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Colorectal cancer (CRC) is the third most common cancer and the second most frequent cause of cancer-related death worldwide. The detection in plasma samples of autoantibodies against specific tumor-associated antigens has been demonstrated to be useful for the early diagnosis of CRC by liquid biopsy. However, new studies related to the humoral immune response in cancer are needed to enable blood-based diagnosis of the disease. Here, our aim was to characterize the humoral immune response associated with the different p53 and p63 proteoforms derived from alternative splicing and previously described as aberrantly expressed in CRC. Thus, here we investigated the diagnostic ability of the twelve p53 proteoforms and the eight p63 proteoforms described to date, and their specific N-terminal and C-terminal end peptides, by means of luminescence HaloTag beads immunoassays. Full-length proteoforms or specific peptides were cloned as HaloTag fusion proteins and their seroreactivity analyzed using plasma from CRC patients at stages I-IV (n = 31), individuals with premalignant lesions (n = 31), and healthy individuals (n = 48). p53γ, Δ40p53β, Δ40p53γ, Δ133p53γ, Δ160p53γ, TAp63α, TAp63δ, ΔNp63α, and ΔNp63δ, together with the specific C-terminal end α and δ p63 peptides, were found to be more seroreactive against plasma from CRC patients and/or individuals with premalignant lesions than from healthy individuals. In addition, ROC (receiver operating characteristic) curves revealed a high diagnostic ability of those p53 and p63 proteoforms to detect CRC and premalignant individuals (AUC higher than 85%). Finally, electrochemical biosensing platforms were employed in POC-like devices to investigate their usefulness for CRC detection using selected p53 and p63 proteoforms. Our results demonstrate not only the potential of these biosensors for the simultaneous analysis of proteoforms’ seroreactivity, but also their convenience and versatility for the clinical detection of CRC by liquid biopsy. In conclusion, we here show that p53 and p63 proteoforms possess differential seroreactivity in CRC patients in comparison to controls, distinctive from canonical proteins, which should improve the diagnostic panels for obtaining a blood-based biomarker signature for CRC detection.

A disposable electrochemical PCR-free biosensor for the selective detection of a fragment encoding the protein Sin a 1, a 2S albumin considered a diagnostic marker for sensitization to mustard, is reported. The methodology is based on the formation of DNA/RNA heterohybrids by sandwich hybridization of a specific fragment of the Sin a 1 allergen coding sequence with appropriately designed RNA probes. Labeling with commercial antibodies specific to the heteroduplexes and secondary antibodies conjugated with horseradish peroxidase (HRP) was carried out onto the surface of magnetic beads (MBs). Amperometric transduction was undertaken on screen-printed electrodes using H2O2 as enzyme substrate and hydroquinone (HQ) a redox mediator. The electrochemical biosensor allows the simple and fast detection (75 min) of Sin a 1 reaching a limit of detection of 3 pM. The bioplatform was successfully applied to the analysis of the targeted Sin a 1 gene specific region using just 50 ng of non-fragmented denatured genomic DNA extracted from yellow mustard seeds.

This paper reports a simple electrochemical strategy for the determination of microRNAs (miRNAs) using a commercial His-Tag-Zinc finger protein (His-Tag-ZFP) that binds preferably (but non-sequence specifically) RNA hybrids over ssRNAs, ssDNAs, and dsDNAs. The strategy involves the use of magnetic beads (His-Tag-Isolation-MBs) as solid support to capture the conjugate formed in homogenous solution between His-Tag-ZFP and the dsRNA homohybrid formed between the target miRNA (miR-21 selected as a model) and a biotinylated synthetic complementary RNA detector probe (b-RNA-Dp) further conjugated with a streptavidin-horseradish peroxidase (Strep-HRP) conjugate. The electrochemical detection is carried out by amperometry at disposable screen-printed carbon electrodes (SPCEs) (- 0.20 V vs Ag pseudo-reference electrode) upon magnetic capture of the resultant magnetic bioconjugates and H2O2 addition in the presence of hydroquinone (HQ). The as-prepared biosensor exhibits a dynamic concentration range from 3.0 to 100 nM and a detection limit (LOD) of 0.91 nM for miR-21 in just ~ 2 h. An acceptable discrimination was achieved between the target miRNA and other non-target nucleic acids (ssDNA, dsDNA, ssRNA, DNA-RNA, miR-122, miR-205, and single central- or terminal-base mismatched sequences). The biosensor was applied to the analysis of miR-21 from total RNA (RNAt) extracted from epithelial non-tumorigenic and adenocarcinoma breast cells without target amplification, pre-concentration, or reverse transcription steps. The versatility of the methodology due to the ZFP's non-sequence-specific binding behavior makes it easily extendable to determine any target RNA only by modifying the biotinylated detector probe.

The evolution of human diseases involves a highly dynamic and interactive system of multiple layers of molecular markers. Therefore, it is now fully accepted that simultaneous analysis of molecular markers from the same or different omics layers leads to novel strategies for their realistic early detection and monitoring, thus improving disease prevention, treatment, and outcomes. In this regard, features such as versatility for profiling multiple and/or multi-omics biomarkers, simplicity, affordable cost, markedly shorter analysis time and smaller sample amount required for analyses compared to conventional or state-of-the-art methodologies, make electrochemical bioplatforms suitable alternatives for routine determinations in clinical and basic research settings. The great advances demonstrated by electrochemical biosensors in recent years have gone hand in hand with the development of new electrochemical substrates, attractive surface chemistries, bioassay formats and amplification strategies, but also with the production and application of new bioreceptors, such as those provided by HaloTag and Phage Display technologies, which has allowed these devices to incur and demonstrate pioneering applications. With all this in mind, this communication will discuss bioelectroanalytical tools recently developed by us and our partners potentially transferable to the clinic due to their simplicity, cost, testing time and decentralized character, which have shown pioneering applications to decisively aid in personalized diagnostics by targeting methylation events in nucleic acids both globally and regionally and deregulated autoantibody expression. These biotools have shown to be able to determine simultaneously and with monobasic sensitivity the most common methylated bases in nucleic acids: 5-methylcytosine 5-mC), 5-hydroxymethylcytosine (5-hmC) and N6-methyladenine (6mA) in DNA and N6-methyladenosine (m6A) in RNA, both globally and regionally and without adopting other amplification strategies than conventional enzymatic labeling [1,2]. A multiplexed platform was proposed with application to the global determination of m6A in total RNA extracted from colorectal cancer cells with different metastatic potential, and to the determination of global methylations of 5-mC, 5-hmC and 6mA in genomic DNA extracted from matched tumor and healthy tissues of CRC patients. The obtained results demonstrated the clinical potential of the developed multiplexed bioplatform to discriminate the aggressiveness of tumor cells as well as tumor versus healthy tissues (accuracy, sensitivity, and specificity of 87.5, 100 and 75 %, respectively, using Ward's clustering method) in only 45 min from small amounts (no more than 100 ng) of denatured genomic DNA and crude total RNA without fragmentation and amplification. As for the electroanalytical bioplatforms developed for autoantibodies determination, they have shown ability to identify and confirm the clinical potential of previously undescribed specific autoantibody signatures against i) tumor-associated antigens, identified in the circulation or in tumor exosomes, for early and differential diagnosis of a given neoplasm [3,4]; ii) phage-derived and aberrant HaloTagged peptides to diagnose Alzheimer's disease (AD) [5]; and iii) SARS-CoV-2 serum-specific immunoglobulins to identify vulnerable population or with natural or acquired immunizations, allowing early detection of exaggerated immune system responses or epidemiological studies on the strain responsible for the infection and the efficacy of available vaccines against the different variants of the virus. It is exciting and rewarding to think that we are tackling and developing highly versatile technologies with the potential to efficiently provide relevant information on key aspects of known and unexpected diseases, such as their onset, severity, and immune response, enabling their management in a fast, simple, affordable, and personalized way in diverse settings. [1] Povedano et al. Anal. Chem. 92 (2020) 5604−5612. [2] Povedano et al. Anal. Chim. Acta 1182 (2021) 338946. https://doi.org/10.1016/j.aca.2021.338946. [3] Garranzo-Asensio et al. Theranostics 10(7) (2020) 3022–3034. [4] Montero-Calle et al. Engineering (2021) in press. https://doi.org/10.1016/j.eng.2021.04.026. [5] Valverde et al. Analysis & Sensing 1 (2021) 161-165. The financial support of PID2019-103899RB-I00 (Spanish Ministerio de Ciencia e Innovación), Research Project, PI17CIII/00045 and PI20CIII/00019 grants from the AES-ISCIII program grants from the AES-ISCIII program and the TRANSNANOAVANSENS-CM Program from the Comunidad de Madrid (Grant S2018/NMT-4349) are gratefully acknowledged.

While wearable and mobile chemical sensors have experienced tremendous growth over the past decade, their potential for tracking and guiding nutrition has emerged only over the past three years. Currently, guidelines from doctors and dietitians represent the most common approach for maintaining optimal nutrition status. However, such recommendations rely on population averages and do not take into account individual variability in responding to nutrients. Precision nutrition has recently emerged to address the large heterogeneity in individuals' responses to diet, by tailoring nutrition based on the specific requirements of each person. It aims at preventing and managing diseases by formulating personalized dietary interventions to individuals on the basis of their metabolic profile, background, and environmental exposure. Recent advances in digital nutrition technology, including calories-counting mobile apps and wearable motion tracking devices, lack the ability of monitoring nutrition at the molecular level. The realization of effective precision nutrition requires synergy from different sensor modalities in order to make timely reliable predictions and efficient feedback. This work reviews key opportunities and challenges toward the successful realization of effective wearable and mobile nutrition monitoring platforms. Non-invasive wearable and mobile electrochemical sensors, capable of monitoring temporal chemical variations upon the intake of food and supplements, are excellent candidates to bridge the gap between digital and biochemical analyses for a successful personalized nutrition approach. By providing timely (previously unavailable) dietary information, such wearable and mobile sensors offer the guidance necessary for supporting dietary behavior change toward a managed nutritional balance. Coupling of the rapidly emerging wearable chemical sensing devices-generating enormous dynamic analytical data-with efficient data-fusion and data-mining methods that identify patterns and make predictions is expected to revolutionize dietary decision-making toward effective precision nutrition.

This work reports the first bioplatform able to determine electrochemically 5-hydroxymethylcytosine (5-hmC) methylation events at localized sites and single-base sensitivity. The described bioplatform relies on a specific antibody (anti-5-hmC), further conjugated with commercial bioreagents loaded with multiple horseradish peroxidase (HRP) molecules, recognizing the epimark in a target DNA, captured through hybridization onto streptavidin-magnetic microbeads (Strep-MBs) modified with a complementary DNA capture probe. The electrochemical detection is performed by amperometry (-0.20 V vs Ag pseudoreference electrode) at disposable screen-printed carbon electrodes (SPCEs) in the presence of H2O2/hydroquinone (HQ) upon magnetic capture of the modified MBs onto the SPCE. The use of the commercial bioreagents ProtA-polyHRP80 and Histostar, very scarcely explored so far in electrochemical biosensors, provides high sensitivities for a synthetic target DNA sequence with a unique 5-hmC in the promoter region of MG...

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.

Edible electrochemical biosensors with remarkable prolonged resistance to extreme acidic conditions are described for direct glucose sensing in gastrointestinal (GI) fluids of different pH ranges and compositions. Such direct and stable glucose monitoring is realized using carbon-paste biosensors prepared from edible materials, such as olive oil and activated charcoal, shown to protect the activity of the embedded glucose oxidase (GOx) enzyme from strongly acidic conditions. The enzymatic resistance to low-pH deactivation allowed performing direct glucose monitoring in strong acidic environments (pH 1.5) over a 90-min period, while the response of conventional screen-printed (SP) biosensors decreased significantly following 10-min incubation in the same fluid. The developed edible biosensor displayed a linear response between 2 and 10 mM glucose with sensitivity depending on the pH of the corresponding GI fluid. In addition, coating the electrode surface with pH-responsive enteric coatings (Eudragit® L100 and Eudragit® E PO), of different types and densities, allows tuning the sensor activation in gastric and intestinal fluids at specific predetermined times. The attractive characteristics and sensing performance of these edible electrochemical biosensors, along with their pH-responsive actuation, hold considerable promise for the development of ingestible devices towards the biosensing of diverse target analytes after prolonged incubation in challenging body fluids. Graphical Abstract Edible biosensors allow direct electrochemical sensing in different gastrointestinal fluids and display remarkable prolonged resistance to extreme acidic conditions.

Transient polymeric coatings with a programmable transiency behavior are used for delayed exposure of fresh surfaces of multi-electrode sensor arrays at preselected times. Such delayed sensor actuation is shown to be extremely attractive for addressing severe biofouling characteristic of electrochemical biosensors in complex biofluids. Controlled coating dissolution and tunable sequential actuation of the individual sensing electrodes are achieved by tailoring the characteristics of the coating (density and thickness). The unique features offered by these delayed sensors allowed direct glucose monitoring in untreated blood and saliva samples over prolonged periods. This attractive delayed-sensor exposure concept, offering time-tunable sequential activation of multiple sensors with remarkable anti-biofouling properties, indicates considerable promise for operating sensors continuously in complex body fluids.

This work describes the first electrochemical immunosensor reported for the determination of IL-13 receptor Rα2 (IL-13Rα2), an emerging relevant biomarker in metastatic colon cancer. The approach involves the formation of sandwich immunocomplexes using specific capture (CAb) and biotinylated detector antibodies (BDAb) further labeled with an streptavidin-horseradish peroxidase (Strep-HRP) polymer, onto carboxylic acid-modified magnetic microbeads (HOOC-MBs). Amperometric detection at disposable carbon screen-printed electrodes (SPCEs) using the (H2O2)/hydroquinone (HQ) system was employed to monitor the affinity reactions. The developed immunosensor exhibits a linear calibration plot over the 3.9–100 ng mL−1 concentration range, a LOD of 1.2 ng mL−1 and excellent selectivity against other non-target proteins. The amperometric immunosensor was applied successfully to quantify for the first time the IL-13Rα2 expression in raw lysates of colon cancer cells and to discriminate the metastatic potential of intact cells through recognition of this target extracellular receptor. In comparison with the commercial Enzyme-Linked ImmunoSorbent Assay (ELISA) kit involving the same immunoreagents, the immunosensor provides a similar LOD in a half-time for the assay.

We report a rapid and sensitive electrochemical strategy for the detection of gene-specific 5-methylcytosine DNA methylation. Magnetic beads (MBs) modified with an antibody for 5-methylcytosines (5-mC) are used for the capture of any 5-mC methylated single-stranded (ss)DNA sequence. A flanking region next to the 5-mCs of the captured methylated ssDNA is recognized by hybridization with a synthetic biotinylated DNA sequence. Amperometric transduction at disposable screen-printed carbon electrodes (SPCEs) is employed. The developed biosensor has a dynamic range from 3.9 to 500 pm and a limit of detection of 1.2 pm for the methylated synthetic sequence of the tumor suppressor gene O-6-methylguanine-DNA methyltransferase (MGMT) promoter region. The method is applied in the 45-min analysis of specific methylation in the MGMT promoter region directly in raw spiked human serum samples and in genomic DNA extracted from U-87 glioblastoma cells and paraffin-embedded brain tumor tissues without any amplification and pretreatment step.

This paper describes two different electrochemical affinity biosensing approaches for the simple, fast and bisulfite and PCR-free quantification of 5-methylated cytosines (5-mC) in DNA using the anti-5-mC antibody as biorecognition element. One of the biosensing approaches used the anti-5-mC as capture bioreceptor and a sandwich type immunoassay, while the other one involved the use of a specific DNA probe and the anti-5-mC as a detector bioreceptor of the captured methylated DNA. Both strategies, named for simplicity in the text as immunosensor and DNA sensor, respectively, were implemented on the surface of magnetic microparticles and the transduction was accomplished by amperometry at screen-printed carbon electrodes by means of the hydrogen peroxide/hydroquinone system. The resulting amperometric biosensors demonstrated reproducibility throughout the entire protocol, sensitive determination with no need for using amplification strategies, and competitiveness with the conventional enzyme-linked immunosorbent assay methodology and the few electrochemical biosensors reported so far in terms of simplicity, sensitivity and assay time. The DNA sensor exhibited higher sensitivity and allowed the detection of the gene-specific methylations conversely to the immunosensor, which detected global DNA methylation. In addition, the DNA sensor demonstrated successful applicability for 1 h-analysis of specific methylation in two relevant tumor suppressor genes in spiked biological fluids and in genomic DNA extracted from human glioblastoma cells. The financial support of the Spanish Ministerio de Economía y Competitividad CTQ2015-64402-C2-1-R and SAF2014-53209-R Research Projects, the PI17CIII/00045 research project from AESI and the NANOAVANSENS Program from the Comunidad de Madrid (S2013/MT-3029) and predoctoral contracts from the Spanish Ministerio de Economía y Competitividad (R.M. Torrente-Rodríguez and E. Povedano) and Universidad Complutense de Madrid (V. Ruiz-Valdepeñas Montiel) are also gratefully acknowledged. Sí

Currently, electrochemical nucleic acid-based biosensing methodologies involving hybridization assays, specific recognition of RNA/DNA and RNA/RNA duplexes, and amplification systems provide an attractive alternative to conventional quantification strategies for the routine determination of relevant nucleic acids at different settings. A particularly relevant objective in the development of such nucleic acid biosensors is the design of as many as possible affordable, quick, and simple methods while keeping the required sensitivity. With this aim in mind, this work reports, for the first time, a thorough comparison between 11 methodologies that involve different assay formats and labeling strategies for targeting the same DNA. The assayed approaches use conventional sandwich and competitive hybridization assays, direct hybridization coupled to bioreceptors with affinity for RNA/DNA duplexes, multienzyme labeling bioreagents, and DNA concatamers. All of them have been implemented on the surface of magnetic beads (MBs) and involve amperometric transduction at screen-printed carbon electrodes (SPCEs). The influence of the formed duplex length and of the labeling strategy have also been evaluated. Results demonstrate that these strategies can provide very sensitive methods without the need for using nanomaterials or polymerase chain reaction (PCR). In addition, the sensitivity can be tailored within several orders of magnitude simply by varying the bioassay format, hybrid length or labeling strategy. This comparative study allowed us to conclude that the use of strategies involving longer hybrids, the use of antibodies with specificity for RNA/DNA heteroduplexes and labeling with bacterial antibody binding proteins conjugated with multiple enzyme molecules, provides the best sensitivity.

Combating the ongoing COVID-19 pandemic has put the spotlight on nutritional support of the immune system through consumption of vitamins C and D. Accordingly, there are urgent demands for an effective on-the-spot multi-vitamin self-testing platform that monitors the levels of these immune-supporting micronutrients for guiding precision nutrition recommendations. Herein, we present a compact bioelectronic dual sensor chip aimed at frequent on-the-spot simultaneous monitoring of the salivary vitamin C and D dynamics. The new bioelectronic chip combines a new electrocatalytic vitamin C amperometric assay along with competitive vitamin D immunoassay on neighboring electrodes, to perform selective and cross-talk free detection of both vitamins in a 10-μL saliva sample within 25 min. The distinct vitamin C or D temporal profiles obtained for different individuals after vitamin supplementation indicate the potential of the new bioelectronic chip strategy for enhancing personalized nutrition towards guiding dietary interventions to meet individual nutrition needs and promote immune system health.

Early and reliable diagnostic of cancer is mandatory to increase patient survival, thus requiring efficient and reliable analytical methods for such a purpose. Within this context, different strategies implying the development of electrochemical biosensors for the sensitive, selective and rapid multiplexed biosensing of genetic or protein cancer-related biomarkers are addressed in this presentation. In particular, novel sensing platforms have been developed for the determination of miRs, interleukin (IL)-8 mRNA, IL-8 protein, and cancer specific receptors. The developed methodologies allow for the determination of the target analytes at clinically relevant levels in complex samples: cancer cells, human tissues cell lysates, serum and raw saliva and can be easily extended to the determination of other relevant biomarkers.

[Background and Purpose]: The humoral immune response in cancer patients can be used for early detection of the disease. Autoantibodies raised against tumor-associated antigens (TAAs) are promising clinical biomarkers for reliable cancer diagnosis, prognosis, and therapy monitoring. In this study, an electrochemical disposable multiplexed immunosensing platform able to integrate difficult- and easy-to-express colorectal cancer (CRC) TAAs is reported for the sensitive determination of eight CRC-specific autoantibodies., [Methods]: The electrochemical immunosensing approach involves the use of magnetic microcarriers (MBs) as solid supports modified with covalently immobilized HaloTag fusion proteins for the selective capture of specific autoantibodies. After magnetic capture of the modified MBs onto screen-printed carbon working electrodes, the amperometric responses measured using the hydroquinone (HQ)/H2O2 system were related to the levels of autoantibodies in plasma., [Results]: The biosensing platform was applied to the analysis of autoantibodies against 8 TAAs described for the first time in this work in plasma samples from healthy asymptomatic individuals (n=3), and patients with high-risk of developing CRC (n=3), and from patients already diagnosed with colorectal (n=3), lung (n=2) or breast (n=2) cancer. The developed bioplatform demonstrated an improved discrimination between CRC patients and controls (asymptomatic healthy individuals and breast and lung cancer patients) compared to an ELISA-like luminescence test., [Conclusions]: The proposed methodology uses a just-in-time produced protein in a simpler protocol, with low sample volume, and involves cost-effective instrumentation, which could be used in a high-throughput manner for reliable population screening to facilitate the detection of early CRC patients at affordable cost., This work was supported by the financial support of the PI17CIII/00045 grant from the AES-ISCIII program to R.B., cofounded by FEDER funds. The financial support of the CTQ2015-64402-C2-1-R (Spanish Ministerio de Economía y Competitividad) and RTI2018-096135-B-I00 (Ministerio de Ciencia, Innovación y Universidades) Research Projects and the TRANSNANOAVANSENS-CM Program from the Comunidad de Madrid (Grant S2018/NMT-4349) to P.Y-S., S.C. and JM.P are gratefully acknowledged. G.D. acknowledges the financial support of PI15/00246 grant of the FIS and Cátedra UAM-Roche en Medicina de Innovación. M.G-A. was supported by a contract of the Programa Operativo de Empleo Juvenil y la Iniciativa de Empleo Juvenil (YEI) with the participation of the Consejería de Educación, Juventud y Deporte de la Comunidad de Madrid y del Fondo Social Europeo. A.M-C. is a recipient of a FPU fellowship from the Ministerio de Educación, Cultura y Deporte. G.S-F. is recipient of a predoctoral contract (grant number 1193818N) supported by The Flanders Research Foundation (FWO). Predoctoral contracts from the Spanish Ministerio de Economía y Competitividad (E.P.) and Universidad Complutense de Madrid (V.R.V-M.) are also gratefully acknowledged.

Alpha-lactalbumin (α-LA) is one of the whey proteins in cows' milk that has been identified as allergenic. In this work, we present, for the first time, a very sensitive magnetic beads (MBs)-based immunosensor for the determination of α-LA. A sandwich configuration involving selective capture and horseradish peroxidase-labeled detector antibodies was implemented on carboxylic acid-modified magnetic beads, captured magnetically under the surface of a disposable screen-printed carbon electrode for amperometric detection using the hydroquinone (HQ)/H2O2 system. The α-LA immunosensor exhibited a wide linear range (37.0-5000pg/ml), a low limit of detection (LOD, 11.0pg/ml) and noteworthy selectivity against other non-target proteins. The MBs-based immunosensing platform was applied successfully for the determination of α-LA in several varieties of milk (raw and UHT cows' milk as well as human milk) and infant formulations. The results were corroborated with those obtained using a commercial ELISA method, thereby substantiating the analytical merits of this unique method.

A highly sensitive disposable amperometric magnetoimmunosensor for the rapid determination of Ara h 2 protein, one of the major peanut allergens, is reported. The approach uses a sandwich configuration involving selective capture and detector antibodies and carboxylic acid-modified magnetic beads (HOOC-MBs). Detector antibodies are labeled with HRP-conjugated secondary antibodies and the MBs bearing the immunoconjugates are magnetically captured on surface of a disposable screen-printed carbon electrode (SPCE). The affinity reactions are monitored amperometrically at −0.20 V (vs a Ag pseudo-reference electrode) in the presence of hydroquinone (HQ) as electron transfer mediator and upon addition of H2O2 as the enzyme substrate. The developed magnetoimmunosensor exhibits a wide range of linearity between 87 and 10,000 pg mL−1 Ara h 2 with a detection limit of 26 pg mL−1 as well as a great selectivity against other non-target proteins. The magnetoimmunosensing platform was successfully applied for the detection of Ara h 2 in different food extracts. After an appropriate sample dilution no matrix effects were observable. The developed methodology was able to detect trace amounts of the peanut allergen (0.0005% or 5.0 mg kg−1) in wheat flour spiked samples. The results correlated properly with those provided by a commercial ELISA kit.

A sensitive and rapid method for the determination of the clinically relevant biomarker human endoglin (CD105) in serum samples is presented, involving a magneto-actuated immunoassay and amperometric detection at disposable screen-printed carbon electrodes (SPCEs). Micro-sized magnetic particles were modified with a specific antibody to selectively capture the target protein which was further sandwiched with a secondary HRP-labeled antibody. The immunocomplexes attached to the magnetic carriers were amperometrically detected at SPCEs using the hydroquinone (HQ)/H2O2/HRP system. The magneto-actuated immunosensing platform was able to detect 5 pmoles of endoglin (in 25μL of sample, 0.2μM) in 30min providing statistically similar results to those obtained using a commercial ELISA kit for the determination of endogenous content of endoglin in human serum samples.

Microribonucleic acids (miRNAs) have been linked with various regulatory functions and diseases and constitute important targets in future medical diagnostics and prognostics. We report here a novel sensitive and rapid bioelectrochemical strategy for miRNA determination. This strategy involves the development of a sensing approach making use of magnetic beads (MBs) modified with a specific DNA-RNA antibody as capture bioreceptor and amperometric detection implying the H2O2/hydroquinone (HQ) system at disposable screen-printed carbon electrodes (SPCEs). The developed biosensor exhibits a dynamic range from 8.2 to 250 pM and a detection limit of 2.4 pM (60 amol) of a synthetic target without any amplification step in 2 h. The usefulness of the approach was evaluated by analyzing total RNA (RNAt) extracted from metastatic cancer cell lines and human tumor tissues, which demonstrated its potential to perform determination of mature miRNAs in these complex samples. Moreover, the feasibility of the developed me...

This paper reports the preparation of versatile electrochemical biosensing platforms for the simple, rapid, and PCR-independent detection of the most frequent DNA methylation marks (5-methylcytosine, 5-mC, and/or 5-hydroxymethylcytosine, 5-hmC) both at global and gene-specific levels. The implemented strategies, relying on the smart coupling of immuno-magnetic beads (MBs), specific DNA probes and amperometric detection at screen-printed carbon electrodes (SPCEs), provided sensitive and selective determination of the target methylated DNAs in less than 90 min with a great reproducibility and demonstrated feasibility for the simultaneous detection of the same or different cytosine epimarks both at global level and in different loci of the same gene or in different genes. The bioplatforms were applied to determine global methylation events in paraffin-embedded colorectal tissues and specific methylation at promoters of tumor suppressor genes in genomic DNA extracted from cancer cells and paraffin-embedded colorectal tissues, and in serum without previous DNA extraction from cancer patients.

This paper reports the development of an amperometric immunosensing platform for the determination of cadherin-17 (CDH-17), an atypical adhesion protein involved in the progression, metastatic potential, and survival of high prevalence gastric, hepatocellular, and colorectal tumors. The methodology developed relies on the efficient capture and enzymatic labeling of the target protein on the magnetic microparticles (MBs) surface using commercial antibodies and amperometric transduction at screen-printed carbon electrodes (SCPEs) through the HRP/H2O2/HQ system. The developed immunosensing platform allows the selective determination of the target protein at low ng mL-1 level (LOD of 1.43 ng mL-1) in 45 min and using a single incubation step. The electrochemical immunosensor was successfully used for the accurate determination of the target protein in a small amount (0.5 μg) of raw lysates of colon cancer cells with different metastatic potential as well as in extracts from paraffin embedded cancer colon tissues of different metastatic grade., The financial support of the Spanish Ministerio de Economía y Competitividad, Grants CTQ2015-70023-R and CTQ2015-64402-C2-1-R, and the NANOAVANSENS Program from the Comunidad de Madrid (Grant S2013/MT-3029), and predoctoral contracts from the Spanish Ministerio de Economia y Competitividad (E.P.) and Universidad Complutense de Madrid (V.R.-V.M.) are grate fully acknowledged. R.B. acknowledges the financial support of the PI17CIII/00045 Grant from the AES-ISCIII program. M.G.-A. was supported by a contract of the Programa Operativo de Empleo Juvenil y la Iniciativa de Empleo Juvenil (YEI) with the participation of the Consejería de Educacion, Juventud y Deporte de la Comunidad de Madrid y del Fondo Social Europeo.

This work reports an amperometric biosensor for the determination of miRNA-21, a relevant oncogene. The methodology involves a competitive DNA-target miRNA hybridization assay performed on the surface of magnetic microbeads (MBs) and amperometric transduction at screen-printed carbon electrodes (SPCEs). The target miRNA competes with a synthetic fluorescein isothiocyanate (FITC)-modified miRNA with an identical sequence for hybridization with a biotinylated and complementary DNA probe (b-Cp) immobilized on the surface of streptavidin-modified MBs (b-Cp-MBs). Upon labeling, the FITC-modified miRNA attached to the MBs with horseradish peroxidase (HRP)-conjugated anti-FITC Fab fragments and magnetic capturing of the MBs onto the working electrode surface of SPCEs. The cathodic current measured at −0.20 V (versus the Ag pseudo-reference electrode) was demonstrated to be inversely proportional to the concentration of the target miRNA. This convenient biosensing method provided a linear range between 0.7 and 10.0 nM and a limit of detection (LOD) of 0.2 nM (5 fmol in 25 μL of sample) for the synthetic target miRNA without any amplification step. An acceptable selectivity towards single-base mismatched oligonucleotides, a high storage stability of the b-Cp-MBs, and usefulness for the accurate determination of miRNA-21 in raw total RNA (RNAt) extracted from breast cancer cells (MCF-7) were demonstrated.

Cancer is the second leading cause of death after cardiovascular diseases and responsible for over 8 million deaths worldwide. [...]

In this work, a new automated bioanalyzer based on the use of enzymatic biosensors as amperometric detectors is reported. This automatic bioanalyzer is configurable both as continuous flow and flow injection analysis systems and enables both on-line and off-line monitoring of ethanol in low-alcohol beer to be performed. The attractive analytical and operational characteristics demonstrated by the automated bioanalyzer make it a promising, simple, rapid, and reliable tool for quality control of this beverage in the beer industry, either during the manufacturing process or in the final product. Moreover its applicability to the analysis of the ethanol content in different non-alcoholic beers working at different modes was successfully demonstrated.

Autoantibodies raised against tumor-associated antigens have shown high promise as clinical biomarkers for reliable diagnosis, prognosis, and therapy monitoring of cancer. An electrochemical disposable biosensor for the specific and sensitive determination of p53-specific autoantibodies has been developed for the first time in this work. This biosensor involves the use of magnetic microcarriers (MBs) modified with covalently immobilized HaloTag fusion p53 protein as solid supports for the selective capture of specific autoantibodies. After magnetic capture of the modified MBs onto screen-printed carbon working electrodes, the amperometric signal using the system hydroquinone/H2O2 was related to the levels of p53-autoantibodies in the sample. The biosensor was applied for the analysis of sera from 24 patients with high-risk of developing colorectal cancer and 6 from patients already diagnosed with colorectal (4) and ovarian (2) cancer. The developed biosensor was able to determine p53 autoantibodies with a...

A novel electrochemical disposable nucleic acid biosensor for simple, rapid, and specific detection of adulterations with horsemeat is reported in this work. The biosensing platform involves immobilization of a 40-mer RNA probe specific for a characteristic fragment of the mitochondrial DNA D-loop region of horse onto the surface of magnetic microcarriers. In addition, signal amplification was accomplished by using a commercial antibody specific to RNA/ DNA duplexes and a bacterial protein conjugated with a horseradish peroxidase homopolymer (ProtA-HRP40). Amperometric detection at −0.20 V vs Ag pseudoreference electrode was carried out at disposable screen-printed carbon electrodes. The methodology achieved a limit of detection (LOD) of 0.12 pM (3.0 attomoles) for the synthetic target and showed ability to discriminate between raw beef and horsemeat using just 50 ng of total extracted mitochondrial DNA (∼16 660 bp in length) without previous fragmentation. The biosensor also allowed discrimination between 100% raw beef and beef meat samples spiked with only 0.5% (w/w) horse meat (levels established by the European Commission) using raw mitochondrial lysates without DNA extraction or polymerase chain reaction (PCR) amplification in just 75 min. These interesting features made the developed methodology an extremely interesting tool for beef meat screening, and it can be easily adapted to the determination of other meat adulterations by selection of the appropriate specific fragments of the mitochondrial DNA region and capture probes.

New amperometric immune-scaffolds for the rapid, sensitive and reliable determination of [...]

A disposable amperometric sensor using magnetic microcarriers has been designed and implemented to be used in combination with the so called Express PCR to detect the presence of hazelnut traces in foodstuffs through the detection of Cor a 9 allergen coding sequence. The developed procedure involves the use of streptavidin-modified magnetic microbeads (Strep-MBs), specific biotinylated capture and detector probes which hybridize with a specific region of the gene encoding the protein Cor a 9, and appropriate primers for PCR amplification. A 50-mer synthetic target DNA or unmodified 100-bp PCR products were selective captured via sandwich hybridization with capture probe modified MBs and biotinylated signaling probes. The resulting biotinylated hybrids were coupled with a commercial streptavidin–peroxidase (Strep-HRP) conjugate and the final modified MBs were magnetically captured onto a screen-printed carbon electrode to perform amperometric detection using the H2O2/HQ system. A LOD of 0.72�pM was obtained for the synthetic target and the applicability studies demonstrated the possibility to detect the denatured PCR amplified samples obtained using only 20�pg of genomic DNA extracted from hazelnut. RSD values obtained, below 10% in all cases, confirmed the good reliability of extraction, amplification and quantification protocols involved in the developed methodology. The strict specificity of the designed primers and selected probes for hazelnut was demonstrated by performing PCR amplification of genomic DNA extracted from different hazelnut varieties and other species of similar families (pistachio, cashew, walnut and tangerine) and analyzing the resultant amplicons by the developed electrochemical sensor. The reliable and sensitive results achieved indicate that Express PCR in conjunction with an electrochemical DNA sensor, used for the first time in this work, provides a suitable sensitive, specific, and cost-effective method for routine food allergens determinations, particularly useful for resource-limited settings. � 2017 Elsevier B.V.

Food safety plays an important role in public health and thus to society as a whole. Food may become unsafe due to the presence of allergens and adulterants amongst others and hence the determination of these analytes has gained great relevance in quality control and safety of food. Electrochemical biosensor devices are emerging as one of the foremost relevant techniques for monitoring food allergens and adulterants due to their quick, specific, sensitive and reliable performances, ease of mass fabrication, economics and field applicability. These electrochemical biosensors, based on the use of existing or recently developed bioreceptors in combination with nanomaterials and surface fabrication techniques are able to offer attractive and efficient platforms which may become a viable option for the development of simpler, faster, cheaper and more robust and reliable analytical methods for the detection of allergens and adulterants in food. This Chapter describes recent analytical strategies developed so far using electrochemical biosensors for the determination of potentially hazardous adulterants and allergenic food residues to ensure food safety. The main progresses achieved to date are presented, highlighting general considerations and potential prospects for the future. The variety of electrochemical biosensors that have appeared in recent years shows that it is a booming research area with still many challenges but also great opportunities to develop sensitive, reliable, robust, and cost effective food allergens and adulterants biosensing methodologies.