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Early diagnosis in primary care settings can increase access to therapies and their efficiency as well as reduce health care costs. In this context, we report in this paper the development of a disposable immunoplatform for the rapid and simultaneous determination of two protein biomarkers recently reported to be involved in the pathological process of neurodegenerative disorders (NDD), tau protein (tau), and TAR DNA-binding protein 43 (TDP-43). The methodology involves implementation of a sandwich-type immunoassay on the surface of dual screen-printed carbon electrodes (dSPCEs) electrochemically grafted with p-aminobenzoic acid (p-ABA), which allows the covalent immobilization of a gold nanoparticle-poly(amidoamine) (PAMAM) dendrimer nanocomposite (3D-Au-PAMAM). This scaffold was employed for the immobilization of the capture antibodies (CAbs). Detector antibodies labeled with horseradish peroxidase (HRP) and amperometric detection at − 0.20 V (vs. Ag pseudo-reference electrode) using the H2O2/hydroquinone (HQ) system were used. The developed methodology exhibits high sensitivity and selectivity for determining the target proteins, with detection limits of 2.3 and 12.8 pg mL−1 for tau and TDP-43, respectively. The simultaneous determination of tau and TDP-43 was accomplished in raw plasma samples and brain tissue extracts from healthy individuals and NDD-diagnosed patients. The analysis can be performed in just 1 h using a simple one-step assay protocol and small sample amounts (5 μL plasma and 2.5 μg brain tissue extracts).

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.

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.

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 highly sensitive amperometric magnetoimmunoassay for rapid determination of lipoprotein(a) (Lp(a)), an important predictor of cardiovascular disease risk, in human serum, is described. It uses a sandwich configuration involving selective capture antibody [antiLp(a)] and biotinylated detector antibody [biotin-antiLp(a)], and a streptavidin-HRP conjugate on carboxy-modified magnetic beads (HOOC-MBs). The resulting MBs bearing the sandwiched immunoconjugates were captured by a magnet placed under the working electrode surface of a disposable screen-printed carbon electrode and the extent of the affinity reaction was monitored amperometrically at −0.20 V (vs a silver pseudo-reference electrode) in the presence of hydroquinone as an electron transfer mediator and upon addition of H2O2 as the enzyme substrate. The method exhibited a wide linear response range (from 0.01 to 0.5 μg mL−1), a detection limit of 4 ng mL−1, and an excellent selectivity over other serum components. The utility of the immunoassay was demonstrated by analyzing a reference serum containing a certified quantity of Lp(a). The performance of this magnetoimmunoassay compares favorably to that of an integrated amperometric immunoassay described earlier.

The preparation and characteristics of a disposable amperometric magnetoimmunosensor, based on the use of functionalized magnetic beads (MBs) and gold screen-printed electrodes (Au/SPEs), for the specific detection and quantification of Staphylococcal protein A (ProtA) and Staphylococcus aureus (S. aureus) is reported. An antiProtA antibody was immobilized onto ProtA-modified MBs, and a competitive immunoassay involving ProtA antigen labelled with HRP was performed. The resulting modified MBs were captured by a magnetic field on the surface of tetrathiafulvalene-modified Au/SPEs and the amperometric response obtained at -0.15 V vs the silver pseudo-reference electrode of the Au/SPEs after the addition of H2O2 was used as transduction signal. The developed methodology showed very low limits of detection (1 cfu S. aureus/mL of raw milk samples), and a good selectivity against the most commonly involved foodborne pathogens originating from milk. These features, together with a short analysis time (2 h), the simplicity, and easy automation and miniaturization of the required instrumentation make the developed methodology a promising alternative in the development of devices for on-site analysis.

8 páginas, 4 figuras, 1 tabla -- PAGS nros. 2413-2420, A disposable magnetogenosensor for the rapid, specific and sensitive detection of Streptococcus pneumoniae is reported. The developed procedure involves the use of streptavidin-modified magnetic beads, a specific biotinylated capture probe that hybridizes with a specific region of lytA, the gene encoding the pneumococcal major autolysin, and appropriate primers for asymmetric polymerase chain reaction (PCR) amplification. Capture probes and amplicons specific for S. pneumoniae were selected by a careful analysis of all lytA alleles available. The selected primers amplify a 235-bp fragment of pneumococcal lytA. A detection limit (LOD) of 5.1 nM was obtained for a 20-mer synthetic target DNA without any amplification protocol, while the LOD for the asymmetric PCR amplicon was 1.1 nM. A RSD value of 6.9% was obtained for measurements carried out with seven different genosensors for 1.1-nM aPCR product. The strict specificity of the designed primers was demonstrated by aPCR amplification of genomic DNA prepared from different bacteria, including some closely related streptococci. Direct asymmetric PCR (daPCR), using cells directly from broth cultures of S. pneumoniae, showed that daPCR products could be prepared with as few as 2 colony-forming units (CFU). Furthermore, this methodology did not show any cross-reaction with closely related streptococci such as Streptococcus mitis (or Streptococcus pseudopneumoniae) even when present in the culture at concentrations up to 105 times higher than that of S. pneumoniae. Preliminary data for rapid detection of pneumococcus directly in clinical samples has shown that it is possible to discriminate between non-inoculated blood and urine samples and samples inoculated with only 103 CFU mL−1 S. pneumoniae, This research was supported by a grant from the Dirección General de Investigación Científica y Técnica (SAF2009-10824). CIBER de Enfermedades Respiratorias (CIBERES) is an initiative of Spanish Instituto de Salud Carlos III. The financial support of Santander/Complutense Research Project PR 27/05-13953, and of the Spanish Ministerio de Ciencia e Innovación Research Project CTQ2009-09351BQU, and the AVANSENS Program from the Comunidad de Madrid (S2009PPQ-1642) are also gratefully acknowledged. S.C. acknowledges a “Juan de la Cierva” research contract

An integrated amperometric fructose biosensor based on a gold electrode (AuE) modified with a self-assembled monolayer (SAM) of 3-mercaptopropionic acid (MPA) on which fructose dehydrogenase (FDH) and the mediator tetrathiafulvalene (TTF) are co-immobilized by cross-linking with glutaraldehyde is reported. Variables concerning the behavior of the biosensor were optimized by taking the slope value obtained for the fructose calibration plot in the 0.1-1.0 mM concentration range as a criterion of selection. At an applied potential of +0.20 V, a good repeatability of such slope values (RSD=6.7%, n=10) was obtained with no need to apply a cleaning or pretreatment procedure to the modified electrode. Moreover, results from five different TTF-FDH-MPA-AuEs yielded a RSD of 5.8%. The useful lifetime of one single biosensor was approximately 30 days, exhibiting a 93% of the original response on the 33rd day. A linear calibration graph was obtained for fructose over the 1.0 x 10(-5)-1.0 x 10(-3) M range, with a limit of detection of 2.4 x 10(-6) M. The effect of potential interferents was evaluated. The TTF-FDH-MPA-AuE also performed well in the flow-injection mode. The biosensor was used for the determination of fructose in real samples, and the results compared with those provided by using a commercial enzyme test kit.