Simultaneous electrochemical immunosensing of relevant cytokines to diagnose and track cancer and autoimmune diseases.

This work reports the first dual magnetic beads (MBs) assisted immunoplatform for the simultaneous determination of BAFF (B cell activation factor) and APRIL (a proliferation-induced ligand), two cytokines related to immunity and tumour invasion, growth and metastasis. The electrochemical immunoplatform involves sandwich-type immunoassays implemented on magnetic microparticles functionalized with neutravidin (NA-MBs) or carboxylic groups (HOOC-MBs), and amperometric detection (Eapp =  - 0.20 V vs. Ag pseudo-reference electrode) at screen-printed dual carbon electrodes (SPdCE) using the H2O2/hydroquinone (HQ) system. The developed immunosensors provide improved sensitivity, with LOD values of 0.33 and 16.4 pg mL-1 for BAFF and APRIL, respectively, and much shorter assay time that those claimed for ELISA kits and allow their simultaneous determination. The dual immunosensor permits discrimination between healthy individuals and patients diagnosed with Systemic Lupus Erythematosus (SLE) or colorectal cancer (CRC) through the determination of both cytokines in 100-times diluted human sera with results in agreement with those provided by the individual ELISA methodologies.

First electrochemical immunosensor for the rapid detection of mustard seeds in plant food extracts.

This paper describes the first biosensor reported to date for the determination of mustard seed traces. The biosensor consists of an amperometric immunosensing platform able to sensitively and selectively determine Sin a 1 content, the major allergen of yellow mustard and the most abundant protein of these seeds. The immunosensing platform exploits the coupling of magnetic microbeads (MBs) modified with sandwich-type immune complexes, comprising polyclonal and monoclonal antibodies, selective to the target protein for its capturing and detection, respectively. In addition, a HRP-conjugated secondary antibody was used for enzymatic labelling of the monoclonal antibody, and amperometric transduction was made at screen-printed carbon electrodes (SPCEs) using the hydroquinone (HQ)/H2O2 system. The electrochemical immunosensor allows the simple and fast detection (a single 1-h incubation step) of Sin a 1 with a limit of detection of 0.82 ng mL-1 (20.5 pg of protein in 25 µL of sample) with high selectivity against structurally similar non-target allergenic proteins (such as Pin p 1 from pine nut). The developed immunoplatform was successfully used for the analysis of peanut, rapeseed, cashew, pine nut and yellow mustard extracts, giving only positive response for the yellow mustard extract with a Sin a 1 content, in full agreement with that provided by conventional ELISA methodology.

Enlightening the advancements in electrochemical bioanalysis for the diagnosis of Alzheimer's disease and other neurodegenerative disorders.

Neurodegenerative disorders (NDD), and particularly Alzheimer's disease (AD), are one of the greatest challenges facing our current medicine and society because of its increasing incidence and the high burden imposed both on patients' families and health systems. Despite this, their accurate diagnosis, mostly conducted by cerebrospinal fluid (CSF) analysis or neuroimaging techniques, costly, time-consuming, and unaffordable for most of the population, remains a complex task. In this situation, electrochemical biosensors are flourishing as promising alternative tools for the simple, fast, and low-cost diagnosis of NDD/AD. This review article provides the relevant clinical details of NDD/AD along with the closely related genetic (genetic mutations, polymorphisms of ApoE and specific miRNAs) and proteomic (amyloid-ß peptides, total and phosphorylated tau protein) biomarkers circulating mostly in CSF. In addition, the article systematically enlightens a general view of the electrochemical affinity biosensors (mostly aptasensors and immunosensors) reported in the past two years for the determination of such biomarkers. The different developed strategies, analytical performances and applications are comprehensively discussed. Recent advancements in signal amplification methodologies involving smart designs and the use of nanomaterials and rational surface chemistries, as well as the challenges that must be struggled and the prospects in electrochemical affinity biosensing to bring more accessibility to NDD/AD diagnosis, prognosis, and follow-up, are also pointed out.

Electrochemical biosensing to move forward in cancer epigenetics and metastasis: A review.

Early detection and effective treatment are crucial to reduce the physical, emotional, and financial pressure exerted by growing cancer burden on individuals, families, communities, and health systems. Currently, it is clear that the accurate analysis of emerging cancer epigenetic and metastatic-related biomarkers at different molecular levels is envisaged as an exceptional solution for early and reliable diagnosis and the improvement of therapy efficiency through personalized treatments. Within this field, electrochemical biosensing has demonstrated to be competitive over other emerging and currently used methodologies for the determination of these biomarkers accomplishing the premises of user-friendly, multiplexing ability, simplicity, reduced costs and decentralized analysis, demanded by clinical oncology, thus priming electrochemical biosensors to spark a diagnostic revolution for cancer prediction and eradication. This review article critically discusses the main characteristics, opportunities and versatility exhibited by electrochemical biosensing, through highlighting representative examples published during the last two years, for the reliable determination of these emerging biomarkers, with great diagnostic, predictive and prognostic potential. Special attention is paid on electrochemical affinity biosensors developed for the single or multiplexed determination of methylation events, non-coding RNAs, ctDNA features and metastasis-related protein biomarkers both in liquid and solid biopsies of cancer patients. The main challenges to which further work must be addressed and the impact of these advances should have in the clinical acceptance of these emerging biomarkers are also discussed which decisively will contribute to understand the molecular basis involved in the epigenetics and metastasis of cancer and to apply more efficient personalized therapies.

Electrochemical tropomyosin allergen immunosensor for complex food matrix analysis.

A shrimp tropomyosin (TPM) immunosensor has been developed and optimized to detect trace amounts of shrimp (in the ppm range), based on a combination of an amperometric transduction, magnetic particles and disposable screen-printed electrodes. The approach is based on the implementation of a sandwich immunoassay format on the surface of magnetic beads and their coupling onto disposable screen-printed electrodes to finally register the amperometric response at -200 mV vs. Ag pseudo-reference electrode, using H2O2 as enzymatic substrate and hydroquinone as redox mediator. The use of carboxyl-functionalized magnetic microbeads (MBs) and in-house made magnetic nanoparticles (MNPs) as solid supports have been evaluated and compared. Our experimental results confirm that the use of MBs, in addition to simplifying the test protocol, improves the resulting sensitivity, so they were selected for the implementation of the immunosensor. In the optimized experimental conditions, the developed immunosensor offered a LOD of 47 pg mL-1 for amperometric determination of shrimp TPM standards and great selectivity against TPM from other sources, thus allowing differentiation between crustaceans (shrimp) and mollusks (squid). Applicability studies demonstrated successful determination both in crude and cooked samples using very simple protocols. Additionally, processed foods based on fish and mollusks that could potentially include crustaceans in their composition have been analyzed using the sensor and compared to the declared ingredients. The sensitivity and specificity showed by the sensor in the analysis of heterogeneous food samples without a previous purification or enrichment stage, also outperforms existing solutions in terms of time and cost effectiveness and permits its direct and smooth implementation in the food industry for routine allergen analyses.

Copper(I)-Catalyzed Click Chemistry as a Tool for the Functionalization of Nanomaterials and the Preparation of Electrochemical (Bio)Sensors.

Proper functionalization of electrode surfaces and/or nanomaterials plays a crucial role in the preparation of electrochemical (bio)sensors and their resulting performance. In this context, copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) has been demonstrated to be a powerful strategy due to the high yields achieved, absence of by-products and moderate conditions required both in aqueous medium and under physiological conditions. This particular chemistry offers great potential to functionalize a wide variety of electrode surfaces, nanomaterials, metallophthalocyanines (MPcs) and polymers, thus providing electrochemical platforms with improved electrocatalytic ability and allowing the stable, reproducible and functional integration of a wide range of nanomaterials and/or different biomolecules (enzymes, antibodies, nucleic acids and peptides). Considering the rapid progress in the field, and the potential of this technology, this review paper outlines the unique features imparted by this particular reaction in the development of electrochemical sensors through the discussion of representative examples of the methods mainly reported over the last five years. Special attention has been paid to electrochemical (bio)sensors prepared using nanomaterials and applied to the determination of relevant analytes at different molecular levels. Current challenges and future directions in this field are also briefly pointed out.

Author Correction: Multi-centennial fluctuations of radionuclide production rates are modulated by the Earth's magnetic field.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Pushing the limits of electrochemistry toward challenging applications in clinical diagnosis, prognosis, and therapeutic action.

Constant progress in the identification of biomarkers at different molecular levels in samples of different natures, and the need to conduct routine analyses, even in limited-resource settings involving simple and short protocols, are examples of the growing current clinical demands not satisfied by conventional available techniques. In this context, the unique features offered by electrochemical biosensors, including affordability, real-time and reagentless monitoring, simple handling and portability, and versatility, make them especially interesting for adaptation to the increasingly challenging requirements of current clinical and point-of-care (POC) diagnostics. This has allowed the continuous development of strategies with improved performance in the clinical field that were unthinkable just a few years ago. After a brief introduction to the types and characteristics of clinically relevant biomarkers/samples, requirements for their analysis, and currently available methodologies, this review article provides a critical discussion of the most important developments and relevant applications involving electrochemical biosensors reported in the last five years in response to the demands of current diagnostic, prognostic, and therapeutic actions related to high prevalence and high mortality diseases and disorders. Special attention is paid to the rational design of surface chemistry and the use/modification of state-of-the-art nanomaterials to construct electrochemical bioscaffolds with antifouling properties that can be applied to the single or multiplex determination of biomarkers of accepted or emerging clinical relevance in particularly complex clinical samples, such as undiluted liquid biopsies, whole cells, and paraffin-embedded tissues, which have scarcely been explored using conventional techniques or electrochemical biosensing. Key points guiding future development, challenges to be addressed to further push the limits of electrochemical biosensors towards new challenging applications, and their introduction to the market are also discussed.

Determination of progesterone in saliva using an electrochemical immunosensor and a COTS-based portable potentiostat.

This paper describes the reliable determination of progesterone (P4) in undiluted saliva making use of a disposable amperometric immunosensors implemented on low-cost and portable device/potentiostat constructed with commercial-off-the-shelf (COTS) components. The immunosensor allows the fast (45 min), selective and sensitive determination (5 pg mL-1 LOD) of P4 using amperometry in stirred solutions. The immunosensor was coupled to the COTS-based potentiostat and amperometry was made into drops of quiescent solutions. No significant differences were apparent between the analytical performance achieved with the immunosensor for P4 using both a conventional and the COST-based potentiostats. The practical applicability of the immunosensor coupled with the COTS-based potentiostat was demonstrated by determining the endogenous P4 content in different undiluted saliva samples with highly variable endogenous contents of the target hormone. The obtained results were in good agreement with those provided by the conventional ELISA methodology and with the contents reported in the literature for samples with similar characteristics. This validated the combined device for the reliable and minimally invasive determination of the target hormone involving a very simple protocol and taking only 45 min.

Precursory worldwide signatures of earthquake occurrences on Swarm satellite data.

The study of the preparation phase of large earthquakes is essential to understand the physical processes involved, and potentially useful also to develop a future reliable short-term warning system. Here we analyse electron density and magnetic field data measured by Swarm three-satellite constellation for 4.7 years, to look for possible in-situ ionospheric precursors of large earthquakes to study the interactions between the lithosphere and the above atmosphere and ionosphere, in what is called the Lithosphere-Atmosphere-Ionosphere Coupling (LAIC). We define these anomalies statistically in the whole space-time interval of interest and use a Worldwide Statistical Correlation (WSC) analysis through a superposed epoch approach to study the possible relation with the earthquakes. We find some clear concentrations of electron density and magnetic anomalies from more than two months to some days before the earthquake occurrences. Such anomaly clustering is, in general, statistically significant with respect to homogeneous random simulations, supporting a LAIC during the preparation phase of earthquakes. By investigating different earthquake magnitude ranges, not only do we confirm the well-known Rikitake empirical law between ionospheric anomaly precursor time and earthquake magnitude, but we also give more reliability to the seismic source origin for many of the identified anomalies.

New perspectives in the study of the Earth's magnetic field and climate connection: The use of transfer entropy.

The debated question on the possible relation between the Earth's magnetic field and climate has been usually focused on direct correlations between different time series representing both systems. However, the physical mechanism able to potentially explain this connection is still an open issue. Finding hints about how this connection could work would suppose an important advance in the search of an adequate physical mechanism. Here, we propose an innovative information-theoretic tool, i.e. the transfer entropy, as a good candidate for this scope because is able to determine, not simply the possible existence of a connection, but even the direction in which the link is produced. We have applied this new methodology to two real time series, the South Atlantic Anomaly (SAA) area extent at the Earth's surface (representing the geomagnetic field system) and the Global Sea Level (GSL) rise (for the climate system) for the last 300 years, to measure the possible information flow and sense between them. This connection was previously suggested considering only the long-term trend while now we study this possibility also in shorter scales. The new results seem to support this hypothesis, with more information transferred from the SAA to the GSL time series, with about 90% of confidence level. This result provides new clues on the existence of a link between the geomagnetic field and the Earth's climate in the past and on the physical mechanism involved because, thanks to the application of the transfer entropy, we have determined that the sense of the connection seems to go from the system that produces geomagnetic field to the climate system. Of course, the connection does not mean that the geomagnetic field is fully responsible for the climate changes, rather that it is an important driving component to the variations of the climate.

Multi-centennial fluctuations of radionuclide production rates are modulated by the Earth's magnetic field.

The production of cosmogenic isotopes offers a unique way to reconstruct solar activity during the Holocene. It is influenced by both the solar and Earth magnetic fields and thus their combined effect needs to be disentangled to infer past solar irradiance. Nowadays, it is assumed that the long-term variations of cosmogenic production are modulated by the geomagnetic field and that the solar field dominates over shorter wavelengths. In this process, the effects of the non-dipolar terms of the geomagnetic field are considered negligible. Here we analyse these assumptions and demonstrate that, for a constant solar modulation potential, the geomagnetic field exerts a strong modulation of multi-centennial to millennial wavelengths (periods of 800 and 2200 yr). Moreover, we demonstrate that the non-dipole terms derived from the harmonic degree 3 and above produce maximum differences of 7% in the global average radiocarbon production rate. The results are supported by the identification, for the first time, of a robust coherence between the production rates independently estimated from geomagnetic reconstructions and that inferred from natural archives. This implies the need to review past solar forcing reconstructions, with important implications both for the assessment of solar-climate relationships as well as for the present and future generation of paleoclimate models.

An electrochemical immunosensor for brain natriuretic peptide prepared with screen-printed carbon electrodes nanostructured with gold nanoparticles grafted through aryl diazonium salt chemistry.

A sensitive amperometric immunosensor has been prepared by immobilization of capture antibodies onto gold nanoparticles (AuNPs) grafted on a screen-printed carbon electrode (SPCE) through aryl diazonium salt chemistry using 4-aminothiophenol (AuNPs-S-Phe-SPCE). The immunosensor was designed for the accurate determination of clinically relevant levels of B-type natriuretic peptide (BNP) in human serum samples. The nanostructured electrochemical platform resulted in an ordered layer of AuNPs onto SPCEs which combined the advantages of high conductivity and improved stability of immobilized biomolecules. The resulting disposable immunosensor used a sandwich type immunoassay involving a peroxidase-labeled detector antibody. The amperometric transduction was carried out at -0.20V (vs the Ag pseudo-reference electrode) upon the addition of hydroquinone (HQ) as electron transfer mediator and H2O2 as the enzyme substrate. The nanostructured immunosensors show a storage stability of at least 25 days, a linear range between 0.014 and 15ngmL-1, and a LOD of 4pgmL-1, which is 100 times lower than the established cut-off value for heart failure (HF) diagnosis. The performance of the immunosensor is advantageously compared with that provided with immunosensors prepared by grafting SPCE with p-phenylendiamine (H2N-Phe-SPCE) and attaching AuNPs by immersion into an AuNPs suspension or by electrochemical deposition, as well as with immunosensors constructed using commercial AuNPs-modified SPCEs. The developed immunosensor was applied to the successful analysis of human serum from heart failure (HF) patients upon just a 10-times dilution as sample treatment.

Nano/microvehicles for efficient delivery and (bio)sensing at the cellular level.

A perspective review of recent strategies involving the use of nano/microvehicles to address the key challenges associated with delivery and (bio)sensing at the cellular level is presented. The main types and characteristics of the different nano/microvehicles used for these cellular applications are discussed, including fabrication pathways, propulsion (catalytic, magnetic, acoustic or biological) and navigation strategies, and relevant parameters affecting their propulsion performance and sensing and delivery capabilities. Thereafter, selected applications are critically discussed. An emphasis is made on enhancing the extra- and intra-cellular biosensing capabilities, fast cell internalization, rapid inter- or intra-cellular movement, efficient payload delivery and targeted on-demand controlled release in order to greatly improve the monitoring and modulation of cellular processes. A critical discussion of selected breakthrough applications illustrates how these smart multifunctional nano/microdevices operate as nano/microcarriers and sensors at the intra- and extra-cellular levels. These advances allow both the real-time biosensing of relevant targets and processes even at a single cell level, and the delivery of different cargoes (drugs, functional proteins, oligonucleotides and cells) for therapeutics, gene silencing/transfection and assisted fertilization, while overcoming challenges faced by current affinity biosensors and delivery vehicles. Key challenges for the future and the envisioned opportunities and future perspectives of this remarkably exciting field are discussed.

Competitive RNA-RNA hybridization-based integrated nanostructured-disposable electrode for highly sensitive determination of miRNAs in cancer cells.

A new method for the detection of miRNAs making use of a competitive RNA/RNA hybridization configuration is described in this work. A biotinylated miRNA (biotin-miRNA) of identical sequence to that of the target miRNA is mixed with the samples to be analyzed allowing competition to be accomplished with the target miRNA for a thiolated RNA probe assembled onto a gold nanoparticles (AuNPs) modified screen-printed electrode. After labeling the hybridized biotin-miRNA with streptavidin-HRP conjugates, amperometric detection at -0.20V was carried out using the H2O2/hydroquinone (HQ) system. The decrease in the amperometric response was proportional to the concentration of model target miRNA-21 in the 100 fM to 25.0 pM range. The integrated sensor provided a very low detection limit (25 fM, 0.25 attomol in 10µL sample) for miRNA-21 without any amplification step, a complete discrimination against single nucleotide mismatched sequences under practical conditions and high storage stability. The usefulness of the developed method was demonstrated by determining the endogenous levels of the mature target miRNA in total RNA (RNAt) extracted from cancerous and non-cancerous cells.

Hybrid 2D-nanomaterials-based electrochemical immunosensing strategies for clinical biomarkers determination.

Owing to the outstanding conductivity and biocompatibility as well as numerous other fascinating properties of two-dimensional (2D)-nanomaterials, 2D-based nanohybrids have shown unparalleled superiorities in the field of electrochemical biosensors. This review highlights latest advances in electrochemical immunosensors for clinical biomarkers based on different hybrid 2D-nanomaterials. Particular attention will be given to hybrid nanostructures involving graphene and other graphene-like 2D-layered nanomaterials (GLNs). Several recent strategies for using such 2D-nanomaterial heterostructures in the development of modern immunosensors, both for tagging or modifying electrode transducers, are summarized and discussed. These hybrid nanocomposites, quite superior than their rival materials, will undoubtedly have an important impact within the near future and not only in clinical areas. Current challenges and future perspectives in this rapidly growing field are also outlined.

Sensitive electrochemical determination of miRNAs based on a sandwich assay onto magnetic microcarriers and hybridization chain reaction amplification.

A novel electrochemical approach for determination of miRNAs involving a sandwich hybridization assay onto streptavidin-magnetic beads (Strep-MBs), hybridization chain reaction (HCR) amplification and amperometric detection at disposable screen-printed carbon electrodes is reported. Using miRNA-21 as the target analyte, a dynamic linear range from 0.2 to 5.0nM with a 60pM (1.5fmol in 25µL) detection limit was obtained. The achieved sensitivity is 24-fold higher than a non-HCR amplification approach involving conventional sandwich type assay onto MBs. Moreover, the whole assay time lasted 1h 45min which is remarkably shorter than other reported methodologies. The methodology exhibited full selectivity against other non-complementary miRNAs as well as an acceptable discrimination between homologous miRNA family members. The applicability of this novel approach was demonstrated by determining mature miRNA-21 in total RNA (RNAt) extracted from tumor cells and human tissues.

Automatic bionalyzer using an integrated amperometric biosensor for the determination of L-malic acid in wines.

A new automatic bioanalyzer for L-malic acid using an integrated amperometric biosensor as detector is reported for the first time in this work. The biosensor is constructed by gold film sputtering deposition on a stainless steel disk electrode and co-immobilization of the enzymes malate dehydrogenase (MDH) and diaphorase (DP) together with the redox mediator tetrathiafulvalene (TTF) by means of dialysis membrane. The analytical performance of the biosensor was evaluated when it was used as amperometric detector in three different analytical methodologies: stirred solutions, semiautomatic FIA system and automatic bioanalyzer. The bienzyme biosensor exhibited great analytical performance in terms of sensitivity, selectivity and reproducibility of the measurements and its usefulness was demonstrated by analyzing wine reference materials with certified content of L-malic acid. The attractive analytical and operational characteristics demonstrated by the automatic bioanalyzer make it a promising simple, rapid and field-based tool for routine wine and fruit control.

Implementation of a new integrated d-lactic acid biosensor in a semiautomatic FIA system for the simultaneous determination of lactic acid enantiomers. Application to the analysis of beer samples.

An integrated amperometric d-lactic acid biosensor involving a gold film deposited by sputtering on a stainless steel disk electrode where the enzymes D-lactic acid dehydrogenase (DLDH) and diaphorase (DP) as well as the redox mediator tetrathiafulvalene (TTF) are coimmobilized by using a dialysis membrane, is reported in this work. Amperometry in stirred solutions at a detection potential of +0.15 V (vs Ag/AgCl reference electrode) provided a linear calibration plot for D-lactic acid over the 1.0×10(-4) to 3.8×10(-3) g L(-1) concentration range, with a limit of detection of 3.1×10(-5) g L(-1). The usefulness of the biosensor was demonstrated by determining D-lactic acid in beer samples with good results. Additionally, the biosensor was implemented together with a commercial L-lactic amperometric biosensor in a semiautomatic flow-injection analysis (FIA) system able to perform a rapid and simple stereo-specific determination of D- and D-lactic without a previous separation step. The operational characteristics of the biosensors under flow conditions were evaluated and its applicability was demonstrated through the simultaneous determination of both enantiomers in beer samples.

Non-invasive determination of glucose directly in raw fruits using a continuous flow system based on microdialysis sampling and amperometric detection at an integrated enzymatic biosensor.

A non-destructive, rapid and simple to use sensing method for direct determination of glucose in non-processed fruits is described. The strategy involved on-line microdialysis sampling coupled with a continuous flow system with amperometric detection at an enzymatic biosensor. Apart from direct determination of glucose in fruit juices and blended fruits, this work describes for the first time the successful application of an enzymatic biosensor-based electrochemical approach to the non-invasive determination of glucose in raw fruits. The methodology correlates, through previous calibration set-up, the amperometric signal generated from glucose in non-processed fruits with its content in % (w/w). The comparison of the obtained results using the proposed approach in different fruits with those provided by other method involving the same commercial biosensor as amperometric detector in stirred solutions pointed out that there were no significant differences. Moreover, in comparison with other available methodologies, this microdialysis-coupled continuous flow system amperometric biosensor-based procedure features straightforward sample preparation, low cost, reduced assay time (sampling rate of 7 h(-1)) and ease of automation.