Generic Platform for the Multiplexed Targeted Electrochemical Detection of Osteoporosis-Associated Single Nucleotide Polymorphisms Using Recombinase Polymerase Solid-Phase Primer Elongation and Ferrocene-Modified Nucleoside Triphosphates.

Osteoporosis is a multifactorial disease influenced by genetic and environmental factors, which contributes to an increased risk of bone fracture, but early diagnosis of this disease cannot be achieved using current techniques. We describe a generic platform for the targeted electrochemical genotyping of SNPs identified by genome-wide association studies to be associated with a genetic predisposition to osteoporosis. The platform exploits isothermal solid-phase primer elongation with ferrocene-labeled nucleoside triphosphates. Thiolated reverse primers designed for each SNP were immobilized on individual gold electrodes of an array. These primers are designed to hybridize to the SNP site at their 3'OH terminal, and primer elongation occurs only where there is 100% complementarity, facilitating the identification and heterozygosity of each SNP under interrogation. The platform was applied to real blood samples, which were thermally lysed and directly used without the need for DNA extraction or purification. The results were validated using Taqman SNP genotyping assays and Sanger sequencing. The assay is complete in just 15 min with a total cost of 0.3€ per electrode. The platform is completely generic and has immense potential for deployment at the point of need in an automated device for targeted SNP genotyping with the only required end-user intervention being sample addition.

Novel nandrolone aptamer for rapid colorimetric detection of anabolic steroids.

The illicit use of anabolic androgenic steroids (AAS) as performance-enhancing drugs remains a global issue threatening not only the credibility of competitive sports but also public health due to the well-documented adverse effects they elicit. AAS abuse is not restricted only to professional sports, but also extends to recreational athletes and adolescents as well as in livestock production as growth-promoting agents. Testosterone and nandrolone are among the AAS most frequently exploited. Gas chromatography-mass spectrometry is the reference method for AAS detection, but it is strictly laboratory-based and cannot be performed on-site. The great potential of aptamers in bioanalytical applications and specifically for the development of simple analytical tools suitable for on-site analysis has been extensively documented. In this report, we describe the selection and identification of aptamers binding nandrolone, exhibiting affinity dissociation constants in the low nanomolar range. A label-free colorimetric assay based on gold nanoparticles was developed using one of these novel aptamers for the detection of nandrolone and/or its metabolites. The assay could be deployed for the rapid, on-site, facile and cost-effective screening of samples and provide qualitative visual results with a red to purple/blue color change being indicative of a positive result.

One-Pot SELEX: Identification of Specific Aptamers against Diverse Steroid Targets in One Selection.

Aptamers are well-established biorecognition molecules used in a wide variety of applications for the detection of their respective targets. However, individual SELEX processes typically performed for the identification of aptamers for each target can be quite time-consuming, labor-intensive, and costly. An alternative strategy is proposed herein for the simultaneous identification of different aptamers binding distinct but structurally similar targets in one single selection. This one-pot SELEX approach, using the steroids estradiol, progesterone, and testosterone as model targets, was achieved by combining the benefits of counter-SELEX with the power of next-generation sequencing and bioinformatics analysis. The pools from the last stage of the selection were compared in order to discover sequences with preferential abundance in only one of the pools. This led to the identification of aptamer candidates with potential specificity to a single steroid target. Binding studies demonstrated the high affinity of each selected aptamer for its respective target, and low nanomolar range dissociation constants calculated were similar to those previously reported for steroid-binding aptamers selected using traditional SELEX approaches. Finally, the selected aptamers were exploited in microtiter plate assays, achieving nanomolar limits of detection, while the specificity of these aptamers was also demonstrated. Overall, the one-pot SELEX strategy led to the discovery of aptamers for three different steroid targets in one single selection without compromising their affinity or specificity, demonstrating the power of this approach of aptamer discovery for the simultaneous selection of aptamers against multiple targets.

The characterization and validation of 17ß-estradiol binding aptamers.

The rapid and sensitive detection of small molecules is garnering increasing importance, and aptamers show great promise in replacing expensive, elaborate detection platforms exploiting chromatographic separation or antibody-based assays. The characterization of aptamer interaction with small molecule targets is not facile, and there is a mature need for a rapid, high-throughput technique for the analysis of aptamer-small molecule kinetics and affinity. In this work we present methodologies for the evaluation of aptamer-small molecule interactions, using the aptamers reported against the steroid 17ß-estradiol as a model system. Microscale thermophoresis, apta-PCR affinity assay and surface plasmon resonance were explored to evaluate the reported aptamers' binding properties in terms of affinity and specificity, and were demonstrated to be successfully applied to the analysis of aptamer-small molecule interactions.

Terapia de pareja: reflexiones sistémicas de un grupo en formación / Couple therapy: systemic reflections in a formative group

Se presentan a continuación algunas ideas y reflexiones planteadas como resultado del trabajo en equipo llevado a cabo durante el curso de terapia de pareja en el proceso de formación como especialistas en terapia familiar de la Universidad Católica Luis Amigó. Inicialmente se plantean algunos datos históricos e ideas acerca de la forma en que los cambios socioculturales han modificado las relaciones, aportando a la consolidación de nuevas formas de ser pareja. Se analizan características de dicha diada y el lugar del amor en su conformación, a la luz de nuevas comprensiones de las dinámicas del mundo contemporáneo y las problemáticas actuales. Concluyendo este recorrido se reconoce la vigencia e importancia de la terapia sistémica como alternativa para el abordaje de las problemáticas de pareja y se describen asuntos propios de dicha intervención, resaltando en ellos el lugar del terapeuta. Se concluye con la idea de la pareja como producto de un proceso de transformación transversalizado por la sociedad y la cultura, con diversos matices y formas de ser, que siendo cambiantes mantienen como constante la idea de pareja como un sistema interaccional complejo, en el que amor, deseo y pasión movilizan sentimientos y emociones vitales que revelan simultáneamente la fragilidad y fuerza del ser humano capaz de transformarlo. Vista de este modo la pareja es un escenario de intervención del terapeuta sistémico y la terapia el encuentro que posibilita el cambio.

Below are some ideas and reflections raised as a result of the team work carried out during the course of couple therapy in the process of training as specialists in family therapy at the Universidad Católica Luis Amigo. Initially, some historical data and ideas about the way in which socio-cultural changes have changed relationships are presented, contributing to the consolidation of new ways of being a couple. We analyze the characteristics of this dyad and the place of love in its conformation, in the light of new understandings of the dynamics of the contemporary world and the current problems. Concluding this tour, the validity and importance of systemic therapy is recognized as an alternative for the treatment of the problems of couples and describes specific issues of this intervention, highlighting in them the place of the therapist. It concludes with the idea of the couple as the product of a process of transformation that is mainstreamed by society and culture, with different nuances and ways of being, which, while being constant, maintain the idea of a constant as a complex interactional system in which Love, desire and passion mobilize feelings and vital emotions that simultaneously reveal the fragility and strength of the human being capable of transforming it. In this way the couple is a scenario of systemic therapist intervention and therapy the encounter that makes change possible.

Automated microsystem for electrochemical detection of cancer markers.

The development of a fully automated microsystem housing an amperometric immunosensor is presented. The microfluidic cell integrates reagent storage and electrochemical immunodetection and was applied for the detection of breast cancer markers. The main advantage of this system is that no external fluidic storage is required and the instrumental setup is thus greatly simplified. The fluidics of the microsystem is computer controlled and requires minimal end-user intervention. The analytical performance of the device was compared with a manually driven system and applied for the amperometric detection of the carcinoembryonic antigen (CEA) and cancer antigen 15-3 (CA15-3). This automation methodology greatly improves the analytical performance of the immunosensor in terms of accuracy and reproducibility as evidenced by a reduction of LOD observed for CEA and CA15-3 with respect to a manually driven system. Finally, the automated microsystem was applied for the analysis of real patient serum samples, demonstrating excellent correlation with a commercial ELISA.