Sandia es un laboratorio multiprograma operado por Sandia Corporation, una compañía de Lockheed Martin, para el Reino Dicho Departamento de Nacional de Energía Nuclear Security Administration bajo el contrato DE-AC04-94AL85000. Los autores agradecen la financiación del Laboratorio Nacional de Sandia Laboratories Dirección de Investigación y Desarrollo (LDRD) del programa.

1. Fabricación Microneedle <ol><li> Uso de modelado tridimensional software de SolidWorks (Dassault Systèmes SA, Vélizy, Francia), el diseño de una matriz de forma piramidal de microagujas huecas (Figura 1) 3-5. </li><li> Diseñar una estructura de apoyo para la matriz de microagujas con Magics RP 13 software (Materialise NV, Lovaina, Bélgica). La estructura de soporte permite que la resina para drenar desde el dispositivo durante la fabricación y proporciona una base sobre la cual las microagujas están construidos. Una estructura de soporte ejemplo se muestra en la Figura 1. </li><li> El apoyo vinculados y archivos de la matriz de microagujas se cargan en el software Perfactory RP (EnvisionTEC GmbH, Gladbeck, Alemania), que controla el proceso de fabricación. Dentro de este paquete de software, seleccione el número de matrices de microagujas que se fabrican y determinar la colocación de dispositivos en la placa de fabricación. </li><li> Ejecute la calibración en el modo de ultravioleta a 180 mW para el pr Perfactory rápidasistema de ototyping de fabricación y verificar la desviación de la energía es de ± 2 mW. </li><li> Una vez que la fabricación, retire las matrices de microagujas de la placa base y desarrollar en isopropanol durante 15 minutos. Seque las matrices con aire comprimido y curar las microagujas a temperatura ambiente durante 50 segundos en el Sistema de Otoflash postvulcanizado (EnvisionTEC GmbH, Gladbeck, Alemania) para asegurar la polimerización completa. </li><li> Validar la fabricación de microagujas a través de microscopía y verifique que cada agujero de microagujas es hueco y sin obstrucciones. Microagujas totalmente fabricados se muestra en la Figura 2. </li></ol> 2. Fabricación de Matrices electrodo de pasta de carbono <ol><li> Utilizar un 60 Modelo W 6,75 CO 2 de trama / vector de sistema láser (Universal Laser Systems, Inc., Phoenix, AZ) para cortar agujeros y exponer los subyacentes individualmente direccionables que conectan los cables de cobre en un cable plano flexible (21.039-0.249), que fue obtenida de una fuente comercial (MoLex conector Corp., Lisle, IL) (Figura 3 (A y B)). Coloque los cables flexibles planas en una plantilla para alinear apropiadamente en la placa de ablación con láser. Utilizar un enfoque de barrido para crear cavidades 500 m de diámetro en la porción aislante del cable flexible. Patrones para la ablación se crean en CorelDraw (Corel, Ottawa, Ontario) y se envía al sistema de láser. </li><li> Limpie los cables flexibles planos modificados con un aerógrafo que la acetona aerosoles a 40 psi. Terminar la limpieza ellos por aclarado con agua desionizada y el isopropanol. Compruebe bajo un microscopio que ninguna película aislante permanece sobre las tiras de cobre expuestas. </li><li> El siguiente paso es crear una cavidad sujeción para el envasado de pastas de carbono. Cinta Melinex (0,002 &quot;de espesor recubierta por una sola cara con adhesivo acrílico sensible a la presión) se realiza la ablación con el mismo patrón que las tiras de los electrodos, orientados sobre las tiras de electrodos ablacionadas y comprimidos a 3000 psi durante 2 minutos para asegurar una conexión adecuada. En esta case, el diámetro de la cavidad es de 750 micras. </li><li> Una capa adicional de cinta Melinex (0,004 &quot;de espesor recubierta por dos lados con adhesivo acrílico sensible a la presión) posteriormente se realiza la ablación en el mismo patrón que la cinta adhesiva de una sola cara y se utiliza después de la alineación para unir las matrices de microagujas a las matrices de electrodos de carbono pasta . </li></ol> 3. Síntesis de las pastas de carbono funcionales y embalaje de la caries de electrodos <ol><li> La pasta de glucosa carbono sensible se basa fuera de una receta anterior y se obtiene mezclando 10 mg de glucosa oxidasa y 2,2 mg de poli (etilenimina) hasta obtener una mezcla homogénea. 16 A esta mezcla, 60 mg de rodio sobre carbón en polvo ( 5% de carga), se añade. 40 mg de aceite mineral se añade y se mezcla posteriormente. Las pastas se almacenan a 4 ° C hasta su uso; las pastas se utilizan hasta una semana después de la preparación. </li><li> La pasta de pH sensible carbono se obtiene por mezcla de aceite mineral al 30% (w / w) y 70% (w / w), grafito powder. Paquete de pegar en la cavidad del electrodo, como se describe en la sección 3.4. Hacer solución de 10 mM de sal Fast Blue RR diazonio (4-benzoilamino-2 ,5-dimethoxybenzenediazonium cloruro hemi (cloruro de zinc) sal) en 0,5 M de ácido fosfórico. 17, Coloque una caída de 20 l de esta solución sobre el electrodo de pasta de envasado para 30 minutos para chemisorb espontáneamente el Fast Blue sal de diazonio de relaciones públicas. Enjuagar con agua desionizada y tienda en tampón o agua desionizada cuando no esté en uso. </li><li> La pasta de lactato de carbono sensible se basa fuera de una receta anterior y se obtiene mezclando 2,5 mg de rodio sobre carbono en polvo y 2,5 mg de lactato oxidasa, alternando entre 5 minutos de sonicación y 5 minutos de agitación durante cinco rotaciones. 18 </li><li> Embalaje de las pastas modificados en el preparado del cable plano flexible se logra mediante la aplicación de las pastas respectivos sobre las cavidades de los electrodos. Usando una fina pieza de plástico (por ejemplo, un borde de un plástico pesan barco) como una paleta y t paqueteél pega hasta una superficie lisa se logra. Repita el proceso con un segundo barco limpio de pesaje hasta que la pasta se elimina el exceso. Lavar con agua desionizada. Un esquema que muestra la ablación con láser para crear cavidades, envasado de pastas de carbono, y la integración microaguja (descrito en la sección 2 y 3) se presenta en la Figura 3. </li></ol> 4. La detección y la calibración del sensor <ol><li> Detección de lactato se logra mediante la medición de la respuesta del sensor chronoamperometric a -0,15 V y grabación de la corriente después de 15 segundos en 0,1 M de tampón fosfato (pH = 7,5). Figura 4 (a) contiene un esquema de la reacción electrocatalítica para la detección de lactato . </li><li> Detección de glucosa se ​​realiza de una manera similar mediante la medición de la respuesta del sensor chronoamperometric a -0,05 V y grabación de la corriente después de 15 segundos en tampón fosfato 0,1 M (pH 7,0). Figura 4 (b) contiene un esquema de la reacción electrocatalítica para detection de glucosa. </li><li> pH se controla mediante la ejecución cíclicos exploraciones voltamétricos desde -0,7 V a 0,8 V a 100 mV / s, y registrar la posición del pico de potencial oxidativo. Un esquema de las reacciones redox para la detección del pH se muestra en la Figura 5. </li><li> Las curvas de calibración para la glucosa y lactato sensores pueden ser creados por adiciones sucesivas de la analito respectivo; mediciones chronoamperometric se realiza después de cada adición analito como se describe en las secciones 5.1 y 5.2. Por otra parte, las mediciones fijas chronoamperometric potenciales se pueden hacer con agitación al tiempo que permite un tiempo suficiente (aproximadamente 10-100 segundos) entre cada adición analito para la estabilización actual. </li><li> Las curvas de calibración de pH puede ser creada mediante la medición de la posición del pico de potencial oxidativo durante una serie de valores de pH conocidos de 5 a 8 en incrementos de 1,0 unidad de pH y registrar voltamogramas cíclicos como se describe en la sección 5.3. </li></ol> 5. Representante Reresultados Al obtener curvas chronoamperometric (por ejemplo, para la detección de glucosa o la detección de lactato) en soluciones quiescentes con carbono modificada pega-llenos microagujas, la corriente disminuirán inmediatamente tras la aplicación del potencial de detección respectivo. Con el tiempo se reduzca a un valor de estado estacionario. Un resultado representativo se muestra en la Figura 6; este resultado se obtuvo a partir de 2 adiciones mM de lactato y de grabación a la microaguja lactato. La solución debe ser brevemente agitó después de cada adición de lactato. La corriente después de 15 segundos se eleva al aumentar la concentración de lactato; la respuesta de corriente puede ser utilizado para determinar la concentración de lactato en una solución desconocida. Alternativamente, la vigilancia continua puede ser utilizada en una solución agitada (o en una solución que fluye) como se demuestra por una solución con una concentración de glucosa en aumento (Figura 5). De nuevo, el aumento en la corriente al aumentar tque la concentración de glucosa puede ser utilizada para normalizar la respuesta de la glucosa a una solución desconocida. Tiempo suficiente se debe permitir que después de cada espiga con el fin de permitir que la solución se estabilice. Voltamogramas cíclicos en la microaguja sensible al pH en tampón fosfato 0,1 M se muestran más de cuatro soluciones diferentes de pH de 5 a 8 en incrementos de 1 unidad de pH en la Figura 6. Los cambios oxidativos pico potenciales con el aumento del pH; este fenómeno se utiliza como un indicador del valor del pH. <img alt="Figura 1" src="/files/ftp_upload/4067/4067fig1.jpg" /> Figura 1. Imágenes del archivo STL de la matriz de microagujas creado en Solidworks (A) y de la pantalla de impresión, que muestra la estructura de soporte (B). <img alt="Figura 2" src="/files/ftp_upload/4067/4067fig2.jpg" /> Figura 2. Micrografías electrónicas de barrido de la matriz de microagujas (A) y una microaguja único dentro de esta matriz (B). Figura 3. Esquema de montaje de cable plano flexible. Los pasos implicados incluyen la modificación del cable plano flexible (A), la ablación de los círculos estampados (B), añadiendo el inicialmente ablated capa Melinex, que está lleno con carbón pasta (C), así como la adición de la segunda capa ablacionada Melinex y acoplar el matriz de microagujas (D). <a href="http://www.jove.com/files/ftp_upload/4067/4067fig3large.jpg" target="_blank">Haga clic aquí para ver más grande la figura</a> . <img alt="Figura 4" src="/files/ftp_upload/4067/4067fig4.jpg" /> Figura 4. Calibración de lactato-sensible pasta con 15 exploraciones segundo chronoamperometric a -0,15 V en 0,1 M de tampón fosfato (pH = 7,5). Cada aumento de la corriente corresponde a una adición de 2 mM de lactato. <img alt="Figura 5" src="/files/ftp_upload/4067/4067fig5.jpg" /> Figura 5. </sTrong Calibración&gt; de pasta sensible a la glucosa ejecutando exploraciones chronoamperometric a -0,05 V. Cada disminución de la respuesta corresponde a una adición de 2 mM de glucosa. El 0,1 M de fosfato solución tampón (pH = 7,0) se agitó durante la calibración. Externa de Ag / AgCl y Pt referencia y electrodos de contador se utilizaron en este estudio. <img alt="Figura 6" src="/files/ftp_upload/4067/4067fig6.jpg" /> Figura 6. Voltamograma cíclico (CV) de carbono, sensible al pH pega en tampón fosfato 0,1 M pH durante 5-8 en incrementos de 1 unidad de pH (pH 8,0 = verde azulado, verde = pH 7,0, púrpura = pH 6,0, rojo = pH 5,0). Un CV quinto se usó para el análisis en comparación con referencia Ag / AgCl y electrodos de Pt de alambre de venta libre.

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No hay conflictos de interés declarado.

Múltiples aspectos del diseño de este sensor de microagujas basado fueron considerados antes de la fabricación del dispositivo. Para poder utilizar este sensor de detección en tiempo real, el tiempo de respuesta del sensor debe ser baja, en este protocolo, cada sensor de prueba mostraron un tiempo de respuesta por debajo de quince segundos. Las pastas se utilizan en este protocolo fueron elegidos también por su selectividad en vivo en ambientes que contienen biomoléculas electroactivos que pueden interfer...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td></tr><tr><td> Cable plano flexible </td><td> Molex </td><td> 3302/10SF </td></tr><tr><td> 0.003 &quot;Side cinta adhesiva </td><td> Melinex </td><td></td></tr><tr><td> 0.004 &quot;cinta de doble cara </td><td> Melinex </td><td></td></tr><tr><td> Lactato oxidasa </td><td> Sigma </td><td> L0638 </td></tr><tr><td> La glucosa oxidasa </td><td> Sigma </td><td> G7141 </td></tr><tr><td> Rodio sobre carbono </td><td> Sigma </td><td> 206164 </td></tr><tr><td> El polvo de grafito </td><td> Sigma </td><td> 385031000 </td></tr><tr><td> poli (etilenimina) </td><td> Acros </td><td> 178570010 </td></tr><tr><td> Aceite mineral </td><td> Sigma </td><td> M5904 </td></tr><tr><td> Glucosa </td><td> Sigma &lt;/ Td&gt; <td> G8270 </td></tr><tr><td> Lactato </td><td> Sigma </td><td> L1750 </td></tr><tr><td> Fast Blue RR sal </td><td> Sigma </td><td> F0500 </td></tr><tr><td> e-Shell 300 </td><td> EnvisionTEC </td><td></td></tr><tr><td> e-Shell 200 </td><td> EnvisionTEC </td><td></td></tr><tr><td> Ag / AgCl electrodo de referencia </td><td> Basi </td><td> MF-2052 </td></tr><tr><td> Pt de alambre </td><td> Basi </td><td></td></tr><tr><td> PGSTAT12 AutolabPotentiostat </td><td> EcoChemie </td><td></td></tr><tr><td> Perfactory RP </td><td> EnvisionTEC </td><td></td></tr><tr><td> Ottoflash postvulcanizado sistema de </td><td> EnvisionTEC </td><td></td></tr><tr><td> El ácido fosfórico </td><td> Pescador </td><td> A366-4 </td></tr><tr><td> 60W Modelo 6.75 de CO 2 raster / vector de sistema láser </td><td> UniversidadSal Laser Systems </td><td> PLS6.75 </td></tr><tr><td> CorelDraw </td><td> Corel </td><td></td></tr><tr><td> Solidworks </td><td> Dassault Systemes </td><td> 2009 </td></tr><tr><td> Magics RP13 </td><td> Materialise </td><td></td></tr></tbody></table>

hollow microneedle-based sensor for multiplexed transdermal electrochemical sensing

El desarrollo de un sistema de monitorización mínimamente invasiva multiplexado para el análisis rápido de las moléculas biológicamente relevantes podrían ofrecer a las personas que sufren de condiciones médicas crónicas evaluación fácil de su estado fisiológico inmediato. Además, podría servir como una herramienta de investigación para el análisis de las condiciones médicas complejas, multifactoriales. A fin de que dicho sensor multianalítico a ser realizado, debe ser mínimamente invasiva, el muestreo de líquido intersticial debe ocurrir sin dolor o daño para el usuario, y el análisis debe ser rápido, así como selectiva. Inicialmente desarrollado para el dolor sin la administración de fármacos, microagujas han sido utilizados para entregar vacunas y agentes farmacológicos (por ejemplo, insulina) a través de la piel. 1-2 Puesto que estos dispositivos acceder al espacio intersticial, microagujas que se integran con microelectrodos se puede utilizar como transdérmica electroquímicas sensores. Detección selectiva de la glucosa, glutamato, lactato, hácido peróxido ydrogen, y ascórbico se ha demostrado utilizando electrodos integrados microaguja-dispositivos con fibras de carbono, modificados pastas de carbono, y revestidos de platino-microagujas polímeros que sirven como elementos de transducción. 3-7,8 Esta tecnología de sensores de microagujas ha permitido un enfoque novedoso y de análisis sofisticado para la detección in situ y simultánea de múltiples analitos. Multiplexación ofrece la posibilidad de monitorear microambientes complejos, que son de otra manera difícil de caracterizar de una manera rápida y mínimamente invasiva. Por ejemplo, esta tecnología podría ser utilizada para el control simultáneo de los niveles extracelulares de, glucosa, lactato y pH 9, que son importantes indicadores metabólicos de estados de enfermedad 7,10-14 (por ejemplo, cáncer de proliferación) y acidosis inducida por el ejercicio. 15

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Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing

Este artículo detalla la construcción de un multiplexado basado en el sensor de microagujas. El dispositivo está siendo desarrollado para el muestreo in situ y el análisis electroquímico de múltiples analitos en una manera rápida y selectiva. Tenemos la visión de la medicina clínica y la investigación biomédica utiliza para estos sensores de microagujas basadas en.

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 ...

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JoVE Journal

Bioengineering

14.2K vistas.  University of North Carolina and North Carolina State University. Este artículo detalla la construcción de un multiplexado basado en el sensor de microagujas. El dispositivo está siendo desarrollado para el muestreo in situ y el análisis electroquímico de múltiples analitos en una manera rápida y selectiva. Tenemos la visión de la medicina clínica y la investigación biomédica utiliza para estos sensores de microagujas basadas en.

Video: Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor (IRIS)

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, environmental/agricultural/biodefense monitoring, nanobiotechnology, and more. For diagnostic applications, monitoring (detecting) the presence, absence, or abnormal expression of targeted proteomic or genomic biomarkers found in patient samples can be used to determine treatment approaches or therapy efficacy. In the research arena, information on molecular affinities and specificities are useful for fully characterizing the systems under investigation.
Many of the current systems employed to determine molecular concentrations or affinities rely on the use of labels. Examples of these systems include immunoassays such as the enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) techniques, gel electrophoresis assays, and mass spectrometry (MS). Generally, these labels are fluorescent, radiological, or colorimetric in nature and are directly or indirectly attached to the molecular target of interest. Though the use of labels is widely accepted and has some benefits, there are drawbacks which are stimulating the development of new label-free methods for measuring these interactions. These drawbacks include practical facets such as increased assay cost, reagent lifespan and usability, storage and safety concerns, wasted time and effort in labelling, and variability among the different reagents due to the labelling processes or labels themselves. On a scientific research basis, the use of these labels can also introduce difficulties such as concerns with effects on protein functionality/structure due to the presence of the attached labels and the inability to directly measure the interactions in real time.
Presented here is the use of a new label-free optical biosensor that is amenable to microarray studies, termed the Interferometric Reflectance Imaging Sensor (IRIS), for detecting proteins, DNA, antigenic material, whole pathogens (virions) and other biological material. The IRIS system has been demonstrated to have high sensitivity, precision, and reproducibility for different biomolecular interactions [1-3]. Benefits include multiplex imaging capacity, real time and endpoint measurement capabilities, and other high-throughput attributes such as reduced reagent consumption and a reduction in assay times. Additionally, the IRIS platform is simple to use, requires inexpensive equipment, and utilizes silicon-based solid phase assay components making it compatible with many contemporary surface chemistry approaches.
Here, we present the use of the IRIS system from preparation of probe arrays to incubation and measurement of target binding to analysis of the results in an endpoint format. The model system will be the capture of target antibodies which are specific for human serum albumin (HSA) on HSA-spotted substrates.

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor IRIS

Quantitative, high-throughput, real-time, and label-free biomolecular detection (DNA, protein, etc.) on SiO2 surfaces can be achieved using a simple interferometric technique which relies on LED illumination, minimal optical components, and a camera. The Interferometric Reflectance Imaging Sensor (IRIS) is inexpensive, simple to use, and amenable to microarray formats.

Biomoleculares de detección utilizando el sensor de reflectancia de imágenes interferométricas (IRIS)

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, ...

Investigación

Bioingeniería

Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers

The generation and detection of mechanical forces is a ubiquitous aspect of cell physiology, with direct relevance to cancer metastasis1, atherogenesis2 and wound healing3. In each of these examples, cells both exert force on their surroundings and simultaneously enzymatically remodel the extracellular matrix (ECM). The effect of forces on ECM has thus become an area of considerable interest due to its likely biological and medical importance4-7.
	Single molecule techniques such as optical trapping8, atomic force microscopy9, and magnetic tweezers10,11 allow researchers to probe the function of enzymes at a molecular level by exerting forces on individual proteins. Of these techniques, magnetic tweezers (MT) are notable for their low cost and high throughput. MT exert forces in the range of ~1-100 pN and can provide millisecond temporal resolution, qualities that are well matched to the study of enzyme mechanism at the single-molecule level12. Here we report a highly parallelizable MT assay to study the effect of force on the proteolysis of single protein molecules. We present the specific example of the proteolysis of a trimeric collagen peptide by matrix metalloproteinase 1 (MMP-1); however, this assay can be easily adapted to study other substrates and proteases.

In this article we describe the use of magnetic tweezers to study the effect of force on enzymatic proteolysis at the single molecule level in a highly parallelizable manner.

Multiplexado de una sola molécula de mediciones de la Fuerza proteólisis utilizando pinzas magnéticas

The  generation and detection of mechanical forces is a ubiquitous aspect of cell physiology,  with direct relevance to cancer metastasis1, atherogenesis2 ...

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 ...

Sensing of Barrier Tissue Disruption with an Organic Electrochemical Transistor

The gastrointestinal tract is an example of barrier tissue that provides a physical barrier against entry of pathogens and toxins, while allowing the passage of necessary ions and molecules. A breach in this barrier can be caused by a reduction in the extracellular calcium concentration. This reduction in calcium concentration causes a conformational change in proteins involved in the sealing of the barrier, leading to an increase of the paracellular flux. To mimic this effect the calcium chelator ethylene glycol-bis(beta-aminoethyl ether)-N,N,N&#39;,N&#39;-tetra acetic acid (EGTA) was used on a monolayer of cells known to be representative of the gastrointestinal tract. Different methods to detect the disruption of the barrier tissue already exist, such as immunofluorescence and&#160;permeability assays. However, these methods are time-consuming and costly and not suited to dynamic or high-throughput measurements. Electronic methods for measuring barrier tissue integrity also exist for measurement of the transepithelial resistance (TER), however these are often costly and complex. The development of rapid, cheap, and sensitive methods is urgently needed as the integrity of barrier tissue is a key parameter in drug discovery and pathogen/toxin diagnostics. The organic electrochemical transistor (OECT) integrated with barrier tissue forming cells has been shown as a new device capable of dynamically monitoring barrier tissue integrity. The device is able to measure minute variations in ionic flux with unprecedented temporal resolution and sensitivity, in real time, as an indicator of barrier tissue integrity. This new method is based on a simple device that can be compatible with high throughput screening applications and fabricated at low cost.

The Organic Electrochemical Transistor is integrated with live cells and used to monitor ion flux across the gastrointestinal epithelial barrier. In this study, an increase in ion flux, related to disruption of tight junctions, induced by the presence of the calcium chelator EGTA (ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetra acetic acid), is measured.

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 ...

A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration of pre-processing steps. Utilizing these devices towards the analysis of DNA hybridization events is important because it offers a technology for real time assessment of biomarkers at the point-of-care for various diseases. However, when the device footprint decreases the dominance of various physical phenomena increases. These phenomena influence the fabrication precision and operation reliability of the device. Therefore, there is a great need to accurately fabricate and operate these devices in a reproducible manner in order to improve the overall performance. Here, we describe the protocols and the methods used for the fabrication and the operation of a microfluidic-based electrochemical biochip for accurate analysis of DNA hybridization events. The biochip is composed of two parts: a microfluidic chip with three parallel micro-channels made of polydimethylsiloxane (PDMS), and a 3 x 3 arrayed electrochemical micro-chip. The DNA hybridization events are detected using electrochemical impedance spectroscopy (EIS) analysis. The EIS analysis enables monitoring variations of the properties of the electrochemical system that are dominant at these length scales. With the ability to monitor changes of both charge transfer and diffusional resistance with the biosensor, we demonstrate the selectivity to complementary ssDNA targets, a calculated detection limit of 3.8 nM, and a 13% cross-reactivity with other non-complementary ssDNA following 20 min&#160;of incubation. This methodology can improve the performance of miniaturized devices by elucidating on the behavior of diffusion at the micro-scale regime and by enabling the study of DNA hybridization events.

We present a microfluidic-based electrochemical biochip for DNA hybridization detection. Following ssDNA probe functionalization, the specificity, sensitivity, and detection limit are studied with complementary and non-complementary ssDNA targets. Results illustrate the influence of the DNA hybridization events on the electrochemical system, with a detection limit of 3.8 nM.

Un biochip electroquímico basado en microfluidos para el análisis de hibridación ADN-Label libre

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration ...

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer (SALB) Method

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related processes while conferring biocompatibility and biofunctionality to solid substrates. The spontaneous adsorption and rupture of phospholipid vesicles is the most commonly used method to form SLBs. However, under physiological conditions, vesicle fusion (VF) is limited to only a subset of lipid compositions and solid supports. Here, we describe a one-step general procedure called the solvent-assisted lipid bilayer (SALB) formation method in order to form SLBs which does not require vesicles. The SALB method involves the deposition of lipid molecules onto a solid surface in the presence of water-miscible organic solvents (e.g., isopropanol) and subsequent solvent-exchange with aqueous buffer solution in order to trigger SLB formation. The continuous solvent exchange step enables application of the method in a flow-through configuration suitable for monitoring bilayer formation and subsequent alterations using a wide range of surface-sensitive biosensors. The SALB method can be used to fabricate SLBs on a wide range of hydrophilic solid surfaces, including those which are intractable to vesicle fusion. In addition, it enables fabrication of SLBs composed of lipid compositions which cannot be prepared using the vesicle fusion method. Herein, we compare results obtained with the SALB and conventional vesicle fusion methods on two illustrative hydrophilic surfaces, silicon dioxide and gold. To optimize the experimental conditions for preparation of high quality bilayers prepared via the SALB method, the effect of various parameters, including the type of organic solvent in the deposition step, the rate of solvent exchange, and the lipid concentration is discussed along with troubleshooting tips. Formation of supported membranes containing high fractions of cholesterol is also demonstrated with the SALB method, highlighting the technical capabilities of the SALB technique for a wide range of membrane configurations.

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method

We present an experimental protocol to form a supported lipid bilayer on solid substrates without using lipid vesicles. We demonstrate a one-step method to form a lipid bilayer on silicon dioxide and gold as well as supported membranes with cholesterol-enriched domain for various biological applications.

Biomembrana Fabrication por la bicapa lipídica (SALB) Método-Solvente asistida

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related ...

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage dependent cells on the 2-dimensional surface of tissue culture plastic. More recently, there is a growing body of data demonstrating more in vivo-like behaviors of cells grown in 3-dimensional culture systems. This manuscript describes in detail the set-up and operation of a hollow fiber bioreactor system for the in vivo-like culture of mammalian cells. The hollow fiber bioreactor system delivers media to the cells in a manner akin to the delivery of blood through the capillary networks in vivo. The system is designed to fit onto the shelf of a standard CO2 incubator and is simple enough to be set-up by any competent cell biologist with a good understanding of aseptic technique. The systems utility is demonstrated by culturing the hepatocarcinoma cell line HepG2/C3A for 7 days. Further to this and in line with other published reports on the functionality of cells grown in 3-dimensional culture systems the cells are shown to possess increased albumin production (an important hepatic function) when compared to standard 2-dimensional tissue culture.

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

The functional behavior of cells in culture can be improved by culturing in more in vivo-like 3-dimensional culture environments16-21. This manuscript describes the set-up and operation of a hollow fiber bioreactor system for in vivo-like mammalian tissue culture.

Biorreactores de fibra hueca para<em&gt; En Vivo</em&gt; -como Cultivo de tejidos de mamíferos

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage ...

Extraction of Plant-based Capsules for Microencapsulation Applications

Microcapsules derived from plant-based spores or pollen provide a robust platform for a diverse range of microencapsulation applications. Sporopollenin exine capsules (SECs) are obtained when spores or pollen are processed so as to remove the internal sporoplasmic contents. The resulting hollow microcapsules exhibit a high degree of micromeritic uniformity and retain intricate microstructural features related to the particular plant species. Herein, we demonstrate a streamlined process for the production of SECs from Lycopodium clavatum spores and for the loading of hydrophilic compounds into these SECs. The current SEC isolation procedure has been recently optimized to significantly reduce the processing requirements which are conventionally used in SEC isolation, and to ensure the production of intact microcapsules. Natural L. clavatum spores are defatted with acetone, treated with phosphoric acid, and extensively washed to remove sporoplasmic contents. After acetone defatting, a single processing step using 85% phosphoric acid has been shown to remove all sporoplasmic contents. By limiting the acid processing time to 30 hr, it is possible to isolate clean SECs and avoid SEC fracturing, which has been shown to occur with prolonged processing time. Extensive washing with water, dilute acids, dilute bases, and solvents ensures that all sporoplasmic material and chemical residues are adequately removed. The vacuum loading technique is utilized to load a model protein (Bovine Serum Albumin) as a representative hydrophilic compound. Vacuum loading provides a simple technique to load various compounds without the need for harsh solvents or undesirable chemicals which are often required in other microencapsulation protocols. Based on these isolation and loading protocols, SECs provide a promising material for use in a diverse range of microencapsulation applications, such as, therapeutics, foods, cosmetics, and personal care products.

This protocol/manuscript describes a streamlined process for the production of sporopollenin exine capsules (SECs) from Lycopodium clavatum spores and for the loading of hydrophilic compounds into these SECs.

Las cápsulas de origen vegetal extracción de microencapsulación para Aplicaciones

Microcapsules derived from plant-based spores or pollen provide a robust platform for a diverse range of microencapsulation applications. Sporopollenin ...

Snap Chip for Cross-reactivity-free and Spotter-free Multiplexed Sandwich Immunoassays

Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands of micro-scale immunoassays performed simultaneously on a single chip. Sandwich assay format improves assay specificity by detecting each target with two antibodies, but suffers from cross-reactivity between reagents thus limiting their multiplexing capabilities. Antibody colocalization microarray (ACM) has been developed for cross-reactivity-free multiplexed protein detection, but requires an expensive spotter on-site for microarray fabrication during assays. In this work, we demonstrate a snap chip technology that transfers reagent from microarray-to-microarray by simply snapping two chips together, thus no spotter is needed during the sample incubation and subsequent application of detection antibodies (dAbs) upon storage of pre-spotted slides, dissociating the slide preparation from assay execution. Both single and double transfer methods are presented to achieve accurate alignment between the two microarrays and the slide fabrication for both methods are described. Results show that &#60;40 &#956;m alignment has been achieved with double transfer, reaching an array density of 625 spots/cm2. A 50-plexed immunoassay has been conducted to demonstrate the usability of the snap chip in multiplexed protein analysis. Limits of detection of 35 proteins are in the range of pg/mL.

We demonstrate a snap chip technology for performing cross-reactivity-free multiplexed sandwich immunoassays by simply snapping two slides. A snap apparatus is used for reliably transferring reagents from microarray-to-microarray. The snap chip can be used for any biochemical reactions requiring colocalization of different reagents without cross-contamination.

Rápido Chip para Cruz-reactivity-libre y observador de tiro multiplexado inmunoensayos de Sandwich

Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands ...

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, ...