Los autores de este trabajo no tienen intereses financieros en competencia.

1. PEDOT: PSS Preparación de la solución <ol><li> Para 50 ml de PEDOT: PSS, añadir glicol de etileno (aumenta la conductividad) en una relación en volumen de 1:4 (glicol de etileno a PEDOT: PSS), 0,5 l / ml de ácido dodecilbencenosulfónico (DBSA) como un agente tensioactivo, y 10 mg / ml de 3-glicidoxipropiltrimetoxisilano (GOPS) como un agente de reticulación para promover la adhesión del polímero a la realización de la lámina de vidrio. </li></ol> 2. OECT Fabricación (Figura 3) <ol><li> Definir los contactos de fuente y drenaje de oro térmicamente evaporados a través de la litografía del despegue: <ol><li> Girar fotorresistente abrigo en un portaobjetos de vidrio limpio normal a 3.000 rpm durante 30 seg. Dimensiones de vidrio son en 3 x 1 pulg </li><li> Definir patrones mediante fotolitografía. Las dimensiones pueden ser modificadas, sin embargo las dimensiones mencionadas aquí, se mostró a conducir a la sensibilidad óptima. A continuación, utilice un desarrollador. </li><li> Evaporar 5 nm y 100 nm de cromo y oro, respectivamente. </li><li> Despegue la fotoprotección en una acbaño de Etone durante 1 hora, dejando el sustrato con la fuente y el drenaje área de contactos de Au solamente. </li></ol></li><li> La longitud deseada de la PEDOT: PSS canal es 1 mm. Esto se logra mediante el estampado utilizando una técnica de desprendimiento de parileno-C (Pa-C): <ol><li> Cargue 3,5 g de Pa-C en la configuración del recubrimiento. Uniformemente evaporar 2 micras de parileno-C en la parte superior del sustrato de Au con contactos. </li><li> Patrón del canal mediante fotolitografía: <ol><li> Haga girar fotoprotector abrigo a 3.000 rpm durante 30 segundos. </li><li> Utilice una máscara para iluminar canal y de contacto dorada de 20 segundos a los rayos UV, la resina fotosensible expuesta será soluble en el revelador. </li><li> Utilice desarrollador para abrir el área del canal en el fotorresistente. </li></ol></li><li> Etch la Pa-C en el área del canal por un paso de plasma 15 min (O 2 (50 sccm) y CHF 4 (3 sccm) de plasma a 160 W) con el fin de abrir el canal y el contacto de oro. </li></ol></li><li> Depositar el PEDOT: PSS solución mezcla de capa de la vuelta al500 rpm durante 45 segundos. Hornear durante 30 segundos a 110 ° C. </li><li> Peel-off de la Pa-C para revelar el PEDOT: PSS canal del sustrato debajo. Este canal debe superponerse ligeramente con la UA contactos establecidos en el Protocolo 1. </li><li> Muestras Hornear durante 1 hora a 140 ° C bajo condiciones atmosféricas. </li></ol> 3. Conjunto de dispositivo <ol><li> Hacer PDMS mediante la mezcla de dos soluciones (por lo general suministrados juntos en un kit) en una relación de 1:10 de solución de curado en solución de base. Hornear durante 1 hora a 120 ° C. </li><li> Diseña bien mediante el uso de una perforadora y cortar un cuadrado alrededor del área deseada. </li><li> Pegar un PDMS bien en la parte superior del canal, para dar lugar a una zona de canal de aproximadamente 6 mm 2. Asegúrese de que los contactos de fuente y drenaje no están cubiertos. </li><li> Hacer un soporte de plástico con un agujero en él (se puede utilizar el soporte para la inserción del cultivo celular) y luego pegarlo en la parte superior de los PDMS. Deje que se seque durante la noche. </li><li> Pruebe el bien para el escape de cargar previamente con water. </li><li> Suelde los cables eléctricos en el contacto de origen y de drenaje con estaño. </li></ol> 4. Cultivo Celular <ol><li> Preparar medios de cultivo celular como sigue: <ol><li> En DMEM, añadir 1% de glutamina, 10% suero bovino fetal, 0,5% de penicilina-estreptomicina, y 0,1% de gentamicina. </li><li> Esterilizar el medio de cultivo celular usando un dispositivo de filtro estéril. </li></ol></li><li> Mantener células Caco-2 entre el paso 49 y 68 a 37 ° C en una atmósfera humidificada de 5% de CO 2, en medios de cultivo celular. </li><li> Divida las células una vez por semana usando tripsina y la semilla a 1,5 x 10 4 células / inserción. </li><li> Cambie medios de cultivo celular dos veces por semana durante 3 semanas. </li></ol> 5. Mediciones con OECT <ol><li> Conectar un alambre de Ag / AgCl a un SourceMeter para su uso como el electrodo de puerta. Sumergir la punta en medios de cultivo celular, que se utiliza como electrolito, como se ilustra en la Figura 1a. </li><li> Conecte los cables de la fuente y el drenaje de tél SourceMeter (configuración de doble canal). </li><li> Aplicar un pulso cuadrado de voltaje positivo (V GS) entre la puerta y la fuente (utilizado como electrodo de referencia: tierra). Un voltaje constante negativa entre el drenaje y la fuente (V DS) se aplica y se mide corriente correspondiente (I DS). </li><li> Utilice una recopilación de datos programa en ejecución PC. Parámetros OECT inscritos en un programa de adquisición personalizado deberían ser las siguientes: V DS = -0.2 V, V GS = 0,3 V, V GS a tiempo = 2 seg, fuera de tiempo = 28 seg. </li><li> Lectura de corriente de fuente-drenaje (I DS) y la corriente de puerta (I GS). Llevar a cabo la medición durante varios minutos para asegurar una señal de línea de base estable. </li></ol> 6. La integración de las células con OECT para la Medición Nota: Antes del experimento, la integridad de la capa de células puede ser verificada mediante la medición de TER con un dispositivo de espectroscopia de impedancia. TER de cada 3 semanas de edad inse células Caco-2rt debe estar por encima de 400 Ω. cm 2. <ol><li> Durante el tiempo libre de la medición OECT: Retire el electrodo de puerta del electrolito, incorporar el inserto de cultivo celular, y vuelva a colocar el electrodo de puerta interior del inserto de cultivo celular. </li><li> Llevar a cabo una medición de línea de base con las células durante varios minutos para asegurar una señal estable. Esta línea de base se utilizará como para el cálculo del valor normalizado correspondiente a una monocapa intacta (0). </li></ol> 7. Preparación y Presentación de los compuestos tóxicos <ol><li> Preparar una solución 1 M de EGTA en agua DI, primero ajustando el pH de la solución a 7,4 con Tris base. </li><li> Añadir la solución EGTA en el volumen adecuado para obtener la concentración deseada (por ejemplo, entre 5 mM y 100 mM) teniendo en cuenta el volumen. La EGTA debe ser añadido en la cámara basal durante el tiempo libre. </li><li> Medir de forma continua durante 90 min como en el Protocolo 5. Si la medición es being lleva a cabo a temperatura ambiente, la estabilidad de las células no se mantendrá constante después de 90 min. </li><li> Al final de la carrera, rayar la capa de células para dar lugar a una destrucción completa de la capa de barrera y medida durante 15 minutos, este punto se podría hacer mediante la eliminación del filtro y sumergir el electrodo de puerta en los medios de comunicación. Esta línea de base se utilizará como para el cálculo del valor normalizado correspondiente a una monocapa destruido. </li></ol>

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Este proyecto es apoyado por el FP7-PEOPLE-2009-RG, Marie Curie Proyecto No. 256367 (CELLTOX) y la propuesta del Consejo Europeo de Investigación ERC-2010-STG No 258966 (IONOSENSE), así como una subvención conjunta por el Conseil Regional de Provenza-Alpes-Costa Azul y CDL Pharma para ST. Damos las gracias a George Malliaras útil para los debates relacionados con el diseño y la función OECT.

Esta técnica proporciona un nuevo método para integrar un transistor electroquímica orgánica con células vivas para medir la integridad del tejido de barrera. Las principales ventajas de esta técnica son la rapidez y sensibilidad, sino también el bajo coste del dispositivo para el control dinámico de tejido de barrera. Como este método utiliza células vivas, un punto crítico es que asegúrese de usar una monocapa, lo que representa una capa de barrera intacta. Los parámetros de l...

El epitelio gastrointestinal es un ejemplo de tejido de barrera, que controla el paso de moléculas entre los diferentes compartimentos del cuerpo. El epitelio se compone de células columnares alargados unidos entre sí por complejos de proteínas que proporcionan una barrera física 1 contra patógenos y toxinas, mientras que permite el paso del agua y los nutrientes necesarios para sostener el cuerpo. Esta selectividad es debido a la polarización de las células epiteliales, lo que crea dos dominios de membrana diferentes: la parte apical de las células expuestas a la luz y la parte basal de las células ancladas en los tejidos subyacentes 2,3. Las uniones estrechas (TJ) son complejos de proteínas presentes en la parte apical de las células epiteliales y son parte de un complejo más amplio conocido como la unión apical 4. Flujo de iones a través del tejido de barrera puede ir ya sea a través de la transcelular (a través de la célula) o a través de un paracelular (entre dos células adyacentes) vía. La suma de losel transporte a través de ambas vías se conoce como la resistencia transepitelial. La unión apical es responsable de la regulación de los iones y moléculas que pasan a través de la barrera de 5,6 a través de una abertura específica y función de cierre. Una disfunción o alteración de estos complejos de proteínas es a menudo relacionado con la enfermedad 7-11. Además, muchos agentes patógenos / toxinas entéricos son conocidos para dirigirse específicamente a este complejo, entrando así al cuerpo y que conduce a la diarrea, muy probablemente como consecuencia de la desregulación masiva de flujo de iones / agua a través de la barrera 12-14. Tejido barrera también puede ser modificado cambiando el microambiente extracelular. Cadherina es una proteína crítica para la adhesión célula-célula, y está implicado en la formación de la unión apical. El calcio es necesario para la conformación estructural correctos de cadherina, y una disminución en el calcio extracelular se ha demostrado que resulta en la destrucción de la unión célula-célula y una abertura posterior de lavía paracelular entre las células 15. En este estudio, EGTA (ácido etilenglicol-bis (beta-aminoetil éter)-N, N, N &#39;, N&#39;-ácido acético TETRA), un quelante de calcio específico, fue utilizado para inducir una brecha en el tejido de barrera, ya que tiene ha demostrado que tienen un efecto rápido y drástico en paracelular flujo de iones a 16,17. Este quelante de calcio se utiliza en una monocapa confluente y diferenciada de la de la línea celular Caco-2. Cultivaron en insertos de cultivo de células, esta línea celular se sabe que se desarrollan las características del tracto gastrointestinal y se utiliza ampliamente por la industria farmacéutica para probar la absorción de fármacos 18,19. Métodos para monitorear la integridad del tejido barrera son abundantes. Estos métodos son a menudo óptico, basándose en la tinción de inmunofluorescencia de proteínas particulares que se sabe que en la unión apical 20, o basándose en la cuantificación de una molécula de trazador fluorescente que es normalmente impermeable al tejido barrera21,22. Sin embargo, los métodos de etiquetado libre (es decir, sin un fluoróforo / cromóforo) son preferibles como el uso de una etiqueta puede incurrir en artefactos, ya menudo aumenta el costo y tiempo de ensayo. Supervisión eléctrica, libre de marca de tejido barrera ha surgido recientemente como un método dinámico de supervisión 23. Por ejemplo recientes avances tecnológicos en la espectroscopia de impedancia eléctrica han permitido el desarrollo de un aparato de exploración disponibles comercialmente 24,25 que pueden medir la resistencia transepitelial (TER), una medición de la conductancia de iones a través de la capa de células. La electrónica orgánica ha creado una oportunidad única para interconectar el mundo de la electrónica y la biología 26,27 28,29 mediante el uso de polímeros conductores que pueden conducir tanto a portadores electrónicos e iónicos. Una nueva técnica para detectar infracciones en el tejido barrera utilizando la OECT 30-32 se introdujo recientemente. Este dispositivo se ha validado con las técnicas existentes nosed para evaluar integridad de la barrera de tejido, incluyendo inmunofluorescencia, ensayos de permeabilidad utilizando Lucifer amarillo y espectroscopia de impedancia usando el Cellzscope. En el caso de todos los compuestos tóxicos ensayados, la OECT se encontró para operar con una sensibilidad igual o mejor, y con el aumento de la resolución temporal, en comparación con las técnicas anteriores. En este dispositivo, de PEDOT: PSS, un polímero conductor que se ha demostrado ser estable y biocompatible 33,34, se utiliza como el material activo en el canal del transistor. La OECT está compuesta de drenaje y fuente electrodos a cada lado de un canal de polímero conductor. Este se coloca entonces en contacto con un electrolito, que forma una parte integral del dispositivo. Un electrodo de puerta se sumerge en el electrolito (Figura 1), y cuando se aplica una tensión de puerta positivo en la puerta, de cationes del electrolito son forzados dentro del canal, desdopado así el polímero conductor y que resulta en un cambio en la fuente-drenaje actual. El device es por lo tanto extremadamente sensibles a los cambios minutos en flujo iónico debido a la amplificación por el transistor. Una capa de células cultivadas en un inserto de cultivo celular se colocó entre el electrodo de puerta y del canal de polímero conductor. La presencia de una capa de células intactas actúa como barrera para los cationes de entrar en el polímero conductor, por lo tanto, en presencia de una monocapa intacta, la corriente disminuye de drenaje (Figura 2: transición de la región de A a B). En la presencia de un compuesto tóxico, el tejido de barrera perderá progresivamente su integridad, dejando que los cationes entran en la película de polímero y aumentando la corriente de drenaje (Figura 2: región C). Con esta técnica, la brecha en el tejido de barrera es visto por la modulación de la corriente de drenaje, que corresponde a la modulación del flujo a través de la monocapa. Este dispositivo es capaz de medir pequeñas variaciones en el flujo iónico con resolución temporal sin precedentes y la sensibilidad en tiempo real. Este Wil tecnologíal ser de interés en el campo de la toxicología de las pruebas de drogas, el diagnóstico de enfermedades o la investigación básica como el modelo de barrera puede ser adaptado fácilmente. Este método también ayudará a reducir la experimentación con animales, ya que permite la validación de los modelos in vitro para sustituir a los ensayos in vivo.

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sensing of barrier tissue disruption with an organic electrochemical transistor

El tracto gastrointestinal es un ejemplo de tejido de barrera que proporciona una barrera física contra la entrada de patógenos y toxinas, mientras que permite el paso de iones y moléculas necesarias. Una violación de esta barrera puede ser causada por una reducción en la concentración de calcio extracelular. Esta reducción en la concentración de calcio causa un cambio conformacional en las proteínas implicadas en el sellado de la barrera, lo que lleva a un aumento del flujo paracelular. Para imitar este efecto los quelantes de calcio ácido etilenglicol-bis (beta-aminoetil éter)-N, N, N &#39;, se utilizó ácido acético N&#39;-tetra (EGTA) en una monocapa de células conocidas para ser representativa del tracto gastrointestinal. Ya existen diferentes métodos para detectar la interrupción de la barrera de tejido, tales como ensayos de inmunofluorescencia y de permeabilidad. Sin embargo, estos métodos son mucho tiempo y costoso y no es adecuado para mediciones dinámicas o de alto rendimiento. Métodos electrónicos para la medición de tejido de barreraintegridad también existe para la medición de la resistencia transepitelial (TER), sin embargo, estos son a menudo costoso y complejo. El desarrollo de métodos rápidos, baratos y sensibles se necesita con urgencia como la integridad de los tejidos de barrera es un parámetro clave en el descubrimiento de fármacos y el diagnóstico de patógenos / toxina. El transistor de electroquímica orgánica (OECT) integrada con el tejido barrera de la formación de células se ha mostrado como un nuevo dispositivo capaz de monitorizar dinámicamente la integridad del tejido barrera. El dispositivo es capaz de medir las variaciones minutos en flujo iónico con resolución temporal sin precedentes y sensibilidad, en tiempo real, como un indicador de la integridad del tejido de barrera. Este nuevo método se basa en un dispositivo simple que puede ser compatible con las aplicaciones de cribado de alto rendimiento y fabricado a bajo costo.

Durante la primera etapa de la medición, la corriente de drenaje puede variar un poco, pero en la mayoría de los casos, debería permanecer estable (Figura 2, apartado a). Si la señal no es estable, el transistor debe ser desechado y sustituido. Esta comprobación de la estabilidad también asegura que las pérdidas iniciales en la conductividad del dispositivo no afectan a la medición posterior. Después de varios minutos de medición, el inserto con las células que forman el tejido de barrera se ...

Watch this Scientific Journal Video about Sensing of Barrier Tissue Disruption with an Organic Electrochemical Transistor at JoVE.com

Sensing of Barrier Tissue Disruption with an Organic Electrochemical Transistor

El transistor orgánico electroquímico está integrado con células vivas y se utiliza para controlar el flujo de iones a través de la barrera epitelial intestinal. En este estudio, un incremento en el flujo de iones, relacionados con la alteración de las uniones estrechas, inducida por la presencia de la EGTA quelante de calcio (ácido etilenglicol-bis (beta-aminoetil éter)-N, N, N &#39;, N&#39;-tetra acético ácido), se mide.

Detección de barrera interrupción del tejido con un transistor orgánico Electroquímica

The gastrointestinal tract is an example of barrier tissue that provides a physical barrier against entry of pathogens and toxins, while allowing the ...

sensing-barrier-tissue-disruption-with-an-organic-electrochemical

Research

JoVE Journal

Bioengineering

11.6K vistas.  Ecole Nationale Superieure des Mines.  El transistor orgánico electroquímico está integrado con células vivas y se utiliza para controlar el flujo de iones a través de la barrera epitelial intestinal. En este estudio, un incremento en el flujo de iones, relacionados con la alteración de las uniones estrechas, inducida por la presencia de la EGTA quelante de calcio (ácido etilenglicol-bis (beta-aminoetil éter)-N, N, N ', N'-tetra acético ácido), se mide. 

Video: Sensing of Barrier Tissue Disruption with an Organic Electrochemical Transistor

Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals suffering from chronic medical conditions facile assessment of their immediate physiological state. Furthermore, it could serve as a research tool for analysis of complex, multifactorial medical conditions. In order for such a multianalyte sensor to be realized, it must be minimally invasive, sampling of interstitial fluid must occur without pain or harm to the user, and analysis must be rapid as well as selective.
Initially developed for pain-free drug delivery, microneedles have been used to deliver vaccines and pharmacologic agents (e.g., insulin) through the skin.1-2 Since these devices access the interstitial space, microneedles that are integrated with microelectrodes can be used as transdermal electrochemical sensors. Selective detection of glucose, glutamate, lactate, hydrogen peroxide, and ascorbic acid has been demonstrated using integrated microneedle-electrode devices with carbon fibers, modified carbon pastes, and platinum-coated polymer microneedles serving as transducing elements.3-7,8
This microneedle sensor technology has enabled a novel and sophisticated analytical approach for in situ and simultaneous detection of multiple analytes. Multiplexing offers the possibility of monitoring complex microenvironments, which are otherwise difficult to characterize in a rapid and minimally invasive manner. For example, this technology could be utilized for simultaneous monitoring of extracellular levels of, glucose, lactate and pH,9 which are important metabolic indicators of disease states7,10-14 (e.g., cancer proliferation) and exercise-induced acidosis.15

This article details the construction of a multiplexed microneedle-based sensor. The device is being developed for in situ sampling and electrochemical analysis of multiple analytes in a rapid and selective manner. We envision clinical medicine and biomedical research uses for these microneedle-based sensors.

Hollow Microneedle basado en sensores para la detección electroquímica multiplexado transdérmica

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals ...

Investigación

Bioingeniería

Bacterial Detection &amp; Identification Using Electrochemical Sensors

Electrochemical sensors are widely used for rapid and accurate measurement of blood glucose and can be adapted for detection of a wide variety of analytes. Electrochemical sensors operate by transducing a biological recognition event into a useful electrical signal. Signal transduction occurs by coupling the activity of a redox enzyme to an amperometric electrode. Sensor specificity is either an inherent characteristic of the enzyme, glucose oxidase in the case of a glucose sensor, or a product of linkage between the enzyme and an antibody or probe.
Here, we describe an electrochemical sensor assay method to directly detect and identify bacteria. In every case, the probes described here are DNA oligonucleotides. This method is based on sandwich hybridization of capture and detector probes with target ribosomal RNA (rRNA). The capture probe is anchored to the sensor surface, while the detector probe is linked to horseradish peroxidase (HRP). When a substrate such as 3,3',5,5'-tetramethylbenzidine (TMB) is added to an electrode with capture-target-detector complexes bound to its surface, the substrate is oxidized by HRP and reduced by the working electrode. This redox cycle results in shuttling of electrons by the substrate from the electrode to HRP, producing current flow in the electrode.

We describe an electrochemical sensor assay method for rapid bacterial detection and identification. The assay involves a sensor array functionalized with DNA oligonucleotide capture probes for ribosomal RNA (rRNA) species-specific sequences. Sandwich hybridization of target rRNA with the capture probe and a horseradish peroxidase-linked DNA oligonucleotide detector probe produces a measurable amperometric current.

Bacterial Detección e Identificación Usando sensores electroquímicos

Electrochemical  sensors are widely used for rapid and accurate measurement of blood glucose and  can be adapted for detection of a wide variety of ...

Electric Cell-substrate Impedance Sensing for the Quantification of Endothelial Proliferation, Barrier Function, and Motility

Electric Cell-substrate Impedance Sensing (ECIS) is an in vitro impedance measuring system to quantify the behavior of cells within adherent cell layers. To this end, cells are grown in special culture chambers on top of opposing, circular gold electrodes. A constant small alternating current is applied between the electrodes and the potential across is measured. The insulating properties of the cell membrane create a resistance towards the electrical current flow resulting in an increased electrical potential between the electrodes. Measuring cellular impedance in this manner allows the automated study of cell attachment, growth, morphology, function, and motility. Although the ECIS measurement itself is straightforward and easy to learn, the underlying theory is complex and selection of the right settings and correct analysis and interpretation of the data is not self-evident. Yet, a clear protocol describing the individual steps from the experimental design to preparation, realization, and analysis of the experiment is not available. In this article the basic measurement principle as well as possible applications, experimental considerations, advantages and limitations of the ECIS system are discussed. A guide is provided for the study of cell attachment, spreading and proliferation; quantification of cell behavior in a confluent layer, with regard to barrier function, cell motility, quality of cell-cell and cell-substrate adhesions; and quantification of wound healing and cellular responses to vasoactive stimuli. Representative results are discussed based on human microvascular (MVEC) and human umbilical vein endothelial cells (HUVEC), but are applicable to all adherent growing cells.

This protocol reviews Electric Cell-substrate Impedance Sensing, a method to record and analyze the impedance spectrum of adherent cells for the quantification of cell attachment, proliferation, motility, and cellular responses to pharmacological and toxic stimuli. Detection of endothelial permeability and assessment of cell-cell and cell-substrate contacts are emphasized.

Eléctrico de la célula-sustrato Impedancia de detección para la cuantificación de la proliferación endotelial, la función de barrera, y la motilidad

Electric Cell-substrate Impedance Sensing (ECIS) is an in vitro impedance measuring system to quantify the behavior of cells within adherent cell layers. ...

Preparation of Silicon Nanowire Field-effect Transistor for Chemical and Biosensing Applications

Surveillance using biomarkers is critical for the early detection, rapid intervention, and reduction in the incidence of diseases. In this study, we describe the preparation of polycrystalline silicon nanowire field-effect transistors (pSNWFETs) that serve as biosensing devices for biomarker detection. A protocol for chemical and biomolecular sensing by using pSNWFETs is presented. The pSNWFET device was demonstrated to be a promising transducer for real-time, label-free, and ultra-high-sensitivity biosensing applications. The source/drain channel conductivity of a pSNWFET is sensitive to changes in the environment around its silicon nanowire (SNW) surface. Thus, by immobilizing probes on the SNW surface, the pSNWFET can be used to detect various biotargets ranging from small molecules (dopamine) to macromolecules (DNA and proteins). Immobilizing a bioprobe on the SNW surface, which is a multistep procedure, is vital for determining the specificity of the biosensor. It is essential that every step of the immobilization procedure is correctly performed. We verified surface modifications by directly observing the shift in the electric properties of the pSNWFET following each modification step. Additionally, X-ray photoelectron spectroscopy was used to examine the surface composition following each modification. Finally, we demonstrated DNA sensing on the pSNWFET. This protocol provides step-by-step procedures for verifying bioprobe immobilization and subsequent DNA biosensing application.

We describe key steps for biosensing by using polysilicon nanowire field-effect transistors, including the preparation of the device and the immobilization and confirmation of a DNA molecular probe on the nanowire surface, as well as conditions for DNA sensing.

Preparación de silicio de nanocables de transistor de efecto de campo de químicos y biosensores Aplicaciones

Surveillance using biomarkers is critical for the early detection, rapid intervention, and reduction in the incidence of diseases. In this study, we ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

Ingeniería de tejidos por intrínseca Vascularización en una<em&gt; En Vivo</em&gt; Cámara Ingeniería de Tejidos

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

Wet Chemistry and Peptide Immobilization on Polytetrafluoroethylene for Improved Cell-adhesion

Endowing materials surface with cell-adhesive properties is a common strategy in biomaterial research and tissue engineering. This is particularly interesting for already approved polymers that have a long standing use in medicine because these materials are well characterized and legal issues associated with the introduction of newly synthesized polymers may be avoided. Polytetrafluoroethylene (PTFE) is one of the most frequently employed materials for the manufacturing of vascular grafts but the polymer lacks cell adhesion promoting features. Endothelialization, i.e., complete coverage of the grafts inner surface with a confluent layer of endothelial cells is regarded key to optimal performance, mainly by reducing thrombogenicity of the artificial interface.
This study investigates the growth of endothelial cells on peptide-modified PTFE and compares these results to those obtained on unmodified substrate. Coupling with the endothelial cell adhesive peptide Arg-Glu-Asp-Val (REDV) is performed via activation of the fluorin-containing polymer using the reagent sodium naphthalenide, followed by subsequent conjugation steps. Cell culture is accomplished using Human Umbilical Vein Endothelial Cells (HUVECs) and excellent cellular growth on peptide-immobilized material is demonstrated over a two-week period.

Cell-adhesiveness is key to many approaches in biomaterial research and tissue engineering. A step-by-step technique is presented using wet-chemistry for the surface modification of the important polymer PTFE with peptides.

Química húmeda y el péptido Inmovilización de politetrafluoroetileno para la adhesión celular mejorada

Endowing materials surface with cell-adhesive properties is a common strategy in biomaterial research and tissue engineering. This is particularly ...

Development of an Electrochemical DNA Biosensor to Detect a Foodborne Pathogen

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, significantly affecting the rate of human mortality and morbidity. Conventional methods for the detection of V. parahaemolyticus such as culture-based methods, immunological assays, and molecular-based methods require complicated sample handling and are time-consuming, tedious, and costly. Recently, biosensors have proven to be a promising and comprehensive detection method with the advantages of fast detection, cost-effectiveness, and practicality. This research focuses on developing a rapid method of detecting V. parahaemolyticus with high selectivity and sensitivity using the principles of DNA hybridization. In the work, characterization of synthesized polylactic acid-stabilized gold nanoparticles (PLA-AuNPs) was achieved using X-ray Diffraction (XRD), Ultraviolet-visible Spectroscopy (UV-Vis), Transmission Electron Microscopy (TEM), Field-emission Scanning Electron Microscopy (FESEM), and Cyclic Voltammetry (CV). We also carried out further testing of stability, sensitivity, and reproducibility of the PLA-AuNPs. We found that the PLA-AuNPs formed a sound structure of stabilized nanoparticles in aqueous solution. We also observed that the sensitivity improved as a result of the smaller charge transfer resistance (Rct) value and an increase of active surface area (0.41 cm2). The development of our DNA biosensor was based on modification of a screen-printed carbon electrode (SPCE) with PLA-AuNPs and using methylene blue (MB) as the redox indicator. We assessed the immobilization and hybridization events by differential pulse voltammetry (DPV). We found that complementary, non-complementary, and mismatched oligonucleotides were specifically distinguished by the fabricated biosensor. It also showed reliably sensitive detection in cross-reactivity studies against various food-borne pathogens and in the identification of V. parahaemolyticus in fresh cockles.

A protocol for the development of an electrochemical DNA biosensor comprising a polylactic acid-stabilized, gold nanoparticles-modified, screen-printed carbon electrode to detect Vibrio parahaemolyticus is presented.

Desarrollo de un Biosensor de ADN electroquímico para la detección de un patógeno de transmisión alimentaria

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, ...

In Vitro Multiparametric Cellular Analysis by Micro Organic Charge-modulated Field-effect Transistor Arrays

Modern electrophysiology has been constantly fueled by the parallel development of increasingly sophisticated tools and materials. In turn, discoveries in this field have driven technological progress in a back-and-forth process that ultimately determined the impressive achievements of the past 50 years. However, the most employed devices used for cellular interfacing (namely, the microelectrode arrays and microelectronic devices based on transistors) still present several limitations such as high cost, the rigidity of the materials, and the presence of an external reference electrode. To partially overcome these issues, there have been developments in a new scientific field called organic bioelectronics, resulting in advantages such as lower cost, more convenient materials, and innovative fabrication techniques.
Several interesting new organic devices have been proposed during the past decade to conveniently interface with cell cultures.&#160;This paper presents the protocol for the fabrication of devices for cellular interfacing based on the organic charge-modulated field-effect transistor (OCMFET). These devices, called micro OCMFET arrays (MOAs), combine the advantages of organic electronics and the peculiar features of the OCMFET to prepare transparent, flexible, and reference-less tools with which it is possible to monitor both the electrical and the metabolic activities of cardiomyocytes and neurons in vitro, thus allowing a multiparametric evaluation of electrogenic cell models.

In Vitro Multiparametric Cellular Analysis by Micro Organic Charge-modulated Field-effect Transistor Arrays

Here, we present the fabrication protocol of an organic charge-modulated field-effect transistor (OCMFET)-based device for in vitro cellular interfacing. The device, called a micro OCMFET array, is a flexible, low-cost, and reference-less device, which will enable the monitoring of the electrical and metabolic activities of electroactive cell cultures.

In vitro Análisis celular multiparamétrico mediante matrices de transistores de efecto de campo moduladas por carga micro orgánica

Modern electrophysiology has been constantly fueled by the parallel development of increasingly sophisticated tools and materials. In turn, discoveries in ...

Disruption of the Blood-Spinal Cord Barrier Using Low-Intensity Focused Ultrasound in a Rat Model

Low-intensity focused ultrasound (LIFU) uses ultrasonic pulsations at lower intensities than ultrasound and is being tested as a reversible and precise neuromodulatory technology. Although LIFU-mediated blood-brain barrier (BBB) opening has been explored in detail, no standardized technique for blood-spinal cord barrier (BSCB) opening has been established to date. Therefore, this protocol presents a method for successful BSCB disruption using LIFU sonication in a rat model, including descriptions of animal preparation, microbubble administration, target selection&#160;and localization, as well as BSCB disruption visualization and confirmation. The approach reported here is particularly useful for researchers who need a fast and cost-effective method to test and confirm target localization and precise BSCB disruption in a small animal model with a focused ultrasound transducer, evaluate the BSCB efficacy of sonication parameters, or explore applications for LIFU at the spinal cord, such as drug delivery, immunomodulation, and neuromodulation. Optimizing this protocol for individual use is recommended, especially for advancing future preclinical, clinical, and translational work.

Disruption of the blood-spinal cord barrier (BSCB) can be successfully achieved with the intravenous administration of microbubbles and the application of low-intensity focused ultrasound (LIFU). This protocol details the opening of the BSCB using LIFU in a rodent model, including equipment setup, microbubble injection, target localization, and BSCB disruption visualization.

Alteración de la barrera hemato-médula espinal mediante ultrasonido focalizado de baja intensidad en un modelo de rata

Low-intensity focused ultrasound (LIFU) uses ultrasonic pulsations at lower intensities than ultrasound and is being tested as a reversible and precise ...

Evaluación de los cambios en el comportamiento de las células inducidos por MOF mediante la detección eléctrica del sustrato de la célula

Electric Cell-Substrate Sensing for Real-Time Evaluation of Metal-Organic Framework Toxicological Profiles

Metal-organic frameworks (MOFs) are hybrids formed through the coordination of metal ions and organic linkers in organic solvents. The implementation of MOFs in biomedical and industrial applications has led to concerns regarding their safety. Herein, the profile of a selected MOF, a zeolitic imidazole framework, was evaluated upon exposure to human lung epithelial cells. The platform for evaluation was a real-time technique (i.e., electric cell-substrate impedance sensing [ECIS]). This study identifies and discusses some of the deleterious effects of the selected MOF on the exposed cells. Furthermore, this study demonstrates the benefits of using the real-time method versus other biochemical assays for comprehensive cell evaluations. The study concludes that observed changes in cell behavior could hint at possible toxicity induced upon exposure&#160;to MOFs of different physicochemical characteristics and the dosage of those frameworks being used. By understanding changes in cell behavior, one foresees the ability to improve safe-by-design strategies of MOFs to be used for biomedical applications by specifically tailoring their physicochemical characteristics.

Autor destacado: Avances en la evaluación de la respuesta de las células epiteliales pulmonares humanas a las estructuras metal-orgánicas 

The following study evaluates the toxicological profile of a selected metal-organic framework utilizing electric cell-substrate impedance sensing (ECIS), a real-time, high-throughput screening technique.

Detección eléctrica de sustratos de células para la evaluación en tiempo real de perfiles toxicológicos de estructuras metal-orgánicas

Metal-organic frameworks (MOFs) are hybrids formed through the coordination of metal ions and organic linkers in organic solvents. The implementation of ...