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Hybridization-based electrochemical DNA sensors have become a methodological mainstay in a wide variety of fields. Among the possible assay formats, sandwich hybridization is the most powerful one. It is so called because the target sequence to be measured is hybridized between two complementary sequences. The analytical performance of this class of sensors is influenced by many construction and operational parameters, which need to be optimized. In this work, we comparatively analyze the analytical performance of different sandwich hybridization sensors, constructed on gold surfaces modified by chemisorption. We find that the structures of the designed probes as well as the topography of the gold support and the nature of the monolayer are key parameters affecting the sensitivity and reproducibility of the genosensors. Other important operational parameters are also described. The results of this study could serve as a guide in the design of genosensors for future applications.

Genosensing technology has mostly based on mixed self-assembled monolayers (SAMs) of thiol-modified oligonucleotides and alkanethiols on gold surfaces. However, the typical backfilling approach, which incorporates the alkanethiol in a second step, gives rise to a heterogeneous distribution of oligonucleotide probes on the surface, negatively affecting to both hybridization efficiency and surface stability. Despite aromatic thiols present a remarkably different behavior from alkanethiols, with higher rigidity and stronger intermolecular interactions, they have been scarcely explored for the fabrication of DNA sensing platforms. We have investigated different approaches involving SAMs of aromatic thiols, namely p-mercaptobenzoic acid (p-MBA) and p-aminothiophenol (p-ATP), to yield DNA sensing layers for sequence-specific detection of target oligonucleotides. The studied monolayers were evaluated by DNA surface coverage and further information was obtained by determining their functionality in a sandwich hybridization assay with enzymatic amplification of the electrochemical read-out. The insertion of thiol-oligonucleotides into p-ATP monolayers previously oxidized, and the covalent binding of amino-oligonucleotides to pure p-MBA monolayers give rise to increased storage stability and better analytical performance. The quantification of RNA from Legionella pneumophila cellular lysates was successfully performed, illustrating the usefulness of these sensing architectures for detecting pathogenic bacteria.

DNA detection is still undergoing major innovations in pursuit of low-cost and simple approaches for decentralized applications. Label-free sensing of DNA hybridization via impedance measurements is a popular strategy to fulfil the goals of cost-efficiency and simplicity. Several materials are often reported for electrode modification to improve the sensitivity of impedance-based sensors. Herein we evaluate the electronic properties of copper phthalocyanine tetrasulfonate (CuPcTs) in Layer-by-Layer (LbL) films for impedimetric sensing of DNA hybridization using silanized Fluorine-doped Tin Oxide (FTO) electrodes. 1 to 5 bilayers were prepared by alternate immersion of the substrate in CuPcTs and poly(allylamine hydrochloride) (PAH). DNA probe immobilization was carried out electrostatically onto the last PAH layer, followed by hybridization with the target sequence leading to the formation of a partial double stranded ( pds ) structure onto the films. Impedance decreased after hybridization proportionally with the concentration of the target sequence at picomolar levels. Not only are these findings useful as a potential biosensing strategy, but also leave an open question about the electronic and synergistic properties of DNA interacting with different materials and surfaces.

Electrochemical genosensors have undergone an enormous development in the last decades, but only very few have achieved a quantification of target content in highly processed food samples. The detection of allergens, and particularly gluten, is challenging because legislation establishes a threshold of 20 ppm for labeling as gluten-free but most genosensors expresses the results in DNA concentration or DNA copies. This paper describes the first attempt to correlate the genosensor response and the wheat content in real samples, even in the case of highly processed food samples. A sandwich-based format, comprising a capture probe immobilized onto the screen-printed gold electrode, and a signaling probe functionalized with fluorescein isothiocyanate (FITC), both hybridizing with the target was used. The hybridization event was electrochemically monitored by adding an anti-FITC peroxidase (antiFITC-HRP) and its substrate, tetramethylbenzidine. Binary model mixtures, as a reference material, and real samples have been analyzed. DNA from food was extracted and a fragment encoding the immunodominant peptide of α2-gliadin amplified by a tailored PCR. The sensor was able to selectively detect toxic cereals for celiac patients, such as different varieties of wheat, barley, rye and oats, from non-toxic plants. As low as 0.001% (10 mg/kg) of wheat flour in an inert matrix was reliably detected, which directly compete with the current method of choice for DNA detection, the real-time PCR. A good correlation with the official immunoassay was found in highly processed food samples.

A novel approach towards the development of biomimetic electrochemical sensors is presented. It is based on magnetic-core/porous-shell molecularly imprinted composites as recognition phase. The selected substrate is 5-hydroxyindole-3-acetic acid (5-HIAA), an urinary metabolite used as carcinoid tumor maker. Using 5-HIAA as template molecule, hemin, 4-vinylpyridine and ethylenglycoldimethacrylate crosslinker are polymerized onto the magnetic core to obtain an artificial enzyme with peroxidase activity. An aliquot of a suspension of the resulting material is deposited onto a carbon screen printed electrode, and the solid material is fixed with a permanent magnet to prepare the sensor. The product of the catalytic oxidation of 5-HIAA at the sensing phase is monitored by differential pulse voltammetry. This sensor allows the detection of 5-HIAA with good sensitivity (0.72 ± 0.01 nA μM−1) and linear response over a concentration range covering the whole window of normal and pathologic urinary levels of 5-HIAA 1–50 μM (0.2–9.6 ppm) with a detection limit of 1.4 μM (0.27 ppm). Urine samples of healthy individuals, spiked with known concentrations of 5-HIAA, yield good recovery values and reproducibility for normal and pathological concentrations (90–100% recovery values, with RSD below 10%).

Enzyme immunoassays are currently the methods of choice for gluten control in foods labelled as gluten free, providing a mechanism for assessing food safety for consumption by coeliac and other allergic patients. However, their limitations, many of them associated to the reactivity of the different antibodies used and their degree of specificity, have prevented the establishment of a standardised method of analysis. We explore new methods for quantitatively determining gluten content in foods based on the use of two recently described aptamers, raised against a 33-mer peptide recognised as the immunodominant fragment from α2-gliadin. The assays use the target peptide immobilised onto streptavidin-coated magnetic beads in combination with a limited amount of biotin-aptamer in a competitive format, followed by streptavidin-peroxidase labelling of the aptamer that remains bound to the magnetic beads. The enzyme activity onto the beads, measured by chronoamperometry in disposable screen-printed electrodes, is inversely related to the target concentration in the test solution. We find that while the assay using the aptamer with the highest affinity towards the target (Gli 4) achieves low detection limits (~0.5 ppm) and excellent analytical performance, when challenged in samples containing the intact protein, gliadin, it fails in detecting the peptide in solution. This problem is circumvented by employing another aptamer (Gli 1), the most abundant one in the SELEX pool, as a receptor. The proposed assays allow the convenient detection of the allergen in different kinds of food samples, including heat-treated and hydrolysed ones. The obtained results correlate with those of commercially available antibody-based assays, providing an alternative for ensuring the safety and quality of nominally gluten-free foods. Graphical Abstract Electrochemical magnetoassay for gluten determination using biotin-aptamers as receptors.

Stable and easily-handled synthetic materials mimicking natural enzymes activity would find important biotechnological applications. This article describes the synthesis and characterization of magnetic molecularly imprinted catalytic polymers that exhibit peroxidase-like activity towards 5-hydroxyindole-3-acetic acid (5-HIAA) oxidation. This multifunctional material is obtained from highly crystalline magnetite nuclei coated with a silica layer to protect the iron nucleus from oxidation and to provide anchoring for hydroxyl surface groups. After acrylic functionalization via sol–gel process, a molecularly imprinted polymer with hemin as catalytic center and 5-HIAA as template has been successfully attached to the structure. The resulting hybrid composite is magnetically separable and possesses excellent catalytic ability for the selective oxidation of the indoleamine metabolite tumor marker, showing Michaelis–Menten kinetics with this molecule but not towards other structural analogs. Therefore, it can be considered an artificial peroxidase enzyme.

The design and performance of an inhibition assay for the electrochemical detection of tobramycin is reported. This platform uses a monovalent system for introducing the enzyme conjugate on a tagged-aptamer that specifically recognizes the aminoglycoside antibiotic tobramycin. Compared with multivalent systems such as biotin–streptavidin, the sensitivity is greatly improved reducing the limit of detection from 5 μM to 0.1 μM in aqueous solution. This labeling strategy along with the use of a nuclease-resistant RNA aptamer allowed the determination of tobramycin in pre-treated human serum samples within the therapeutic range, using magnetic microparticles as a solid support for the aptassay, and differential pulse voltammetry as detection technique.

A new nanomotor-based surface-patterning technique based on the movement of a magnetically powered enzyme-functionalized flexible nanowire swimmer offers the ability to create complex helical metal microstructures.

A magnetic microsphere-based hybridization assay for the electrochemical detection of pathogen-specific DNA sequences is reported. Enzyme-linked sandwich hybridization assays with different tags (biotin, fluorescein or digoxigenin) and enzyme conjugates (streptavidin-alkaline phosphatase, streptavidin-peroxidase, antifluorescein-peroxidase or antidigoxigenin-peroxidase) are comparatively evaluated. Sensitivity and limit of detection depend on both the choice of the enzyme and the tag. The highest sensitivity and lowest limit of detection were obtained when antibody Fab fragments conjugated to peroxidase were used as reporter molecule. The digoxigenin system yields the lowest detection limit, mainly due to producing much lower background signals.

An RNA aptamer is proposed as a recognition element for the detection of tobramycin in human serum. A displacement assay was developed using faradaic-electrochemical impedance spectroscopy (F-EIS) as a detection technique. Two modified aptamers, a partially (ATA) and a fully O-methylated aptamer (FATA) were evaluated and compared. The affinity constant, K(D), for both aptamers was estimated by F-EIS resulting virtually identical within the experimental error. The selectivity towards other aminoglycosides was also studied. The analytical characteristics were evaluated in aqueous solution using both aptamers and FATA was selected for human serum experiments. Using a 1:0.5 dilution of the serum, a linear range between 3 μM and 72.1 μM was obtained, which included the therapeutic range of the antibiotic.

A comparative evaluation of an electrochemical sandwich genoassay for pathogenic bacteria based on immobilized hairpin DNA probes and three different enzyme labels (horseradish peroxidase, alkaline phosphatase and a biomimetic peroxidase-like DNAzyme) is reported. The natural enzymes were used as streptavidin conjugates, coupled to the surface duplex by using a biotin-labeled signaling probe, whereas the DNAzyme was directly incorporated to the sequence of the signaling probe. HRP provides enhanced sensitivity although the choice of a catalytic reporter DNA sequence could simplify the assay.

The use of nucleic acid with a specific sequence and a highly ordered secondary structure such as hairpins, quadruplexes and pseudoknots as biological recognition elements and switches in biosensors is rapidly increasing because of their improved features (e.g. selectivity) when compared with the traditional linear probes. Owing to the novelty, a critical outlook of their characteristics and a compilation of the latest advances are lacking. This article describes the potential of those nucleic acids probes whose molecular recognition ability relies on a conformational change (e.g. folding/unfolding mechanism) in electrochemical sensing. It provides an overview of the toolbox of assays using these probes for genosensors and aptasensors, highlighting its performance characteristics and the prospects and challenges for biosensor design.

We have studied how the modification of the RNA aptamer evolved against neomycin B at 2' position of ribose with a methyl group influences the affinity of the interaction. Using surface plasmon resonance (SPR) and faradaic impedance spectroscopy (FIS) an affinity constant in the muM range was calculated. The results showed that the modification of the aptamer does not significantly alter the affinity of the aptamer for the antibiotic. This finding opens up the possibility of designing modified RNA aptamers resistant to endonucleases without variation of the analytical features. In addition to this, we propose a competitive assay for the detection of neomycin B using SPR as a transduction technique. A range of quantification between 10 nM and 100 microM was obtained, which shows the feasibility of detecting small molecules using aptamers with high sensitivity.

The electrochemical oxidation of guanosine and xanthosine, which only differ in the presence of an oxygen atom at C2 position instead of an amine group, is studied on pyrolytic graphite electrodes at different electrolysis time scales in solutions of physiological pH. The oxidation products adsorbed on the electrode surface were electrochemical characterized in terms of variation of the formal potential with pH and catalytic activity towards the oxidation of NADH. The results were compared with those previously described for the oxidation of guanosine and adenosine in alkaline solutions. A common oxidation product is described, the oxidized form of 9-β-D-ribofuranosyluric acid, which is proposed as the point of convergence in the oxidation routes of all three purine nucleosides.

We have studied the potential electrocatalytic activity towards the oxidation of NADH of several oxidation products of guanine and its derivative guanosine-5′-monophosphate (5′-GMP) on pyrolytic graphite electrodes (PGE). The distribution of products generated strongly depends on the experimental conditions. Our investigations focused on the oxidation products that are adsorbed on the electrode surface, are redox active and, exhibited electrocatalytic activity toward the oxidation of NADH. These compounds were electrochemically and kinetically characterized in terms of dependence of the formal potential on pH and electron transfer rate constant ( k s ). The voltammetric and catalytic behavior of both guanine and 5′-GMP oxidation products was compared with that of other guanine derivatives we have previously studied. Some mechanistic aspects concerning the generation of the catalysts are also discussed.

We herein explore a novel route for oxidative stress in DNA by using electrochemistry as mimicking tool. Essentially, the electrochemical oxidation of guanine and adenine nucleosides and oligo-homo-nucleotides at pyrolytic graphite electrodes in neutral and alkaline aqueous solutions was studied. Under these experimental conditions all these compounds give rise to an adsorbed product not previously described, which was electrochemically and kinetically characterized. The virtually identical kinetic and electrochemical features exhibited by the oxidized compounds generated from all precursors strongly suggest a common species arising from both adenine and guanine derivatives. Supported by DFT calculations, we propose two convergent pathways for the electrochemical oxidation of adenosine and guanosine. Those calculations indicate that the common oxidized base product forms stable H-bond base pairs with both thymine and cytosine. Keywords: Cyclic voltammetry, Density functional calculations, Electrochemistry, Guanosine, Substitution mutation

A simple and sensitive adsorptive- differential pulse- voltammetric method for the determination of flufenamic acid (FFA) is presented. The method is based on the preconcentration of FFA on carbon paste electrodes modified in situ with cationic surfactants, dodecyltrimethylammonium chloride (DTAC) or cetyltrimethylammonium chloride (CTAC), at submicellar concentrations. The best results were obtained with DTAC-modified electrodes. After optimization, with these electrodes and using an accumulation step at 0 V for 3 minutes in 0.1 M phosphate solutions pH 7, the peak current of the main oxidation process of FFA varies linearly with its concentration from 1×10−9 M to 5×10−5 M, with a limit of detection of 0.64 nM (0.2 ng/mL). The proposed method has been applied to the determination of FFA in spiked serum samples, treated with acetonitrile to separate the proteins. In the concentration range studied (3 μM to 65 μM) the recovery was near 100 % and the lowest concentration attainable in these samples is below 3 μM (0.8 ppm).

s-Adenosyl-L-methionine (SAMe) is an adenosine analogue with therapeutical activity against affective disorders and liver dysfunctions. It can be oxidized on graphite electrode yielding a strongly adsorbed electroactive oxidation product for which a quinone-imine structure is proposed. This compound is capable of electrocatalyzing the NADH oxidation at low potentials, lowering the overvoltage by about 300 mV. An amperometric method for NADH determination at +0.1 V (Ag|AgCl|KClsat) is developed using an oxidized-SAMe-modified electrode in pH 9. Linear calibration plots were obtained with a detection limit of 2.4 nM. The electrode response time and the relative standard deviation of the slope of the calibration plot for 5 different modified electrodes were 12 s and 5.6% respectively. The catalytic scheme also provides the first method to determine SAMe itself by adsorptive differential pulse voltammetry. The linear range was found to be 42.4–424 nM with a reproducibility of 6.9%. The method was applied to SAMe determination in a pharmaceutical formulation.

This article summarises our work on the development of voltammetric sensors based on molecularly imprinted polymers. Several recognition elements and integration strategies were used:1.membranes electropolymerised at the electrode surface; 2.casting of polymeric membranes by drop-coating a solution of pre-formed polymer (polyphosphazene) and template in a low-boiling-point solvent on to the electrode surface; 3.preparation of composite membranes containing conductive material (graphite or carbon black), acrylic-type molecularly imprinted polymers (small particle size), and PVC as binder; and 4.in-situ polymerisation of a thin layer of acrylic imprinted polymer deposited on the electrode surface by spin coating. All the options evaluated offer the possibility of controlling electrode characteristics such as hydrophobic/hydrophilic character, permeability, or film thickness, which are essential for obtaining good sensor performance.

Molecularly imprinted polymers (MIPs) are becoming an important class of synthetic materials mimicking molecular recognition by natural receptors. This review examines the literature on non-covalent MIP-based electrochemical sensors over the last 10 years. With insight into the different sensing phases, electrochemical transductions and integration strategies, we evaluate achievements and difficulties to date and assess future prospects.

A voltammetric sensor for the determination of diclofenac was developed, based on the molecular recognition of the analyte by molecularly imprinted methacrylate-ethyleneglycol dimethacrylate co-polymers. Pre-polymerisation solutions were deposited onto the surface of a glassy carbon electrode and a polymer film was obtained after spin coating control of thickness and in situ thermal polymerisation. After the template extraction from the resultant film, re-binding of diclofenac is performed from acetonitrile solutions containing the analyte. The amount of bonded diclofenac was then evaluated by differential pulse voltammetry in different electrolytes. The best results were obtained in 0.025 M citrate solution pH 6 containing 10% of acetonitrile. This medium favours the release of diclofenac from the polymer binding sites. In this way, the voltammetric transduction of the molecular recognition event is achieved. Voltammetric selectivity measurements revealed negligible interferences from diclofenac family of anti-inflammatory drugs, such as niflumic or meclofenamic acids.

Graphite electrodes modified with a drop-coated layer of polyethyleneimine (PEI) and adenosine diphosphate (ADP) displayed an electrocatalytic response to NADH after the adenine moiety of ADP was electrochemically oxidised. NADH can be detected amperometrically in alkaline solution (pH 9.0) at low applied potentials (+50 mV (Ag/AgCl)). Using a stationary electrode arrangement, linear response for NADH concentrations between 1.0×10 −8 and 1.0×10 −4 M was found, with a response time of 12 s and a detection limit of 8×10 −9 M. The electrode was applied to the amperometric monitoring of the reaction between lactate and NAD + catalysed by lactate dehydrogenase (LDH). A flow injection-amperometric method for the determination of LDH activity in human serum was developed. The method allows a fast and accurate discrimination between pathological and normal LDH activity levels, with a sampling rate of 40 h −1 . Quantitative results for a random set of human serum samples were found to be in good agreement with the standard spectrophotometric method.

Molecularly imprinted polyphosphazenes are presented as sensitive coatings for the detection of rifamycin SV in water. These membranes were combined with voltammetric transduction using glassy carbon (GC) working electrodes. The receptor layers were obtained by the drop-coating technique using tetrahydrofuran (THF) solutions containing 0.27 % (m/v) of the polymer and different amounts of template (0.03–0.17 %, m/v). Evaporation of THF results on a water insoluble membrane, which shows good adherence to the GC electrode surface. After removal of the template from the solid polymer membrane, the binding of rifamycin SV was examined by equilibrating the membrane in an aqueous solution of the template. The amount of bound rifamycin SV was measured by cyclic voltammetry or differential pulse voltammetry. Similar experiments were carried out with imprinted membranes using rifampicin as template. Less effective binding was obtained for rifamycin SV in the rifampicin-imprinted polymer. The rifamycin SV sensitive electrode, constructed using the corresponding molecularly imprinted polymer, allows rifamycin SV measurement in the concentration interval between 2.5×10−7 and 6.3×10−6 M.