling h-uPA quantification at nanomolar levels (105.8 ng/mL for P1 and 32.5 ng/mL for P2), this case study showed the potential of synthetic bicyclic peptides applicability and how bioreceptor affinity can influence the performance of the final sensing platform.

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.

Pharmacogenomics of soft tissue sarcomas: New horizons to understand efficacy and toxicity.

Clinical responses to anticancer therapies in advanced soft tissue sarcoma (STS) are unfortunately limited to a small subset of patients. Much of the inter-individual variability in treatment efficacy and risk of toxicities is as result of polymorphisms in genes encoding proteins involved in drug pharmacokinetics and pharmacodynamics. Therefore, the detection of pharmacogenomics (PGx) biomarkers that might predict drug response and toxicity can be useful to explain the genetic basis for the differences in treatment efficacy and toxicity among STS patients. PGx markers are frequently located in transporters, drug-metabolizing enzyme genes, drug targets, or HLA alleles. Along this line, genetic variability harbouring in the germline genome of the patients can influence systemic pharmacokinetics and pharmacodynamics of the treatments, acting as predictive biomarkers for drug-induced toxicity and treatment efficacy. By linking drug activity to the functional complexity of cancer genomes, also systematic pharmacogenomic profiling in cancer cell lines and primary STS samples represents area of active investigation that could eventually lead to enhanced efficacy and offer a powerful biomarker discovery platform to optimize current treatments and improve the knowledge about the individual's drug response in STS patients into the clinical practice.

Growth arrest-specific 5 lncRNA as a valuable biomarker of chemoresistance in osteosarcoma.

Osteosarcoma is the most common primary malignant bone tumour in children and teenagers, and it is characterised by drug resistance and high metastatic potential. Increasing studies have highlighted the critical roles of long noncoding RNAs (lncRNAs) as oncogenes or tumour suppressors as well as new biomarkers and therapeutic targets in osteosarcoma. The growth arrest-specific 5 (GAS5) lncRNA can function as a tumour suppressor in several cancers. The present study aimed to validate GAS5 and other chemoresistance-associated lncRNAs as biomarkers in a cohort of primary osteosarcoma samples, to obtain predictive information on resistance or sensitivity to treatment. The GAS5 and a panel of lncRNAs related to chemoresistance [SNGH1, FOXD2-AS1, deleted in lymphocytic leukemia (DLEU2) and LINC00963] were evaluated in a cohort of osteosarcoma patients enrolled at the Careggi University Hospital. Total RNA was extracted from formalin-fixed paraffin-embedded (FFPE) tissue sections and the expression levels of the lncRNAs were quantified by qPCR. A bioinformatic analysis on deposited RNA-seq data was performed to validate the qPCR results. Clustering analysis shows that GAS5 could be linked to the expression of isoforms 02 and 04 of the lncRNA DLEU2, whereas the DLEU2 isoform 08 is linked to the lncRNA LINC00963. We found that GAS5 is significantly increased in patients with a good prognosis and is expressed differently between chemosensitive and chemoresistant osteosarcoma patients. However, the results obtained are not concordant with the in-silico analysis performed on the TARGET osteosarcoma dataset. In the future, we would enlarge the case series, including different disease settings.

Nitroimidazole-Based Ruthenium(II) Complexes: Playing with Structural Parameters to Design Photostable and Light-Responsive Antibacterial Agents.

5-Nitroimidazole (5NIMH), chosen as a molecular model of nitroimidazole derivatives, which represent a broad-spectrum class of antimicrobials, was incorporated into the ruthenium complexes [Ru(tpy)(phen)(5NIM)]PF6 (1) and [Ru(tpy)(dmp)(5NIM)]PF6 (2) (tpy = terpyridine, phen = phenanthroline, dmp = 2,9-dimethyl-1,10-phenanthroline). Besides the uncommon metal coordination of 5-nitroimidazole in its imidazolate form (5NIM), the different architectures of the spectator ligands (phen and dmp) were exploited to tune the "mode of action" of the resulting complexes, passing from a photostable compound where the redox properties of 5NIMH are preserved (1) to one suitable for the nitroimidazole phototriggered release (2) and whose antibacterial activity against B. subtilis, chosen as cellular model, is effectively improved upon light exposure. This study may provide a fundamental knowledge on the use of Ru(II)-polypyridyl complexes to incorporate and/or photorelease biologically relevant nitroimidazole derivatives in the design of a novel class of antimicrobials.

The Role of Peptides in the Design of Electrochemical Biosensors for Clinical Diagnostics.

Peptides represent a promising class of biorecognition elements that can be coupled to electrochemical transducers. The benefits lie mainly in their stability and selectivity toward a target analyte. Furthermore, they can be synthesized rather easily and modified with specific functional groups, thus making them suitable for the development of novel architectures for biosensing platforms, as well as alternative labelling tools. Peptides have also been proposed as antibiofouling agents. Indeed, biofouling caused by the accumulation of biomolecules on electrode surfaces is one of the major issues and challenges to be addressed in the practical application of electrochemical biosensors. In this review, we summarise trends from the last three years in the design and development of electrochemical biosensors using synthetic peptides. The different roles of peptides in the design of electrochemical biosensors are described. The main procedures of selection and synthesis are discussed. Selected applications in clinical diagnostics are also described.

Stimulation of Ca2+ -ATPase Transport Activity by a Small-Molecule Drug.

The sarco(endo)plasmic reticulum Ca2+ -ATPase (SERCA) hydrolyzes ATP to transport Ca2+ from the cytoplasm to the sarcoplasmic reticulum (SR) lumen, thereby inducing muscle relaxation. Dysfunctional SERCA has been related to various diseases. The identification of small-molecule drugs that can activate SERCA may offer a therapeutic approach to treat pathologies connected with SERCA malfunction. Herein, we propose a method to study the mechanism of interaction between SERCA and novel SERCA activators, i. e. CDN1163, using a solid supported membrane (SSM) biosensing approach. Native SR vesicles or reconstituted proteoliposomes containing SERCA were adsorbed on the SSM and activated by ATP concentration jumps. We observed that CDN1163 reversibly interacts with SERCA and enhances ATP-dependent Ca2+ translocation. The concentration dependence of the CDN1163 effect provided an EC50 =6.0±0.3 µM. CDN1163 was shown to act directly on SERCA and to exert its stimulatory effect under physiological Ca2+ concentrations. These results suggest that CDN1163 interaction with SERCA can promote a protein conformational state that favors Ca2+ release into the SR lumen.

Soft Tissue Sarcoma: An Insight on Biomarkers at Molecular, Metabolic and Cellular Level.

Soft tissue sarcomas (STSs) are a heterogeneous group of rare tumors. Although constituting only 1% of all human malignancies, STSs represent the second most common type of solid tumors in children and adolescents and comprise an important group of secondary malignancies. Over 100 histologic subtypes have been characterized to date (occurring predominantly in the trunk, extremity, and retroperitoneum), and many more are being discovered due to molecular profiling. STS mortality remains high, despite adjuvant chemotherapy. New prognostic stratification markers are needed to help identify patients at risk of recurrence and possibly apply more intensive or novel treatments. Recent scientific advancements have enabled a more precise molecular characterization of sarcoma subtypes and revealed novel therapeutic targets and prognostic/predictive biomarkers. This review aims at providing a comprehensive overview of the most relevant cellular, molecular and metabolic biomarkers for STS, and highlight advances in STS-related biomarker research.

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.

Determination of Glyphosate in Water from a Rural Locality in México and Its Implications for the Population Based on Water Consumption and Use Habits.

Glyphosate is a broad-spectrum herbicide widely used worldwide. Indeed, it is the herbicide most applied to all Mexican crops. Due to the overuse and poor disposal of the waste, this herbicide can reach the aquatic environments such as groundwater and surface water. Thus, there is a clear need to implement monitoring and surveillance programs for evaluating and controlling the exposure to this herbicide in rural populations. The goal of this study was to quantify the presence of glyphosate in different water bodies (groundwater, surface and drinking water) as well as to identify the uses and managements of water resources by rural communities to evaluate the potential human exposure to glyphosate in the Tenampulco region of the Mexican state of Puebla. Measurements were performed by a rapid and cost-effective ELISA-based method in groundwater and surface water from various sampling sites of the Tenampulco region. Glyphosate was detected in all groundwater samples to be below the maximum limit for glyphosate in water in Mexico. Nevertheless, these results indicate an exposure of glyphosate in these agricultural communities and the need to establish a monitoring program.