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An electrochemical biosensing platform for serum autoantibodies (AAbs) detection is reported in this work, exploiting for the first time six Alzheimer's disease (AD)-specific phage-derived and frameshift aberrant HaloTag peptides as receptors, immobilized on magnetic microbeads (MBs) surface and captured on disposable electrodes to perform amperometric detection. Operational analytical characteristics and clinical diagnostic ability of the bioplatform were probed in optimized key experimental conditions by analysing serum AAbs of AD patients and healthy subjects. The value of 100 % obtained for AUC, sensitivity, and selectivity from the all peptides combined ROC curve, indicate full AD-diagnostic capability of the methodology, which was further implemented, as proof of concept, in a POC multiplexing platform to detect the signature in a single test over clinically actionable times (1 h 15 min), opening great promise for the type of diagnosis and AD patients’ monitoring follow-up currently pursued. The financial support of RTI2018-096135-B−I00 (Spanish Ministerio de Ciencia, Innovación y Universidades) and PID2019-103899RB−I00 (Spanish Ministerio de Ciencia e Innovación), Research Projects, PI17CIII/00045 and PI20CIII/00019 grants from the AES-ISCIII program and the TRANSNANOAVANSENS-CM Program from the Comunidad de Madrid (Grant S2018/NMT-4349) are gratefully acknowledged. A. Montero-Calle and P. San Segundo-Acosta acknowledge the support of the FPU predoctoral contracts by the Spanish Ministerio de Educación, Cultura y Deporte. A. Valverde acknowledges a predoctoral contract from Complutense University of Madrid. B. Arévalo acknowledges a predoctoral contract from the Spanish Ministerio de Economía y Competitividad (PRE2019-087596). G.S-F. is a recipient of a predoctoral contract (grant number 1193818N) supported by The Flanders Research Foundation (FWO). Sí

Due to their widespread use in veterinary medicine, the presence of tetracyclines (TCs) and their metabolites in foodstuffs from treated animal represents a growing concern for human health and nutrition and, therefore, many countries have established maximum levels for their presence in foods. The compliance with these legislations requires sensitive and selective methods for the determination of this antibiotics’ family in different matrices. In this sense, electrochemical sensors and biosensors are competitive methodologies versus other approaches commonly used for this purpose such as chromatography and ELISA methods, mainly in terms of low-cost equipment, minimal sample treatment, reduced turnaround time, and compatibility with multiplexed and point-of-care determinations. With this background in mind, this article reviews in a general but comprehensive way recent contributions of electrochemical (bio)sensors developed for the determination of TCs and applied to TC analysis in environmental samples, food, and biological matrices. The highlighted representative methods show the key role played both by the materials (mostly nanomaterials and polymers) employed to impart surface electrocatalytic properties and significant signal amplification, and by different (bio)receptors to provide electrochemical sensing with the sensitivity and selectivity demanded by the determination of TCs in environmental, clinical, and food samples. Main challenges to overcome and future prospects to turn over all the benefits of these simple, rapid, sensitive, selective, and cost effective electroanalytical (bio)tools in single or multiplexed TCs analysis, even at decentralized settings and after minimal sample treatment, are also pointed out.

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.

Nowadays, materials, solvents, and techniques are being used in accordance with the principles of green analytical chemistry for the development and application of electrochemical (bio)sensors. Electrodes prepared from nontoxic materials, eco-friendly solvents and reagents, and methodologies that make it possible the reduction of the sample size and the amount of waste products are being used with the main objective of minimizing the environmental impact. These aspects are briefly discussed in this short review through addressing the state of the art of electrochemical (bio)sensors in connection with the green chemistry. The relevant aspects related to the different items are considered with some illustrative examples taken from the recent literature. With the aim of providing suitable complete information, a table summarizing the fundamentals and analytical characteristics of some configurations is included in the Supporting Information .

Nowadays, there is increasing demand to overcome the major constraints involved in cumbersome and protracted procedures used in conventional laboratory methods for molecular detection and develop methodologies suitable to near real-time, continuous, and direct monitoring of the target analyte in the analyzed sample at decentralized settings. In this context, biosensors using electrochemistry as transduction technique are particularly attractive to monitor binding-induced changes in the rigidity of a redox-tagged nucleic acid or peptide-switching probes (switch-based biosensors) or a capture antibody attached to an interrogating electrode. Sensors based on biomolecular switches, DNAs, aptamers, and peptides that reversibly shift between two or more conformations (or conformational ensembles) in response to the binding of a specific target ligand have been applied to the determination of a wide range of relevant analytes (nucleic acids, proteins, small molecules, and ions) and are rapid (responding in minutes), sensitive, reagentless, easily reusable, and less prone to fouling issues. They are capable to report in near real time the target binding event without any subsequent processing step, directly in challenging samples or continuously in flowing samples, and even in vivo using affordable and miniaturization-compatible instrumentation. Only a few redox-labeled capture antibody–based immunosensing strategies have been reported to date for the reagentless and continuous electrochemical determination of relevant analytes.

Harmful algal blooms (HABs) are more frequent as climate changes and tropical toxic species move northward, especially along the Iberian Peninsula, a rich aquaculture area. Monitoring programs, detecting the presence of toxic algae before they bloom, are of paramount importance to protect ecosystems, aquaculture, human health and local economies. Rapid, reliable species identification methods using molecular barcodes coupled to biosensor detection tools have received increasing attention as an alternative to the legally required but impractical microscopic counting-based techniques. Our electrochemical detection system has improved, moving from conventional sandwich hybridization protocols using different redox mediators and signal probes with different labels to a novel strategy involving the recognition of RNA heteroduplexes by antibodies further labelled with bacterial antibody binding proteins conjugated with multiple enzyme molecules. Each change has increased sensitivity. A 150-fold signal increase has been produced with our newest protocol using magnetic microbeads (MBs) and amperometric detection at screen-printed carbon electrodes (SPCEs) to detect the target RNA of toxic species. We can detect as few as 10 cells L&minus, 1 for some species by using a fast (~2 h), simple (PCR-free) and cheap methodology (~2 EUR/determination) that will allow this methodology to be integrated into easy-to-use portable systems.

The preparation of an integrated biosensor for the easy, fast, and sensitive determination of miRNAs is described based on a direct hybridization format and a label-free voltammetric detection. The biosensor involves a disposable carbon electrode substrate doubly nanostructured with reduced graphene oxide (rGO) and AuNPs modified with pyrene carboxylic acid (PCA) and 6-ferrocenylhexanethiol (Fc-SH), respectively. A synthetic amino terminated DNA capture probe was covalently immobilized on the CO2H moieties of PCA/rGO, while Fc-SH was used as a signaling molecule. Differential pulse voltammetry was employed to record the decrease in the oxidation peak current of Fc after the hybridization due to the hindering of the electron transfer upon the formation of the DNA-RNA duplex on the electrode surface. The stepwise biosensor preparation was characterized by surface and electrochemical techniques showing the role played by each biosensor component as well as the reliability of the target miRNA determination. The determination of the oncogene miRNA-21 synthetic target allowed quantification in the low femtomolar range (LOD of 5 fM) with a high discrimination of single-base mismatched sequences in a single 30-min incubation step. The bioplatform allowed the determination of the target miRNA in a small amount of total RNA extracted from breast cancer (BC) cells or directly in serum samples collected from BC patients without the need for prior extraction, purification, amplification, or reverse transcription of the genetic material and with no matrix effect. Graphical abstract.

Over the past decade, artificial nanomaterials that exhibit properties similar to those of enzymes are gaining attraction in electrochemical biosensing as highly stable and low-cost alternatives to enzymes. This review article discusses the main features of the various nanomaterials (metal oxide, metal, and carbon-based materials) explored so far to mimic different kinds of enzymes. The unprecedented opportunities imparted by these functional nanomaterials or their nanohybrids, mostly providing peroxidase-like activity, in electrochemical affinity biosensing are critically discussed mainly in connection with their use as catalytic labels or electrode surface modifiers by highlighting representative strategies reported in the past 5 years with application in the food, environmental, and biomedical fields. Apart from outlining the pros and cons of nanomaterial-based enzyme mimetics arising from the impressive development they have experienced over the last few years, current challenges and future directions for achieving their widespread use and exploiting their full potential in the development of electrochemical biosensors are discussed. Graphical abstract.

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.

This paper reports a dual electrochemical biosensor involving carboxylated- or neutravidin-functionalized magnetic microbeads and dual screen-printed carbon electrodes for the simultaneous determination of rheumatoid factor (RF) and anti-cyclic citrullinated peptide (CCPA) autoantibodies used as biomarkers for the detection of rheumatoid arthritis autoimmune disease. Sandwich-type biosensors involving Fc fragments of IgG Fc(IgG) and biotinylated cyclic cytrullinated peptide (CCP-biotin) to form CCP-biotin-Neutr-MBs for the specific immobilization of RF and CCPA, respectively, as well as conjugation with HRP-IgM and HRP-IgG for RF and CCPA, respectively, were prepared. Amperometric detection was performed at -0.20 V vs. Ag pseudo-reference electrode using the H2O2/hydroquinone (HQ) system upon capturing the bioconjugates onto the corresponding working electrode (WE1 or WE2) of SPCdEs. The dual biosensor exhibits high sensitivity for RF and CCPA with LOD values of 0.8 and 2.5 IU mL-1, respectively. The simultaneous determination can be completed in about two hours using a simple protocol and a sample volume (25 μL) four times smaller than that required by the ELISA method. The dual electrochemical biosensor was used for the determination of both target biomarkers in human serum.

Two different disposable amperometric biosensors for the rapid detection and quantification of Brettanomyces bruxellensis (Brett) and total yeast content in wine were constructed. The biosensing approach implied the use of magnetic capturing elements based on core-shell Fe3O4@SiO2 superparamagnetic nanoparticles (NanoCaptors) functionalized with specific bioreceptors: Concanavalin A (Con A) for total yeast analysis, and anti-Brett polyclonal antibodies for Brett. A Con A-peroxidase conjugate and carbon screen printed electrodes were used as signaling element and sensing interfaces, respectively. The biosensor based on antibody-modified magnetic nanoparticles allowed the amperometric detection of B. bruxellensis in buffer solution and red wine samples in the range of 10 - 106 CFU/mL, with low detection limits of 6 CFU/mL and 8 CFU/mL, respectively. In addition, the use of Con A-linked magnetic nanoparticles as capturing elements allowed determination of total yeast content in buffer solution and red wine in the range of 10 - 106 CFU/mL, with detection limit of 5 CFU/mL. These electrochemical biosensors also exhibited high reproducibility, selectivity and storage stability.

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.

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.

A sensitive and disposable amperometric immunosensor for Saccharomyces cerevisiae was constructed by using carbon screen-printed electrodes modified with propionic acid-functionalized graphene oxide as transduction element. The affinity-based biosensing interface was assembled by covalent immobilization of a specific polyclonal antibody on the carboxylate-enriched electrode surface via a water-soluble carbodiimide/N-hydroxysuccinimide coupling approach. A concanavalin A-peroxidase conjugate was further used as signaling element. The immunosensor allowed the amperometric detection of the yeast in buffer solution and white wine samples in the range of 10–107 CFU/mL. This electroanalytical device also exhibited low detection limit and high selectivity, reproducibility, and storage stability. The immunosensor was successfully validated in spiked white wine samples.

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.

This review overviews and revalues the role that the electrochemical techniques and the continuously evolving electrode designs play in the tremendous progress experienced by electrochemical biosensing. These aspects are scarcely covered in the recent literature on electrochemical biosensors and are discussed in this article through selected representative examples together with other approaches involving the use of signal amplification strategies, electrocatalytic materials, ratiometric designs or antifouling chemistries. Regarding electrodes, the continuous advances made in screen-printed, micro-, paper-based and flexible and wearable materials are critically overviewed. Concerning electrochemical techniques, the use of selected widely known techniques (electrochemical impedance spectroscopy, stripping voltammetry, and potentiometry) to successfully address different applications in response to current biosensing challenges is highlighted through their smart coupling with new experimental designs, bioreceptors and materials.

This review article sheds useful insight in the use of Janus nanoparticles for (bio)sensing in connection with optical and electrochemical transduction. After a brief introduction of the main properties, types and fabrication strategies of Janus nanoparticles, selected applications for their use in electrochemical and optical biosensing are critically discussed. Highlighted examples illustrate the great versatility and interesting possibilities offered by these smart multifunctional nanoparticles for (bio)sensing of relevant analytes operating both in static and dynamic modes. Progress made so far demonstrate their suitability for designing single- or multiplexed (bio)sensing strategies for target analytes of different nature (organic and inorganic compounds, proteins, cells and oligomers) with relevance in clinical (H2O2, glucose, cholesterol, CEA, human IgG, propranolol, bacterial and tumor cells) and environmental (lead and organophosphorous nerve agents) fields. Key future challenges and envisioned opportunities of the use of Janus nanoparticles in the (bio)sensing field are also discussed.

The assembly of a novel disposable amperometric immunosensor for the detection of the red wine spoilage yeast Brettanomyces bruxellensis is reported. The nanostructured sensing interface was prepared by first coating carbon screen printed electrodes with a gold nanoparticles-reduced graphene oxide hybrid nanomaterial, which was then modified with 3-mercaptopropionic acid to further immobilize specific antibodies for B. bruxellensis via a carbodiimide-coupling reaction. The functionalized electrode allowed the amperometric detection of B. bruxellensis in buffered solutions and red wine samples in the range of 10–106 CFU/mL and 102–106 CFU/mL, with low detection limits of 8 CFU/mL and 56 CFU/mL, respectively. The electrochemical immunosensor also exhibited high reproducibility, selectivity, and storage stability.

Made available in DSpace on 2018-11-26T15:37:44Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-03-15 Spanish Ministry of Economy and Competitiveness Comunidad de Madrid, Programme NANOAVANSENS A novel nanocomposite material consisting of reduced graphene oxide/Rh nanoparticles was prepared by a one-pot reaction process. The strategy involved the simultaneous reduction of RhCl3 and graphene oxide with NaBH4 and the in situ deposition of the metal nanoparticles on the 2D carbon nanomaterial planar sheets. Glassy carbon electrode coated with this nanocomposite was employed as nanostructured support for the cross-linking of the enzyme laccase with glutaraldehyde to construct a voltammperometric biosensor for 17 beta-estradiol in the 0.9-11 pM range. The biosensor showed excellent analytical performance with high sensitivity of 25.7 A mu M-1 cm(-1), a very low detection limit of 0.54 pM and high selectivity. The biosensor was applied to the rapid and successful determination of the hormone in spiked synthetic and real human urine samples. (C) 2016 Elsevier B.V. All rights reserved. Univ Complutense Madrid, Fac Chem, Dept Analyt Chem, E-28040 Madrid, Spain State Univ Sao Paulo, Inst Chem, POB 780, BR-13560970 Sao Carlos, SP, Brazil Mackenzie Presbiterian Univ, Engn Sch, BR-01302907 Sao Paulo, SP, Brazil State Univ Sao Paulo, Inst Chem, POB 780, BR-13560970 Sao Carlos, SP, Brazil Spanish Ministry of Economy and Competitiveness: CTQ2014-58989-P Spanish Ministry of Economy and Competitiveness: CTQ2011-24355 Spanish Ministry of Economy and Competitiveness: CTQ2015-71936-REDT Spanish Ministry of Economy and Competitiveness: CTQ2012-34238 Comunidad de Madrid, Programme NANOAVANSENS: S2013/MIT-3029

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.

A disposable enzyme−free biosensing platform for the sensitive and selective voltammetric determination of miRNAs is reported. The bioplatform implies a sandwich−type hybridization configuration involving the use of two synthetic DNA probes that hybridize contiguously with the target miRNA−21. A thiolated capture probe was immobilized through thiol chemistry on disposable carbon electrodes modified with a hybrid nanomaterial composed of reduced graphene oxide (rGO) and gold nanoparticles (AuNPs). A biotinylated detection probe was conjugated with ferrocene-capped AuNPs modified with streptavidin (Fc−AuNPs−Strep) which were used as labeling nanocarriers. The extent of the hybridization event was followed by differential pulse voltammetric measurement of the Fc oxidation peak. Under the optimized conditions, the developed biosensor provides attractive characteristics for the determination of the synthetic target miRNA, with a linear range between 10 fM and 2 pM and a limit of detection (LOD) of 5 fM, fully discrimination towards a highly homologous miRNA (with just one mismatched base) and a storage stability of at least two months. The biosensor was able to determine accurately the target miRNA directly in scarcely diluted serum from breast cancer (BC) patients with no need for a previous total RNA (RNAt) extraction and in a very small amount of RNAt extracted from breast adenocarcinoma cells without the need for amplification or reverse transcription to complementary DNA.

[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.

A novel and disposable electrochemical biosensor for PCR-free and selective detection of Sola l 7, a non-specific lipid transfer protein (nsLTP) found in tomato seeds associated to severe symptoms of tomato-allergic patients, is reported in this work. The methodology involves the formation of DNA/RNA heterohybrids by sandwich hybridization of a specific fragment of the Sola l 7 allergen coding sequence with appropriate RNA probes designed and described for the first time in this work. Labeling was carried out with commercial antibodies specific to the heteroduplexes and secondary antibodies conjugated with HRP onto the surface of magnetic beads. Amperometric transduction was performed upon magnetic capture of the resulting magnetic bioconjugates on screen-printed electrodes using the system H2O2/HQ. A comparison of the sandwich hybridization format with a direct approach as well as between different labeling strategies was performed. The LOD value achieved was 0.2 pM (5 amol in 25 μL). The biosensor was successfully applied to the selective analysis of the targeted Sola l 7 specific region directly in just 100 ng of non-fragmented denatured genomic DNA extracted from tomato seeds., The financial support of the Spanish Ministerio de Economía y Competitividad Research Projects, CTQ2015-64402-C2-1-R, SAF2017-86483-R and the TRANSNANOAVANSENS Program from the Comunidad de Madrid (Grant P2018/NMT-4349) are gratefully acknowledged. M.F.B. is grateful to FCT grant SFRH/BPD/78845/2011,financed by POPH–QREN–Tipologia4.1–Formação Avançada, subsidized by Fundo Social Europeu and Ministério da Ciência, Tecnologiae Ensino Superior. The author M.A.P.B. is grateful to the authors J.M.R., M.F.B., S.C., J.M.P. for the scientific assistance and suggestions shared throughout the supervision of her Master’s project and Master’s thesis at University of Minho. The author M.A.P.B. also acknowledges the Department of Biology (DB) and the Centre of Molecular and Environmental Biology (CBMA) from University of Minho (UM), Braga,Portugal, by providing all the conditions leading to the Master's thesis in“Biophysics and Bionanosystems”. Financial support of M.A.P.B. forthis work was provided by a fellowship within the programme Erasmus+ scholarship/Portugal, which allowed performing the laboratorial work at the University Complutense of Madrid, Madrid, Spain. The author J.M.R. acknowledges CBMA-UM and DB-UM, Portugal, by the conditions provided. J.M.R. is also grateful to the Green Chemistry Laboratory (LAQV)–Research center Chemistry and Technology Network (REQUIMTE), and the Department of Chemistry and Biochemistry (DCB) from Faculty of Sciences, University of Porto(FCUP), Porto, Portugal, where currently he is researcher. J.M.R. acknowledges the financial support of the strategic programmes UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) and PTDC/SAUNUT/30448/2017 (POCI-01-0145-FEDER-030448) and M.F.B. the financial support of the projects PTDC/QUI-QAN/30735/2017 and UID/QUI/50006/2019, funded by national funds through Fundação para a Ciência e Tecnologia [Foundation for Science and Technology](FCT-I.P.)/Ministério da Ciência, Tecnologia e Ensino Superior [Ministry of Science, Technology and Higher Education] (MCTES), and Fundo Europeu de Desenvolvimento Regional [European RegionalDevelopment Fund] (FEDER), under the scope of the COMPETE2020–Programa Operacional Competitividade e Internacionalização[COMPETE2020–Competitiveness and InternationalizationOperational Program, POCI]. S.B. acknowledges the financial support from MINECO through the“Juan de la Cierva” program.

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.

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.

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.

Au nanoparticles modified with 4-mercaptopyridine and 6-mercapto-1-hexanol were used as coordination agents to prepare a novel hybrid nanomaterial with Ag:4,4'-bipyridine nanobelts. This nanohybrid was employed to modify glassy carbon electrodes and to construct a horseradish peroxidase-based mediatorless amperometric biosensor for H2O2. The electrode, poised at -100mV, exhibited a rapid response within 4s and a linear calibration range from 90pM to 6.5nM H2O2. The biosensor showed a high sensitivity of 283A/Mcm(2) and a very low detection limit of 45pM at a signal-to-noise ratio of 3. The enzyme biosensor showed high stability when stored at 4°C under dry conditions, retaining over 96% and 78% of its initial activity after 15 and 30days of storage at 4°C, respectively.

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...

An electrochemical magnetoimmunosensor for the determination of estrogen receptor α (ERα) protein in complex samples (serum and cell lysates) able to discriminate between ERα positive and negative breast cancer cells is reported. Specifically functionalized magnetic microbeads with sandwich immunocomplexes and amperometric detection at disposable screen-printed carbon electrodes (SPCEs) resulted in highly selective and sensitive ERα detection with a detection limit of 19 pg mL−1. This magnetoimmunosensing platform was successfully applied to the quantitation of ERα in spiked human serum and cell lysates samples without any matrix effect with an advantageous performance in terms of simplicity and assay times over commercial ELISA assays. The biosensor capability for assessing ERα in intact breast cancer cells makes it competitive with conventional strategies providing rapidly quantitative and reliable results on this relevant biomarker currently used in the clinical practice for diagnosis, follow-up and monitoring of metastatic breast cancer. Keywords: Magnetoimmunosensor, Amperometry, ERα, Lysates, Intact cancer cells, Breast cancer

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.

This work reports the first amperometric biosensor involving the use of neutravidin-functionalized magnetic microbeads (NA-MBs) modified with a biotinylated-anti-dsDNA (b-dsDNA) as efficient magnetic microcarriers to selectively capture anti-dsDNA autoantibodies (IgG, IgA and IgM AAbs) present in the sera of patients with rheumatoid arthritis (RA). Subsequently, the attached anti-dsDNA AAbs are detected with a mixture of conventional HRP-labeled secondary antibodies (HRP-anti-human IgG/IgM/IgA mixture). The biorecognition event is monitored by amperometric transduction using the hydroquinone (HQ)/H2O2 system upon capturing the modified MBs on the surface of screen-printed carbon electrodes (SPCEs). The developed bioplatform exhibits a linear calibration plot ranging from 1 to 200 IU mL−1 with a LOD of 0.3 IU mL−1 for anti-dsDNA AAbs standards. In addition, the biosensor allows performing the determination of the anti-dsDNA AAbs levels directly in 100-times diluted serum samples from patients diagnosed with RA and in just 75 min. The obtained results are in agreement with those provided by an ELISA kit and allow discrimination between positive and negative samples.

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.

International audience; This paper describes a disposable amperometric biosensor for simple and sensitive determination of miRNA-21. The bioplatform consists of gold nanoparticles-nanostructured electrode surfaces on which a direct hybridization assay involving the immobilization of a specific thiolated DNA capture probe and recognition of the formed DNA−miRNA-21 heteroduplex by a specific antibody is implemented. The antibody is further recognized through its Fc region by a commercial bacterial protein containing 40 units of horseradish peroxidase (HRP) (ProtA-polyHRP40). The amperometric detection of the hybridization process using the H 2 O 2 /hydroquinone system allows quantification of the target miRNA in the 0.096−25 pM linear range with a detection limit of 29 fM (0.29 amol in 10 μL of sample). The bioplatform offers excellent selectivity against noncomplementary sequences and very acceptable against sequences with an unpaired base (only 30% of the response obtained for the target miRNA). In addition, the bioplatform was shown to be useful for the determination of the endogenous content of the target oncomiR directly in blood serum from breast cancer patients and in breast cancer cells using only 10 ng of total extracted RNA.

Electrochemical nucleic-acid-based biosensing strategies involving the use of nanoparticles as electrode modifiers and as advanced labels are attractive options for the determination of substances that are relevant clinically, from an environmental perspective, and to food analysis, as these strategies are able to overcome some of the well-known limitations of conventional methodologies for routine applications. In this article, we provide a selective overview of current strategies for nucleic acid electrochemical biosensing based on nanoparticles, in order to demonstrate the relevance and potential of these strategies to readers familiar with this field and to non-experts. The benefits provided by the use of nanoparticles, including enhanced analytical performance of the resulting electrochemical biosensors, as well as the main challenges to be solved and potential future advances in this field are discussed.

A novel electroanalytical strategy for copper and ascorbic acid detection was developed by using a nanostructured electrode surface mechanized with a DNAzyme-based molecular gate. This sensing interface was constructed by first electrodeposition of a mesoporous silica thin film on Au electrodes and further assembly of a Cu(II)-specific DNAzyme. The biosensing assay was based on the Cu(II) and ascorbic acid responsible activation of the DNAzyme, which acted as a molecular switch able to control the diffusion of the Fe(CN)63−/4− electrochemical probe through the nanochannels of the mesoporous film. Keywords: Biosensor, DNAzyme, Copper, Mesoporous silica film, Ascorbic acid

Gold–mesoporous silica Janus nanoparticles were prepared through a masking strategy and further employed as building blocks for the preparation of bienzyme biosensors through toposelective immobilization of the corresponding enzymes. The system was evaluated by immobilizing glucose oxidase and horseradish peroxidase on the Au and mesoporous silica faces, respectively. The anisotropic nanoparticles were deposited on glassy carbon electrodes coated with single-walled carbon nanotubes in order to construct an amperometric biosensor for glucose detection. The enzymatic electrode showed excellent electroanalytical performance with a fast response in about 6 s, a linear range of 490 nM–600 μM, a high sensitivity of 4.3 mA M−1 , and a low detection limit of 360 nM for glucose. This biosensor was successfully employed to determine the content of this monosaccharide in commercial soft drink samples.

Made available in DSpace on 2018-11-26T15:27:46Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-08-20 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Spanish Ministry of Science and Innovation Community of Madrid, Programme NANOAVANSENS A novel reduced graphene oxide/Sb2O5 hybrid nanomaterial was prepared by a one-pot reaction process. The nanomaterial was characterized by different spectroscopic, microscopic and electrochemical techniques, demonstrating that the graphene sheets were doped with a Sb2O5 thin film. A glassy carbon electrode coated with the hybrid material was further employed as support for the covalent immobilization of laccase to develop an electrochemical biosensor for estriol. The enzyme biosensor showed high sensitivity (275 mA/M) and fast analytical response (4 s) for the hormone, with a limit of detection of 11 nM in the range of 25 nM to 1.03 p,M. The biosensor showed high selectivity for the analytical detection of estriol. (C) 2015 Elsevier Ltd. All rights reserved. Univ Complutense Madrid, Fac Chem, Dept Analyt Chem, E-28040 Madrid, Spain State Univ Sao Paulo, Inst Chem, BR-13560970 Sao Carlos, SP, Brazil Mackenzie Presbiterian Univ, Sch Engn, BR-01302907 Sao Paulo, SP, Brazil State Univ Sao Paulo, Inst Chem, BR-13560970 Sao Carlos, SP, Brazil FAPESP: 2011/23047-7 FAPESP: 2014/02457-0 Spanish Ministry of Science and Innovation: CFQ2011-24355 Spanish Ministry of Science and Innovation: CTQ2012-34238 Spanish Ministry of Science and Innovation: CTQ2014-58989 Community of Madrid, Programme NANOAVANSENS: S2013/MIT-3029

A novel nanostructured architecture for the construction of electrochemical enzyme biosensors is here described. It implies the electrostatic layer-by-layer assembly of four-generation ethylenediamine core polyamidoamine G-4 dendrimers on glassy carbon electrodes coated with a graphene oxide-carboxymethylcellulose hybrid nanomaterial. This modified surface was further employed for the covalent immobilization of the model enzyme tyrosinase through a glutaraldehyde-mediated cross-linking. The prepared enzyme electrode allowed the amperometric detection of catechol in the 2–400 nM range. The biosensor showed excellent analytical performance with high sensitivity of 6.3 A/M and low detection limit of 0.9 nM. The enzyme electrode retained over 93 % of the initial activity after 40 days at 4 °C.

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.

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.

This work describes the development of a simple and ultrasensitive electroanalytical methodology for the determination of microribonucleic acids (miRNAs) making use of nanostructured disposable electrodes. The strategy involves direct hybridization of the target miRNA (miRNA–21) with a specific thiolated RNA probe self-assembled together with mercaptohexanol (MCH) onto gold nanoparticles (AuNP)-modified screen-printed electrodes (SH-RNA/MCH-AuNP-SPCEs). In addition, the resulting RNA/miRNA homoduplexes are recognized with the viral protein p19 and labeled with a HRP-conjugated anti-Maltose Binding Protein (MBP) monoclonal antibody. Amperometric detection was carried out at −0.20 V (vs. the Ag pseudoreference electrode) using the H2O2/hydroquinone (HQ) system. The prepared biosensors showed a linear dependence between the measured cathodic current and the concentration of the target miRNA over the 0.5–50 pmol L−1 range with a calculated detection limit (LOD) of 142 fmol L−1 without any target miRNA amplification. The assay lasted 60 min once the SH-RNA/MCH-AuNP-SPCEs were prepared. The SH-RNA/MCH-AuNP-SPCEs bioplatforms exhibit at least 2 months-storage stability and possess attractive practical advantages compared with other methods involving either p19 protein or amplification techniques. The usefulness of the method was demonstrated by determining the endogenous level of the target miRNA in total RNA (RNAt) extracted from healthy and cancerous breast cells.

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.

Nanotechnology is becoming increasingly important in the field of (bio)sensors. The performance and sensitivity of electrochemical biosensors can be greatly improved by the integration of nanomaterials into their construction. In this sense, carbon nanomaterials have been widely used for preparation of biosensors due to their ability to enhance electron-transfer kinetics, high surface-to-volume ratios, and biocompatibility. Fullerenes are a very promising family of carbon nanomaterials and have attracted great interest in recent years in the design of novel biosensing systems due to fullerenes’ exceptional properties. These include multiple redox states, stability in many redox forms, easy functionalization and signal mediation. This paper outlines the state-of-the-art and future directions in the use and functionalization of fullerene-C60 and its derivatives, both as electrode modifiers and advanced labels in electrochemical catalytic and affinity biosensors through selected applications.

Background: Studies on the interactions of DNA with small molecular drugs are currently performed both to explore their mechanism of action and to develop new drugs. Electrochemical biosensors offer a very promising alternative to more complex conventional techniques for drug determination due to rapidness, low cost, simplicity, high sensitivity and compatibility with use in different settings. In this review, selected electrochemical nucleic acid-based biosensing methods described so far for the determination of pharmaceuticals and illicit drugs are briefly overviewed, discussing their basics and main features. A section pointing out general conclusions and future directions in this field is also provided. Results: The 42 selected contributions described electrochemical platforms to determine drugs of interest by monitoring their specific interactions with nucleic acids (DNA and aptamers), DNA damage and specific DNA-protein interactions. The highlighted approaches reported the use of electrodes unmodified or modified with nanomaterials and/or polymers in which DNA-drug interaction was followed by electrochemical detection of DNA puric bases, active drug or diffusion-free markers, and monitoring changes in the surface layer morphology/permeability and charge transfer resistance using different electrochemical techniques. Conclusion: Although electrochemical nucleic acid biosensing approaches constitute an interesting option for drugs determination in terms of cost, simplicity and miniaturized instrumentation, validating exhaustively their performance in real samples against conventional methodologies and implementing them into portable and automatic high throughput devices, together with exploring novel electrode modifications with nanomaterials and polymers and studying in more detail their multiplexing ability for analysis of a large number of analytes, is still needed.