María Díaz-González

Development of an immunosensor for the determination of rabbit IgG using streptavidin modified screen-printed carbon electrodes.

Voltammetric enzyme immunosensors based on the employment of streptavidin modified screen-printed carbon electrodes (SPCEs) for the detection of rabbit IgG, as a model analyte, were described. Alkaline phosphatase (AP) and 3-indoxyl phosphate (3-IP) were used as the enzymatic label and substrate, respectively. The adsorption of streptavidin was performed by deposition of a drop of a streptavidin solution overnight at 4 degrees C on the pre-oxidized surface of the SPCEs. The analytical characteristics of these sensors were evaluated using biotin conjugated to AP. The immunosensor devices were based on a specific reaction of rabbit IgG with its biotinylated antibodies, which were immobilised on the modified screen-printed carbon electrodes through the streptavidin:biotin reaction. The immunosensors were used for a direct determination of AP labelled rabbit IgG, and for free rabbit IgG detection using a sequential competitive immunoassay. A calibration curve in the range of 5 x 10(-11) to 1 x 10(-9)M of rabbit IgG was obtained with a estimated detection limit of 5 x 10(-11)M (7.0ng/ml). These immunosensors were stable for 5 months if they were stored at 4 degrees C.

Achieving Extremely Concentrated Aqueous Dispersions of Graphene Flakes and Catalytically Efficient Graphene-Metal Nanoparticle Hybrids with Flavin Mononucleotide as a High-Performance Stabilizer

The stable dispersion of graphene flakes in an aqueous medium is highly desirable for the development of materials based on this two-dimensional carbon structure, but current production protocols that make use of a number of surfactants typically suffer from limitations regarding graphene concentration or the amount of surfactant required to colloidally stabilize the sheets. Here, we demonstrate that an innocuous and readily available derivative of vitamin B2, namely the sodium salt of flavin mononucleotide (FMNS), is a highly efficient dispersant in the preparation of aqueous dispersions of defect-free, few-layer graphene flakes. Most notably, graphene concentrations in water as high as ∼50 mg mL(-1) using low amounts of FMNS (FMNS/graphene mass ratios of about 0.04) could be attained, which facilitated the formation of free-standing graphene films displaying high electrical conductivity (∼52000 S m(-1)) without the need of carrying out thermal annealing or other types of post-treatment. The excellent performance of FMNS as a graphene dispersant could be attributed to the combined effect of strong adsorption on the sheets through the isoalloxazine moiety of the molecule and efficient colloidal stabilization provided by its negatively charged phosphate group. The FMNS-stabilized graphene sheets could be decorated with nanoparticles of several noble metals (Ag, Pd, and Pt), and the resulting hybrids exhibited a high catalytic activity in the reduction of nitroarenes and electroreduction of oxygen. Overall, the present results should expedite the processing and implementation of graphene in, e.g., conductive inks, composites, and hybrid materials with practical utility in a wide range of applications.

Comparative Voltammetric Behavior of Indigo Carmine at Screen‐Printed Carbon Electrodes

The redox activity of indigo carmine (IC) at two different commercial screen-printed carbon electrode strips (SPCE) was investigated in this work by different voltammetric techniques. Cyclic voltammograms of this molecule in 0.1 M HClO4 solutions show two well-defined diffusion-controlled signals, one reversible at a formal potential (E1/2) of −0.57 V vs. Pt pseudoreference electrode (−0.15 V vs. Ag), and a second anodic peak at a potential (Ep, a) of +0.13 V vs. Pt (+0.57 V vs. Ag). Both electrodes exhibit good analytical performance towards this analyte, if they were applied in a disposable manner. Their use in consecutive experiments offers substantial differences, since an oxidation wave due to adsorption of indigo carmine superimposed on the anodic peak of the redox pair at one of these SPCEs. In addition, this adsorption component appears to foul the electrode surface. This wave is greatly enhanced if the working electrode is previously activated by applying high positive potentials for a certain period of time. The other SPCE does not exhibit any fouling process, thus allowing the performance of several consecutive experiments with the same strip. With these electrodes, calibration plots were obtained by cyclic, differential pulse, square-wave and alternating current voltammetry. Wide linear ranges of more than two orders of magnitude and limits of the detection down to 10−7 M were achieved. Relative standard deviations of the peak current in a range of 0.9–7%, recorded in indigo carmine solutions of different concentrations, at one single electrode, give an estimation of the good precision of this data.

DNA hybridization biosensors using polylysine modified SPCEs

Abstract Two electrochemical DNA hybridization biosensors (genosensors) for the detection of a 30-mer sequence unique to severe acute respiratory syndrome (SARS) virus are described in this work. Both genosensors rely on the hybridization of the oligonucleotide target with its complementary probe, which is immobilized on positively charged polylysine modified screen-printed carbon electrodes (SPCEs), through electrostatic interactions. In one design, a biotinylated target is used and the detection of the hybridization reaction is monitored using alkaline phosphatase labeled streptavidin (S-AP). This enzyme catalyzes the hydrolysis of the substrate 3-indoxyl phosphate (3-IP) to indigo, which is then solubilized to indigo carmine and detected by means of cyclic voltammetry (CV). In the other design, the target is labeled using an Au(I) complex, sodium aurothiomalate, and the duplex formation is detected by measuring, for first time, the current generated by the hydrogen evolution catalyzed by the gold label. Using 30min of hybridization time, a detection limit of 8pM is calculated for the enzymatic genosensor. Although this good sensitivity cannot be reached with the metal label (0.5nM), the use of this label allows a considerable decrease of the analysis time. Both genosensors do not require the modification of the oligonucleotide probe and using stringent experimental conditions (60min of hybridization time and 50% formamide in the hybridization buffer) can discriminate between a complementary oligonucleotide and an oligonucleotide with a three-base mismatch.

Fabrication of biofunctionalized microfluidic structures by low-temperature wax bonding.

In this work, a new fabrication technology for microfluidics based on the use of wax is described. Microfluidic structures are assembled using wax as both a thermoplastic adhesive layer between two glass substrates and a spacer layer defining the microchannels. Wax patterns with dimensions down to 25 μm are easily produced on glass substrates using specially developed decal-transfer microlithography. A complete microfluidic system is created by bonding the wax patterned layer with an additional glass substrate. On the basis of the special melting behavior of waxes, an effective glass-wax bonding is achieved at 40 °C by applying a soft pressure and without the requirement of any glass pretreatment. Wax bonding provides an effective sealing of the fluidic networks even on nonflat glass substrates (i.e., containing metal electrodes). The mild conditions required for the bonding process enables the fabrication of lab-on-a-chip devices incorporating biomolecules, as is demonstrated with the implementation of a simple heterogeneous immunoassay in a microfluidic device with amperometric detection.

Aqueous Exfoliation of Transition Metal Dichalcogenides Assisted by DNA/RNA Nucleotides: Catalytically Active and Biocompatible Nanosheets Stabilized by Acid–Base Interactions

The exfoliation and colloidal stabilization of layered transition metal dichalcogenides (TMDs) in an aqueous medium using functional biomolecules as dispersing agents have a number of potential benefits toward the production and practical use of the corresponding two-dimensional materials, but such a strategy has so far remained underexplored. Here, we report that DNA and RNA nucleotides are highly efficient dispersants in the preparation of stable aqueous suspensions of MoS2 and other TMD nanosheets at significant concentrations (up to 5-10 mg mL-1). Unlike the case of common surfactants, for which adsorption on 2D materials is generally based on weak dispersive forces, the exceptional colloidal stability of the TMD flakes was shown to rely on the presence of relatively strong, specific interactions of Lewis acid-base type between the DNA/RNA nucleotide molecules and the flakes. Moreover, the nucleotide-stabilized MoS2 nanosheets were shown to be efficient catalysts in the reduction of nitroarenes (4-nitrophenol and 4-nitroaniline), thus constituting an attractive alternative to the use of expensive heterogeneous catalysts based on noble metals, and exhibited an electrocatalytic activity toward the hydrogen evolution reaction that was not impaired by the possible presence of nucleotide molecules adsorbed on their active sites. The biocompatibility of these materials was also demonstrated on the basis of cell proliferation and viability assays. Overall, the present work opens new vistas on the colloidal stabilization of 2D materials based on specific interactions that could be useful toward different practical applications.