Beatriz Prieto-Simón

Aptamer-DNAzyme hairpins for biosensing of Ochratoxin A

We report an aptasensor for biosensing of Ochratoxin A (OTA) using aptamer-DNAzyme hairpin as biorecognition element. The structure of this engineered nucleic acid includes the horseradish peroxidase (HRP)-mimicking DNAzyme and the OTA specific aptamer sequences. A blocking tail captures a part of these sequences in the stem region of the hairpin. In the presence of OTA, the hairpin is opened due to the formation of the aptamer-analyte complex. As a result, self-assembly of the active HRP-mimicking DNAzyme occurs. The activity of this DNAzyme is linearly correlated with OTA concentration up to 10 nM, showing a limit of detection of 2.5 nM.

Biosensors to detect marine toxins: Assessing seafood safety

This article describes the different types of marine toxins and their toxic effects, and reviews the bio/analytical techniques for their detection, putting special emphasis to biosensors. Important health concerns have recently appeared around shellfish (diarrheic, paralytic, amnesic, neurologic and azaspiracid) and fish (ciguatera and puffer) poisonings produced by different types of phycotoxins, making evident the urgent necessity of counting on appropriate detection technologies. With this purpose, several analysis methods (bioassays, chromatographic techniques, immunoassays and enzyme inhibition-based assays) have been developed. However, easy-to-use, fast and low-cost devices, able to deal with complicated matrices, are still required. Biosensors offer themselves as promising biotools, alternative and/or complementary to conventional analysis techniques, for fast, simple, cheap and reliable toxicity screening. Nevertheless, despite the wide range of seafood toxins and the already rooted biosensing systems, the literature on biosensors for phycotoxins is scarce. This article discusses the existing biosensor-based strategies and their advantages and limitations. Finally, the article gives a general overlook about the regulation toxin levels and monitoring programmes currently established around the world concerning seafood safety.

Highly sensitive detection of pathogen Escherichia coli O157:H7 by electrochemical impedance spectroscopy

The presence of enterohemorrhagic Escherichia coli bacteria in food can cause serious foodborne disease outbreaks. Early detection and identification of these pathogens is extremely important for public health and safety. Here we present a highly sensitive label-free immunosensor for the detection of pathogenic E. coli O157:H7. Anti-E. coli antibodies were covalently immobilised onto gold electrodes via a self-assembled monolayer (SAM) of mercaptohexadecanoic acid and the pathogenic bacteria were detected by electrochemical impedance spectroscopy (EIS). Surface Plasmon Resonance (SPR) was used to monitor the antibody immobilisation protocol and antibody patterned surfaces were used to demonstrate the specificity of the antibody coated surfaces against the pathogenic bacteria. The immunosensor showed a very low limit of detection (2CFU/mL) and a large linear range (3 × 10-3 × 10(4)CFU/mL). Finally, the selectivity of the sensor was demonstrated and no significant adsorption of Salmonella typhimurium was observed.

Enzyme-Linked Immunosorbent Assay (ELISA) based on superparamagnetic nanoparticles for aflatoxin M1 detection

Five different clones of antibodies developed against the aflatoxin M(1) were investigated by using the classical indirect and direct competitive Enzyme-Linked Immunosorbent Assay (ELISA) formats, and also the direct competitive ELISA based on the use of the superparamagnetic nanoparticles. The purpose of this study was to assess if not so friendly time classical ELISA procedures can be further improved, by reducing the coating, blocking and competition time. Here we showed that a complete dc-ELISA (coating, blocking and competition step) based on the use of superparamagnetic nanoparticles can be performed in basically 40 min, if coating step (20 min) should be taken into account. Moreover, the standard analytical characteristics of the proposed method fulfil the requirements for detecting AFM(1) in milk, in a wide linear working range (4-250 ng/L). The IC(50) value is 15 ng/L. The matrix effect and the recovery rate were assessed, using the European Reference Material (BD282, zero level of AFM(1)), showing an excellent percentage of recovery, close to 100%.

A review of the use of genetically engineered enzymes in electrochemical biosensors.

This article gives an overview of the electrochemical biosensors that incorporate genetically modified enzymes. Firstly, the improvements on the sensitivity and selectivity of biosensors that integrate mutated enzymes are summarised. Next, new trends focused on the oriented immobilisation of mutated enzymes through specific functional groups located at their surface are reviewed. Finally, the effect of enzyme mutations on the electron transfer distance and kinetics of electrochemical biosensors is described.

New advances in electrochemical biosensors for the detection of toxins: Nanomaterials, magnetic beads and microfluidics systems. A review

The use of nanotechnology in bioanalytical devices has special advantages in the detection of toxins of interest in food safety and environmental applications. The low levels to be detected and the small size of toxins justify the increasing number of publications dealing with electrochemical biosensors, due to their high sensitivity and design versatility. The incorporation of nanomaterials in their development has been exploited to further increase their sensitivity, providing simple and fast devices, with multiplexed capabilities. This paper gives an overview of the electrochemical biosensors that have incorporated carbon and metal nanomaterials in their configurations for the detection of toxins. Biosensing systems based on magnetic beads or integrated into microfluidics systems have also been considered because of their contribution to the development of compact analytical devices. The roles of these materials, the methods used for their incorporation in the biosensor configurations as well as the advantages they provide to the analyses are summarised.

Biomolecule Immobilization in Biosensor Development: Tailored Strategies Based on Affinity Interactions

The exponential development of biosensors as powerful analytical tools in the last four decades mainly relies on the high sensitivity and selectivity offered when detecting the target analyte. The transducer and the biological receptor are the bases of the biosensor development. Nevertheless, the bioreceptor immobilisation is also important, playing a key role in the retention of the biological activity, and thus affecting the sensitivity. Parameters such as shelf-life and surface regeneration also depend on the biomolecule immobilisation. Researchers are focusing their efforts towards random and oriented immobilisation procedures. Adsorption, entrapment, cross-linking and electrostatic interactions provide randomly immobilised biomolecules, sometimes partially hindering their biological activity. Covalent binding and affinity interactions may enable oriented biomolecule immobilisations, providing controlled, reproducible and highly active modified surfaces. This paper reviews the main immobilisation strategies used in the biosensors development, putting special emphasis on our contribution to mild and oriented immobilisation techniques.

Rapid high-throughput analysis of ochratoxin A by the self-assembly of DNAzyme-aptamer conjugates in wine

We report a new label-free colorimetric aptasensor based on DNAzyme-aptamer conjugate for rapid and high-throughput detection of Ochratoxin A (OTA, a possible human carcinogen, group 2B) in wine. Two oligonucleotides were designed for this detection. One is N1 for biorecognition, which includes two adjacent sequences: the OTA-specific aptamer sequence and the horseradish peroxidase (HRP)-mimicking DNAzyme sequence. The other is a blocking DNA (B2), which is partially complementary to a part of the OTA aptamer and partially complementary to a part of the DNAzyme. The existence of OTA reduces the hybridization between N1 and B2. Thus, the activity of the non-hybridized DNAzyme is linearly correlated with the concentration of OTA up to 30 nM with a limit of detection of 4 nM (3σ). Meanwhile, a double liquid-liquid extraction (LLE) method is accordingly developed to purify OTA from wine. Compared with the existing HPLC-FD or immunoassay methods, the proposed strategy presents the most appropriate balance between accuracy and facility, resulting in a considerable improvement of real-time quality control, and thereby, preventing chronic poisoning caused by OTA contained red wine.

Electrochemical biosensors featuring oriented antibody immobilization via electrografted and self-assembled hydrazide chemistry.

Appropriate site-directed chemistry is essential to maximize the performance of immunosensors. We present two new functionalization strategies that preserve proper folding and binding potential of antibodies by forcing their oriented immobilization. Both strategies are based on the formation of hydrazone bonds between aldehyde groups on the Fc moieties of periodate-oxidized antibodies and hydrazide groups on functionalized gold electrodes. Those hydrazide groups are introduced by electrografting of diazonium salts or by self assembly of mono- and dithiolated hydrazide linkers, resulting in films with tailored functional groups and, thus, antibody distribution and spacing. Their barrier properties and permeability toward electroactive species are evaluated. To demonstrate the potential of these new functionalization strategies, detection of bacteriophage MS2 is performed through either a direct assay using electrochemical impedance spectroscopy (EIS) or through a sandwich assay using differential pulse voltammetry (DPV). Diazonium and monothiolated self-assembled monolayer-modified electrodes enable the detection of less than 1 plaque forming unit (pfu)/mL in a direct EIS assay. However, nonspecific adsorption renders measurements in river water samples difficult. In contrast, sandwich-assays on electrodes with electrografted diazonium salts and monothiolated self-assembled monolayers do not show significant matrix effects using river water samples, but the limits of detection are 10(8) times higher than those of the direct assay. Best results are achieved for immunosensors based on mixed monolayers of hydrazide and hydroxyl diothiolated linkers (15 pfu/mL). These new functionalization techniques are facile to implement. They afford the possibility to tune the surface composition and tailor the electrochemical properties of electrochemical sensors. These advantages should translate into broad interest in this type of surface chemistry for biosensor development.

Evaluation of different mediator-modified screen-printed electrodes used in a flow system as amperometric sensors for NADH

This work presents a comparative study between two different methods for the preparation of mediator-modified screen-printed electrodes, to be used as detectors in a reliable flow injection system for the determination of the nicotinamide adenine dinucleotide (NADH) coenzyme. The best strategy was selected for the final development of compact biosensors based on dehydrogenase enzymes. For the first immobilisation strategy, different redox mediators were electropolymerised onto the SPE surface. The second immobilisation strategy was carried out using polysulfone-graphite composites, which were deposited by screen-printing technology onto the screen-printed electrode (SPE) surface. Both methods achieved an effective and reliable incorporation of redox mediators to the SPE configuration. Finally, a flow system for ammonium determination was developed using a glutamate dehydrogenase (GlDH)-Meldolas Blue (MB)-polysulfone-composite film-based biosensor. The stability of the redox mediators inside the composite films as well as the negligible fouling effect observed on the electrode surface improve the repeatability and reproducibility of the sensors, important features for continuous analysis in flow systems. Furthermore, the optimised bio/sensors, incorporated in a flow injection system, showed good sensitivities and short response times. Such a good analytical performance together with the simple and fast sensor construction are interesting characteristics to consider the polysulfone-composite films as attractive electrochemical transducer materials for the development of new dehydrogenase-based SPEs.