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This work reports the first bioplatform to assist in the early and reliable diagnosis of autoimmune diseases by quadruple determination of autoantibodies (Abs) produced against extractable nuclear antigens (ENAs): La/SSB-Abs, Ro/SSA-Abs, U1snRNP70-Abs and smRNP-Abs. The bioplatform involves indirect immunoassays on the surface of magnetic microcarriers (independent batches for each of the target autoantibodies) and amperometric transduction using the H2O2/hydroquinone (HQ) system on a disposable multiple electrode platform. The magnetic microcarriers were modified with the corresponding antigens using His-tag and carbodiimide/succinimide chemistries and employed for the selective capture of the corresponding autoantibodies. Thereafter, they were enzymatically labelled with a secondary antibody conjugated with horseradish peroxidase (HRP) and magnetically captured on each of the working surfaces of the quadruple platform. The evaluation of the analytical and operational characteristics of the bioplatform for the amperometric determination of standards, performed under optimized experimental conditions, confirmed the bioplatform competitiveness in terms of sensitivity and point-of-care application compared to commercially available ELISA methodologies for the single determination of target Abs. The developed bioplatform was applied to the analysis of serum samples from healthy individuals and from patients with two prevalent autoimmune diseases (systemic lupus erythematosus, SLE, and Sjögren's syndrome, SS). The obtained results proved the potential of the bioplatform for the differential diagnosis of these two autoimmune diseases through the accurate, simple, and rapid multidetermination of the four target Abs.

Abstract The determination of metals in clinical samples is an important challenge since their levels are relevant to ensure personal exposure to safe environments, as well as to assist in the prevention and treatment of disease. The low concentrations of metal ions in clinical samples and their coexistence with other metals and/or molecules of diverse nature implies the need for sensitive and selective analytical methodologies, which can be provided by conventional techniques. However, most of these techniques do not satisfy other highly demanded requirements such as high sampling frequency, multiplexed ability, and in‐field analysis. In this context, the simplicity, speed of response, and compatibility with single or multiplexed point‐of‐care determinations have led to the successful incursion of electrochemical (bio)sensing into the clinical field for the determination of these analytes. Electrochemical (bio)devices have come on strong by exploiting and combining the unique attributes provided by electrochemical techniques (primarily voltammetry but also giving way to impedimetry in electrochemical affinity biosensing), electrode materials (Hg, Bi, Sb, Au, GCE, CPE, ITO, FTO), and substrates (conventional, screen‐printed, flexible, paper, wearable or tattoo‐type), suitable modifiers (nanomaterials, polymers, mesoporous films), bioreceptors (aptamers, peptides, biomolecular switches, DNAzymes, and DNA molecules), and nucleic acid amplification strategies (rolling circle and exonucleases‐assisted target recycling). Therefore, the purpose of this review is to provide a bird's eye view of the state of the art, discussing the remarkable opportunities and potentialities demonstrated in the last decade by electrochemical sensors and electrochemical affinity biosensors for the analysis of metal ions in challenging clinical samples and forecasting future directions in this exciting field.

Natural enzymes are used as special reagents for the preparation of electrochemical (bio)sensors due to their ability to catalyze processes, improving the selectivity of detection. However, some drawbacks, such as denaturation in harsh experimental conditions and their rapid de- gradation, as well as the high cost and difficulties in recycling them, restrict their practical applications. Nowadays, the use of artificial enzymes, mostly based on nanomaterials, mimicking the functions of natural products, has been growing. These so-called nanozymes present several advantages over natural enzymes, such as enhanced stability, low cost, easy production, and rapid activity. These outstanding features are responsible for their widespread use in areas such as catalysis, energy, imaging, sensing, or biomedicine. These materials can be divided into two main groups: metal and carbon-based nanozymes. The latter provides additional advantages compared to metal nanozymes, i.e., stable and tuneable activity and good biocompatibility, mimicking enzyme activities such as those of peroxidase, catalase, oxidase, superoxide dismutase, nuclease, or phosphatase. In this review article, we have focused on the use of carbon-based nanozymes for the preparation of electrochemical (bio)sensors. The main features of the most recent applications have been revised and illustrated with examples selected from the literature over the last four years (since 2020).

The crosstalk between cancer cells and the tumor microenvironment has been implicated in cancer progression and metastasis. Fibroblasts and immune cells are widely known to be attracted to and modified by cancer cells. However, the role of pericytes in the tumor microenvironment beyond endothelium stabilization is poorly understood. Here, we report that pericytes promoted colorectal cancer (CRC) cell proliferation, migration, invasion, stemness, and chemoresistance in vitro, as well as tumor growth in a xenograft CRC model. We demonstrate that coculture with human CRC cells induced broad transcriptomic changes in pericytes, mostly associated with TGF‐β receptor activation. The prognostic value of a TGF‐β response signature in pericytes was analyzed in CRC patient data sets. This signature was found to be a good predictor of CRC relapse. Moreover, in response to stimulation by CRC cells, pericytes expressed high levels of TGF‐β1, initiating an autocrine activation loop. Investigation of secreted mediators and underlying molecular mechanisms revealed that IGFBP‐3 is a key paracrine factor from activated pericytes affecting CRC cell migration and invasion. In summary, we demonstrate that the interplay between pericytes and CRC cells triggers a vicious cycle that stimulates pericyte cytokine secretion, in turn increasing CRC cell tumorigenic properties. Overall, we provide another example of how cancer cells can manipulate the tumor microenvironment.

Rheumatoid arthritis (RA) is a systemic chronic autoimmune inflammatory disease that is characterized by the destruction of bone and production of autoantibodies such as rheumatoid factor (RF) and anticitrullinated protein antibodies (ACPAs). The high prevalence of this disease and the need of affordable tools for its early detection led us to prepare the first electrochemical immunoplatform for the simultaneous determination of four RA biomarkers, the autoantibodies: RF, anti-peptidyl-arginine deiminase enzyme (anti-PAD4), anti-cyclic citrullinated peptide (anti-CCP), and anti-citrullinated vimentin (anti-MCV). Functionalized magnetic beads (MBs) were used to immobilize the specific antigens, and sandwich-type immunoassays were implemented for the amperometric detection of the four autoantibodies, using the horseradish peroxidase (HRP)/H2O2/hydroquinone (HQ) system. The immunoplatform was applied to the determination of the biomarkers in human serum of twenty-two patients diagnosed with RA and four healthy individuals, and the results were validated against ELISA tests and the certified values.

The study of the human microbiome is a multidisciplinary area ranging from the field of technology to that of personalized medicine. The possibility of using microbiota biomarkers to improve the diagnosis and monitoring of diseases (e.g., cancer), health conditions (e.g., obesity) or relevant processes (e.g., aging) has raised great expectations, also in the field of bioelectroanalytical chemistry. The well-known advantages of electrochemical biosensors—high sensitivity, fast response, and the possibility of miniaturization, together with the potential for new nanomaterials to improve their design and performance—position them as unique tools to provide a better understanding of the entities of the human microbiome and raise the prospect of huge and important developments in the coming years. This review article compiles recent applications of electrochemical (bio)sensors for monitoring microbial metabolites and disease biomarkers related to different types of human microbiome, with a special focus on the gastrointestinal microbiome. Examples of electrochemical devices applied to real samples are critically discussed, as well as challenges to be faced and where future developments are expected to go.

Serum level of CCL5 chemokine is considered an emerging biomarker for multiple sclerosis (MS). Due to the lack of specific assays for this disease, the development of a point-of-care test for rapid detection of MS could lead to avoiding diagnostics delays. In this paper, we report the first electrochemical immunoplatform for quantification of the CCL5 biomarker at the clinically required levels, able to discriminate between patients diagnosed with MS and healthy individuals. The immunosensing device involves protein capture from biological samples by complexation with biotinylated specific antibodies immobilized onto neutravidin-functionalized microparticles and sandwich assay with anti-CCL5 antibody and IgG labelled with horseradish peroxidase (HRP) for the enzyme-catalyzed amperometric detection of H2O2 using hydroquinone (HQ) as the redox mediator. The method shows excellent analytical performance for clinical application with a wide linear range of concentrations (0.1–300 ng·mL−1 CCL5, R2 = 0.998) and a low detection limit (40 pg·mL−1 CCL5). The biosensing platform was applied to the determination of the CCL5 endogenous content in 100-fold diluted sera both from healthy individuals and patients diagnosed with MS, with no further sample treatment in just two hours. The results were successfully compared with those obtained by the ELISA methodology.

Chemokines are a class of cytokine whose special properties, together with their involvement and relevant role in various diseases, make them a restricted group of biomarkers suitable for diagnosis and monitoring. Despite their importance, biodetection techniques dedicated to the selective determination of one or more chemokines are very scarce. For some years now, the critical diagnosis of inflammatory diseases by detecting both cytokine and chemokine biomarkers, has had a strong impact on the development of multiple detection platforms. However, it would be desirable to implement methodologies with a higher degree of selectivity for chemokines, in order to provide more precise information. In addition, better development of biosensor technology applied to this specific field would make it possible to address the main challenges of detection methods for several diseases with a high incidence in the population, avoiding high costs and low sensitivity. Taking this into account, this review aims to present the state of the art of chemokine biodetection techniques and emphasize the role of these systems in the prevention, monitoring and treatment of various diseases associated with chemokines as a starting point for future developments that are also analyzed throughout the article.

Multifunctional nanomaterials, defined as those able to achieve a combined effect or more than one function through their multiple functionalization or combination with other materials, are gaining increasing attention in the last years in many relevant fields, including cargo targeted delivery, tissue engineering, in vitro and/or in vivo diseases imaging and therapy, as well as in the development of electrochemical (bio)sensors and (bio)sensing strategies with improved performance. This review article aims to provide an updated overview of the important advances and future opportunities exhibited by electrochemical biosensing in connection to multifunctional nanomaterials. Accordingly, representative aspects of recent approaches involving metal, carbon, and silica-based multifunctional nanomaterials are selected and critically discussed, as they are the most widely used multifunctional nanomaterials imparting unique capabilities in (bio)electroanalysis. A brief overview of the main remaining challenges and future perspectives in the field is also provided.

The multifaceted key roles of cytokines in immunity and inflammatory processes have led to a high clinical interest for the determination of these biomolecules to be used as a tool in the diagnosis, prognosis, monitoring and treatment of several diseases of great current relevance (autoimmune, neurodegenerative, cardiac, viral and cancer diseases, hypercholesterolemia and diabetes). Therefore, the rapid and accurate determination of cytokine biomarkers in body fluids, cells and tissues has attracted considerable attention. However, many currently available techniques used for this purpose, although sensitive and selective, require expensive equipment and advanced human skills and do not meet the demands of today’s clinic in terms of test time, simplicity and point-of-care applicability. In the course of ongoing pursuit of new analytical methodologies, electrochemical biosensing is steadily gaining ground as a strategy suitable to develop simple, low-cost methods, with the ability for multiplexed and multiomics determinations in a short time and requiring a small amount of sample. This review article puts forward electrochemical biosensing methods reported in the last five years for the determination of cytokines, summarizes recent developments and trends through a comprehensive discussion of selected strategies, and highlights the challenges to solve in this field. Considering the key role demonstrated in the last years by different materials (with nano or micrometric size and with or without magnetic properties), in the design of analytical performance-enhanced electrochemical biosensing strategies, special attention is paid to the methods exploiting these approaches.

The excellent capabilities demonstrated over the last few years by electrochemical affinity biosensors should be largely attributed to their coupling with particular nanostructures including dendrimers, DNA-based nanoskeletons, molecular imprinted polymers, metal-organic frameworks, nanozymes and magnetic and mesoporous silica nanoparticles. This review article aims to give, by highlighting representative methods reported in the last 5 years, an updated and general overview of the main improvements that the use of such well-ordered nanomaterials as electrode modifiers or advanced labels confer to electrochemical affinity biosensors in terms of sensitivity, selectivity, stability, conductivity and biocompatibility focused on food and environmental applications, less covered in the literature than clinics. A wide variety of bioreceptors (antibodies, DNAs, aptamers, lectins, mast cells, DNAzymes), affinity reactions (single, sandwich, competitive and displacement) and detection strategies (label-free or label-based using mainly natural but also artificial enzymes), whose performance is substantially improved when used in conjunction with nanostructured systems, are critically discussed together with the great diversity of molecular targets that nanostructured affinity biosensors are able to quantify using quite simple protocols in a wide variety of matrices and with the sensitivity required by legislation. The large number of possibilities and the versatility of these approaches, the main challenges to face in order to achieve other pursued capabilities (development of antifouling, continuous operation, wash-, calibration- and reagents-free devices, regulatory or Association of Official Analytical Chemists, AOAC, approval) and decisive future actions to achieve the commercialization and acceptance of these devices in our daily routine are also noted at the end.

Screen-printing technology has revolutionized many fields, including that of electrochemical biosensing. Due to their current relevance, this review, unlike other papers, discusses the relevant aspects of electrochemical biosensors manufactured using this technology in connection to both paper substrates and wearable formats. The main trends, advances, and opportunities provided by these types of devices, with particular attention to the environmental and biomedical fields, are addressed along with illustrative fundamentals and applications of selected representative approaches from the recent literature. The main challenges and future directions to tackle in this research area are also pointed out.

Nowadays, electrochemical biosensors are reliable analytical tools to determine a broad range of molecular analytes because of their simplicity, affordable cost, and compatibility with multiplexed and point-of-care strategies. There is an increasing demand to improve their sensitivity and selectivity, but also to provide electrochemical biosensors with important attributes such as near real-time and continuous monitoring in complex or denaturing media, or in vivo with minimal intervention to make them even more attractive and suitable for getting into the real world. Modification of biosensors surfaces with antibiofouling reagents, smart coupling with nanomaterials, and the advances experienced by folded-based biosensors have endowed bioelectroanalytical platforms with one or more of such attributes. With this background in mind, this review aims to give an updated and general overview of these technologies as well as to discuss the remarkable achievements arising from the development of electrochemical biosensors free of reagents, washing, or calibration steps, and/or with antifouling properties and the ability to perform continuous, real-time, and even in vivo operation in nearly autonomous way. The challenges to be faced and the next features that these devices may offer to continue impacting in fields closely related with essential aspects of people’s safety and health are also commented upon.

The presence of allergens and adulterants in food, which represents a real threat to sensitized people and a loss of consumer confidence, is one of the main current problems facing society. The detection of allergens and adulterants in food, mainly at the genetic level (characteristic fragments of genes that encode their expression) or at functional level (protein biomarkers) is a complex task due to the natural interference of the matrix and the low concentration at which they are present. Methods for the analysis of allergens are mainly divided into immunological and deoxyribonucleic acid (DNA)-based assays. In recent years, electrochemical affinity biosensors, including immunosensors and biosensors based on synthetic sequences of DNA or ribonucleic acid (RNA), linear, aptameric, peptide or switch-based probes, are gaining special importance in this field because they have proved to be competitive with the methods commonly used in terms of simplicity, test time and applicability in different environments. These unique features make them highly promising analytical tools for routine determination of allergens and food adulterations at the point of care. This review article discusses the most significant trends and developments in electrochemical affinity biosensing in this field over the past two years as well as the challenges and future prospects for this technology.

This article critically discusses the latest advances in the use of voltammetric, amperometric, potentiometric, and impedimetric biosensors for forensic analysis. Highlighted examples that show the advantages of these tools to develop methods capable of detecting very small concentrations of analytes and provide selective determinations through analytical responses, without significant interferences from other components of the samples, are presented and discussed, thus stressing the great versatility and utility of electrochemical biosensors in this growing research field. To illustrate this, the determination of substances with forensic relevance by using electrochemical biosensors reported in the last five years (2015−2019) are reviewed. The different configurations of enzyme or affinity biosensors used to solve analytical problems related to forensic practice, with special attention to applications in complex samples, are considered. Main prospects, challenges to focus, such as the fabrication of devices for rapid analysis of target analytes directly on-site at the crime scene, or their widespread use and successful applications to complex samples of interest in forensic analysis, and future efforts, are also briefly discussed.

Magnetic Janus particles bring together the ability of Janus particles to perform two different functions at the same time in a single particle with magnetic properties enabling their remote manipulation, which allows headed movement and orientation. This article reviews the preparation procedures and applications in the (bio)sensing field of static and self-propelled magnetic Janus particles. The main progress in the fabrication procedures and the applicability of these particles are critically discussed, also giving some clues on challenges to be dealt with and future prospects. The promising characteristics of magnetic Janus particles in the (bio)sensing field, providing increased kinetics and sensitivity and decreased times of analysis derived from the use of external magnetic fields in their manipulation, allows foreseeing their great and exciting potential in the medical and environmental remediation fields.

Nowadays, analyzing circulating tumor DNA (ctDNA), a very small part of circulating free DNA (cfDNA) carried by blood, is considered to be an interesting alternative to conventional single-site tumor tissue biopsies, both to assess tumor burden and provide a more comprehensive snapshot of the time-related and spatial heterogeneity of cancer genetic/epigenetic scenery. The determination of ctDNA and/or mapping its characteristic features, including tumor-specific mutations, chromosomal aberrations, microsatellite alterations, and epigenetic changes, are minimally invasive, powerful and credible biomarkers for early diagnosis, follow-up, prediction of therapy response/resistance, relapse monitoring, and tracking the rise of new mutant subclones, leading to improved cancer outcomes This review provides an outline of advances published in the last five years in electrochemical biosensing of ctDNA and surrogate markers. It emphasizes those strategies that have been successfully applied to real clinical samples. It highlights the unique opportunities they offer to shift the focus of cancer patient management methods from actual decision making, based on clinic-pathological features, to biomarker-driven treatment strategies, based on genotypes and customized targeted therapies. Also highlighted are the unmet hurdles and future key points to guide these devices in the development of liquid biopsy cornerstone tools in routine clinical practice for the diagnosis, prognosis, and therapy response monitoring in cancer patients.

Graphene quantum dots (GQDs) and carbon dots (CDs) are among the latest research frontiers in carbon-based nanomaterials. They provide interesting attributes to current electrochemical biosensing due to their intrinsic low toxicity, high solubility in many solvents, excellent electronic properties, robust chemical inertness, large specific surface area, abundant edge sites for functionalization, great biocompatibility, low cost, and versatility, as well as their ability for modification with attractive surface chemistries and other modifiers/nanomaterials. In this review article, the use of GQDs and CDs as signal tags or electrode surface modifiers to develop electrochemical biosensing strategies is critically discussed through the consideration of representative approaches reported in the last five years. The advantages and disadvantages arising from the use of GQDs and CDs in this context are outlined together with the still required work to fulfil the characteristics needed to achieve suitable electrochemical enzymatic and affinity biosensors with applications in the real world.

Adequate selection of the electrode surface and the strategies for its modification to enable subsequent immobilization of biomolecules and/or nanomaterials integration play a major role in the performance of electrochemical affinity biosensors. Because of the simplicity, rapidity and versatility, electrografting using diazonium salt reduction is among the most currently used functionalization methods to provide the attachment of an organic layer to a conductive substrate. This particular chemistry has demonstrated to be a powerful tool to covalently immobilize in a stable and reproducible way a wide range of biomolecules or nanomaterials onto different electrode surfaces. Considering the great progress and interesting features arisen in the last years, this paper outlines the potential of diazonium chemistry to prepare single or multianalyte electrochemical affinity biosensors on screen-printed electrodes (SPEs) and points out the existing challenges and future directions in this field.

A biosensor for 3-hydroxybutyrate (3-HB) involving immobilization of the enzyme 3-hydroxybutyrate dehydrogenase onto a screen-printed carbon electrode modified with reduced graphene oxide (GO) and thionine (THI) is reported here. After addition of 3-hydroxybutyrate or the sample in the presence of NAD+ cofactor, the generated NADH could be detected amperometrically at 0.0 V vs. Ag pseudo reference electrode. Under the optimized experimental conditions, a calibration plot for 3-HB was constructed showing a wide linear range between 0.010 and 0.400 mM 3-HB which covers the clinically relevant levels for diluted serum samples. In addition, a limit of detection of 1.0 µM, much lower than that reported using other biosensors, was achieved. The analytical usefulness of the developed biosensor was demonstrated via application to spiked serum samples.

Rapid and reliable diagnosis of infectious diseases caused by pathogens, and timely initiation of appropriate treatment are critical determinants to promote optimal clinical outcomes and general public health. Conventional in vitro diagnostics for infectious diseases are time-consuming and require centralized laboratories, experienced personnel and bulky equipment. Recent advances in electrochemical affinity biosensors have demonstrated to surpass conventional standards in regards to time, simplicity, accuracy and cost in this field. The tremendous potential offered by electrochemical affinity biosensors to detect on-site infectious pathogens at clinically relevant levels in scarcely treated body fluids is clearly stated in this review. The development and application of selected examples using different specific receptors, assay formats and electrochemical approaches focusing on the determination of specific circulating biomarkers of different molecular (genetic, regulatory and functional) levels associated with bacterial and viral pathogens are critically discussed. Existing challenges still to be addressed and future directions in this rapidly advancing and highly interesting field are also briefly pointed out.

Management and prognosis of disease requires the accurate determination of specific biomarkers indicative of normal or disease-related biological processes or responses to therapy. Moreover since multiple determinations of biomarkers have demonstrated to provide more accurate information than individual determinations to assist the clinician in prognosis and diagnosis, the detection of several clinical biomarkers by using the same analytical device hold enormous potential for early detection and personalized therapy and will simplify the diagnosis providing more information in less time. In this field, electrochemical immunosensors have demonstrated to offer interesting alternatives against conventional strategies due to their simplicity, fast response, low cost, high sensitivity and compatibility with multiplexed determination, microfabrication technology and decentralized determinations, features which made them very attractive for integration in point-of-care (POC) devices. Therefore, in this review, the relevance and current challenges of multiplexed determination of clinical biomarkers are briefly introduced, and an overview of the electrochemical immunosensing platforms developed so far for this purpose is given in order to demonstrate the great potential of these methodologies. After highlighting the main features of the selected examples, the unsolved challenges and future directions in this field are also briefly discussed.

Growing demand for developing ultrasensitive electrochemical bioassays has led to the design of numerous signal amplification strategies. In this context, carbon-based nanomaterials have been demonstrated to be excellent tags for greatly amplifying the transduction of recognition events and simplifying the protocols used in electrochemical biosensing. This relevant role is due to the carbon-nanomaterials’ large surface area, excellent biological compatibility and ease functionalization and, in some cases, intrinsic electrochemistry. These carbon-based nanomaterials involve well-known carbon nanotubes (CNTs) and graphene as well as the more recent use of other carbon nanoforms. This paper briefly discusses the advantages of using carbon nanostructures and their hybrid nanocomposites for amplification through tagging in electrochemical biosensing platforms and provides an updated overview of some selected examples making use of labels involving carbon nanomaterials, acting both as carriers for signal elements and as electrochemical tracers, applied to the electrochemical biosensing of relevant (bio)markers.

Early diagnosis is often the key to successful patient treatment and survival. The identification of various disease signaling biomarkers which reliably reflect normal and disease states in humans in biological fluids explain the burgeoning research field in developing new methodologies able to determine the target biomarkers in complex biological samples with the required sensitivity and selectivity and in a simple and rapid way. The unique advantages offered by electrochemical sensors together with the availability of high affinity and specific bioreceptors and their great capabilities in terms of sensitivity and stability imparted by nanostructuring the electrode surface with different carbon nanomaterials have led to the development of new electrochemical biosensing strategies that have flourished as interesting alternatives to conventional methodologies for clinical diagnostics. This paper briefly reviews the advantages of using carbon nanostructures and their hybrid nanocomposites as electrode modifiers to construct efficient electrochemical sensing platforms for diagnosis. The review provides an updated overview of some selected examples involving attractive amplification and biosensing approaches which have been applied to the determination of relevant genetic and protein diagnostics biomarkers.

Ultrasensitive biosensing is currently a growing demand that has led to the development of numerous strategies for signal amplification. In this context, the unique properties of magnetic particles; both of nano- and micro-size dimensions; have proved to be promising materials to be coupled with disposable electrodes for the design of cost-effective electrochemical affinity biosensing platforms. This review addresses, through discussion of selected examples, the way that nano- and micro-magnetic particles (MNPs and MMPs; respectively) have contributed significantly to the development of electrochemical affinity biosensors, including immuno-, DNA, aptamer and other affinity modes. Different aspects such as type of magnetic particles, assay formats, detection techniques, sensitivity, applicability and other relevant characteristics are discussed. Research opportunities and future development trends in this field are also considered.