Ordered Mesoporous Electrodes for Sensing Applications.

Electrochemical sensors have become increasingly relevant in fields such as medicine, environmental monitoring, and industrial process control. Selectivity, specificity, sensitivity, signal reproducibility, and robustness are among the most important challenges for their development, especially when the target compound is present in low concentrations or in complex analytical matrices. In this context, electrode modification with Mesoporous Thin Films (MTFs) has aroused great interest in the past years. MTFs present high surface area, uniform pore distribution, and tunable pore size. Furthermore, they offer a wide variety of electrochemical signal modulation possibilities through molecular sieving, electrostatic or steric exclusion, and preconcentration effects which are due to mesopore confinement and surface functionalization. In order to fully exploit these advantages, it is central to develop reproducible routes for sensitive, selective, and robust MTF-modified electrodes. In addition, it is necessary to understand the complex mass and charge transport processes that take place through the film (particularly in the mesopores, pore surfaces, and interfaces) and on the electrode in order to design future intelligent and adaptive sensors. We present here an overview of MTFs applied to electrochemical sensing, in which we address their fabrication methods and the transport processes that are critical to the electrode response. We also summarize the current applications in biosensing and electroanalysis, as well as the challenges and opportunities brought by integrating MTF synthesis with electrode microfabrication, which is critical when moving from laboratory work to in situ sensing in the field of interest.

Fully Inkjet-Printed Biosensors Fabricated with a Highly Stable Ink Based on Carbon Nanotubes and Enzyme-Functionalized Nanoparticles.

Enzyme inks can be inkjet printed to fabricate enzymatic biosensors. However, inks containing enzymes present a low shelf life because enzymes in suspension rapidly lose their catalytic activity. Other major problems of printing these inks are the non-specific adsorption of enzymes onto the chamber walls and stability loss during printing as a result of thermal and/or mechanical stress. It is well known that the catalytic activity can be preserved for significantly longer periods of time and to harsher operational conditions when enzymes are immobilized onto adequate surfaces. Therefore, in this work, horseradish peroxidase was covalently immobilized onto silica nanoparticles. Then, the nanoparticles were mixed into an aqueous ink containing single walled carbon nanotubes. Electrodes printed with this specially formulated ink were characterized, and enzyme electrodes were printed. To test the performance of the enzyme electrodes, a complete amperometric hydrogen peroxide biosensor was fabricated by inkjet printing. The electrochemical response of the printed electrodes was evaluated by cyclic voltammetry in solutions containing redox species, such as hexacyanoferrate (III/II) ions or hydroquinone. The response of the enzyme electrodes was studied for the amperometric determination of hydrogen peroxide. Three months after the ink preparation, the printed enzyme electrodes were found to still exhibit similar sensitivity, demonstrating that catalytic activity is preserved in the proposed ink. Thus, enzyme electrodes can be successfully printed employing highly stable formulation using nanoparticles as carriers.

One Step Histological Detection and Staining of the PTEN Tumor Suppressor Protein by a Single Strand DNA.

Antibodies are the most used technological tool in histochemistry. However, even with monoclonal antibodies, their standardization is difficult due to variation of biological systems as well as to variability due to the affinity and amplification of the signal arising from secondary peroxidase detection systems. In this article we combined two synthetic molecules to facilitate the standardization of a detection protocol of protein markers in histological sections. The first molecule was an aptamer, a 50-base single-stranded DNA fragment, which recognizes a PTEN tumor suppressor. The second molecule used was also another single stranded 18-base aptamer DNA fragment, which forms a quadruplex structure guanine box. This G-quadruplex recognizes and attaches a molecule of hemin, increasing the catalytic capacity for the hydrogen peroxide. Our results show how the correct structural design of DNA combining an aptamer together with the peroxidase-like DNAzyme allows to detect proteins in histological sections. This tool offers the standardization of the detection of prognostic markers in cancer, in quality and quantity, due to its synthetic nature and its 1:1 antigen:enzyme ratio. This is the first time that reproducible results have been presented in histological sections staining a cancer marker using a single-stranded DNA molecule with dual function.

Magnetic mesoporous silica nanospheres with dual probe & release fluorescent functionality.

The combination of different nanomaterials through step-by-step synthesis procedures has turned into a promising alternative to fabricate high-quality nanosystems in order to satisfy the increasingly demanding requirements of the biomedical field. In this work, we report a detailed study on the synthesis and characterization of a complex nanosystem composed of nanoparticles with a single magnetic nanoparticle core and a shell of dense and mesoporous silica arranged in layers. The procedure designed to fabricate these systems lead us to the formation of a dispersion of non-agglomerated spherical nanoparticles of nearly 100 nm. The structural characterization performed over the final samples confirmed both the prevalence of single-core systems and the presence of the mesoporous silica shell in the outer layer. The performance of the nanosystem in a specific technological application was tested by sequentially loading two different fluorescents molecules by covalent and non-covalent bonding strategies. Due to the distinct loading strategies, the resulting nanosystem presented a magnetically-assisted probe & release functionality as analyzed in a magnetophoretic experiment.

Chemical Stability of Mesoporous Oxide Thin Film Electrodes under Electrochemical Cycling: from Dissolution to Stabilization.

Mesoporous oxide thin films (MOTF) present very high surface areas and highly controlled monodisperse pores in the nanometer range. These features spurred their possible applications in separation membranes and permselective electrodes. However, their performance in real applications is limited by their reactivity. Here, we perform a basic study of the stability of MOTF toward dissolution in aqueous media using a variety of characterization techniques. In particular, we focus in their stability behavior under the influence of ionic strength, adsorption of electrochemical probes, and applied electrode potential. Mesoporous silica thin films present a limited chemical stability after electrochemical cycling, particularly under high ionic strength, due to their high specific surface area and the interactions between the electrochemical probes and the surface. In contrast, TiO2 or Si0.9Zr0.1O2 matrices present higher stability; thus, they are an adequate alternative to produce accessible, sensitive, and robust permselective electrodes or membranes that perform under a wide variety of conditions.

Electrochemical Determination of ß-Lactoglobulin Employing a Polystyrene Bead-Modified Carbon Nanotube Ink.

In this article, we introduce the use of a carboxy-functionalized waterborne carbon nanotube ink for the fabrication of an amperometric biosensor aimed at the quantification of ß-lactoglobulin. Detection of this protein from cow's milk was performed by a sandwich immunoassay onto printed carbon nanotube electrodes. The electrodes were printed using a carbon nanotube ink modified with polystyrene beads containing a high amount of carboxylic groups for protein immobilization. This strategy showed enhanced sensing performance compared to the use of oxidative treatments for the functionalization of electrodes. These electrodes showed an excellent electrochemical behavior, and proteins could be immobilized on their surface via the carbodiimide reaction. These antibody-immobilized carbon nanotube electrodes allowed for the detection of ß-lactoglobulin in sub-ppm concentrations.

Improvement of bovine semen quality by removal of membrane-damaged sperm cells with DNA aptamers and magnetic nanoparticles.

In cattle, cryopreservation of semen and sex-sorting kill up to 50% of spermatozoa and decrease the success of assisted insemination (AI). Therefore, significant efforts are being carried out to improve the quality of semen prior to AI. In this work we used the Cell-SELEX technique to select single strand DNA aptamers able to recognize with high affinity and specificity damaged sperm cells generated by heat-treatment. We first isolated aptamers with a conserved two motifs of 6 nucleotides of length that bind to the membrane of heat-treated spermatozoa. Then, we used synthetic biotin-labeled aptamers containing the conserved motif to recognize membrane-damaged cells and separate them from viable cells by the use of avidin-coated superparamagnetic iron oxide nanoparticles (SPION). This procedure improved the quality of semen by significantly increasing the percentage of healthy sperm cells without affecting the rate of blastocyst cleavage. This technique was successfully applied to both unsorted and sex-sorted sperm suspension.

Electrochemical magnetic microbeads-based biosensor for point-of-care serodiagnosis of infectious diseases.

Access to appropriate diagnostic tools is an essential component in the evaluation and improvement of global health. Additionally, timely detection of infectious agents is critical in early diagnosis and treatment of infectious diseases. Conventional pathogen detection methods such as culturing, enzyme linked immunosorbent assay (ELISA) or polymerase chain reaction (PCR) require long assay times, and complex and expensive instruments making them not adaptable to point-of-care (PoC) needs at resource-constrained places and primary care settings. Therefore, there is an unmet need to develop portable, simple, rapid, and accurate methods for PoC detection of infections. Here, we present the development and validation of a portable, robust and inexpensive electrochemical magnetic microbeads-based biosensor (EMBIA) platform for PoC serodiagnosis of infectious diseases caused by different types of microorganisms (parasitic protozoa, bacteria and viruses). We demonstrate the potential use of the EMBIA platform for in situ diagnosis of human (Chagas disease and human brucellosis) and animal (bovine brucellosis and foot-and-mouth disease) infections clearly differentiating infected from non-infected individuals or animals. For Chagas disease, a more extensive validation of the test was performed showing that the EMBIA platform displayed an excellent diagnostic performance almost indistinguishable, in terms of specificity and sensitivity, from a fluorescent immunomagnetic assay and the conventional ELISA using the same combination of antigens. This platform technology could potentially be applicable to diagnose other infectious and non-infectious diseases as well as detection and/or quantification of biomarkers at the POC and primary care settings.

Diagnosis of foot-and-mouth disease by electrochemical enzyme-linked immunoassay.

The development of an inmunosensor for the point-of-care detection of the foot-and-mouth cattle disease is presented. The detector is based on an ELISA method with electrochemical detection. A non-structural protein, 3ABC, is used to selectively detect antibodies is used to selectively detect anti-3ABC antibodies produced after infection. The biological test is performed onto a screen printed electrodes. A dedicated small, portable potentiostat is employed for the control of the sensors, as well as data acquisition, processing, and storage.

Exoneration reduces adult conflict's effects on preschoolers' cognitions, behavioral distress, and physiology.

In this experiment, the authors investigated the influence of exoneration from blame on children's overt behavioral distress and physiological reactivity following the presentation of overheard adult conflict. The participants were 48 children (48-71 months of age) and their mothers. Through random assignment, the authors presented 16 children with statements that exonerated them from an overheard disagreement between two adults, did not address 16 during a similar disagreement, and presented 16 with a neutral discussion of difficulties. Exonerated children responded with less distress than did nonaddressed children, but did not differ from children presented the neutral discussion, except for overt behavioral distress. Nonaddressed children most often blamed themselves for the argument. Exonerating statements may protect children from attributional error and resultant physiological arousal during adult conflict.