Sandia é laboratório multiprogram operado por Sandia Corporation, uma empresa da Lockheed Martin, para o Reino demonstrados Departamento de Administração da Energia Nacional de Segurança Nuclear no âmbito do contrato DE-AC04-94AL85000. Os autores agradecem o financiamento do Laboratório Sandia National Laboratories &quot;Direção de Pesquisa e Desenvolvimento de programa (LDRD).

1. Fabricação de microagulhas <ol><li> Usando modelagem tridimensional software Solidworks (Dassault Systemes SA, Velizy, França), criar um piramidal em forma de matriz de microagulhas oco (Figura 1) 3-5. </li><li> Projetar uma estrutura de suporte para a matriz de microagulhas usando Magics RP 13 software (Materialise NV, Leuven, Bélgica). A estrutura de suporte permite que a resina para drenar a partir do dispositivo durante a fabricação e fornece uma base sobre a qual as microagulhas são construídas. Uma estrutura de suporte exemplo é mostrado na Figura 1. </li><li> O apoio ligado e arquivos de matriz microagulhas são enviados para o RP Perfactory software (EnvisionTEC GmbH, Gladbeck, Alemanha), que controla o processo de fabricação. Dentro deste pacote de software, escolha o número de matrizes microneedle a ser fabricados e determinar a colocação de dispositivos na placa de fabricação. </li><li> Executar calibração em modo de ultravioleta a 180 mW para a rápida Perfactory prototyping sistema de fabricação e verificar o desvio de energia está dentro de ± 2 mW. </li><li> Uma vez que a fabricação está completa, remove as matrizes microneedle a partir da placa de base e desenvolver-se em isopropanol, durante 15 minutos. Secam-se as matrizes com ar comprimido e curar as microagulhas à temperatura ambiente durante 50 segundos no Sistema Postcuring Otoflash (EnvisionTEC GmbH, Gladbeck, Alemanha) para assegurar a polimerização completa. </li><li> Validar fabricação microneedle via microscopia e verificar que cada furo de microagulhas é oca e sem obstruções. Microagulhas totalmente-fabricadas são mostrados na Figura 2. </li></ol> 2. Fabricação de matrizes de carbono eletrogel <ol><li> Utilizar um modelo de 60 W 6,75 CO 2 raster / vector sistema de laser (Universal Laser Systems, Inc., Scottsdale, AZ) para cortar furos e expor as subjacentes individualmente endereçáveis ​​fios de cobre de ligação de um cabo flexível plana (21039-0249), que foi obtido a partir de uma fonte comercial (Molex conector Corp, Lisle, IL) (Figura 3 (A e B)). Colocar os cabos planos flexíveis em um gabarito para alinhar adequadamente los na placa de ablação por laser. Usar uma abordagem rastering para criar 500 cavidades diâmetro uM na porção de isolamento do cabo flexível. Padrões para ablação são criados no CorelDraw (Corel, Ottawa, Ontario) e enviado para o sistema de laser. </li><li> Limpe os planos modificados cabos flexíveis com um aerógrafo que sprays acetona a 40 psi. Termine a limpeza-los por lavagem com água deionizada e isopropanol. Verificar sob um microscópio de que nenhum filme isolante permanece sobre as tiras de cobre expostas. </li><li> O passo seguinte é o de criar uma cavidade de retenção para a embalagem de pastas de carbono. Fita Melinex (0,002 espessura &quot;revestida em um único lado com adesivo acrílico sensível à pressão) é cauterizado com o mesmo padrão que as tiras de eléctrodos, orientadas ao longo das tiras de eléctrodos cauterizado, e comprimido a 3000 psi durante 2 minutos para assegurar uma ligação adequada. Em esta case, o diâmetro da cavidade é de 750 mM. </li><li> Uma camada adicional de fita Melinex (0,004 &quot;espessura revestida em dois lados com adesivo acrílico sensível à pressão) é subsequentemente ablated no mesmo padrão que a fita de lado único adesivo e é usado após o alinhamento para ligação das matrizes microneedle para as matrizes de carbono eletrogel . </li></ol> 3. Síntese das Pastas de carbono funcionais e Embalagem de cavidades Eletrodo <ol><li> A glicose de pasta de carbono sensível é baseado fora de uma receita anterior, e é obtida por mistura de 10 mg de glicose oxidase e 2,2 mg de poli (etilenimina) até uma mistura homogénea. 16 A esta mistura, 60 mg de ródio sobre carbono em pó ( 5% de carga) é adicionado. 40 mg de óleo mineral é adicionado e, subsequentemente, misturado. As pastas são armazenadas a 4 ° C até à sua utilização, as pastas são utilizados até uma semana após a preparação. </li><li> O pH de pasta de carbono sensível é obtida por mistura de óleo mineral a 30% (w / w) e 70% (w / w) de grafite powder. Embale cole na cavidade do eletrodo como descrito na seção 3.4. Fazer solução de 10 Fast Blue RR sal de diazónio mM (4-benzoilamino-2 ,5-dimethoxybenzenediazonium cloreto de hemi sal (cloreto de zinco)) em 0,5 M de ácido fosfórico. 17, colocar uma gota 20 uL desta solução sobre o eléctrodo de pasta embalado para 30 minutos para espontaneamente chemisorb o Fast Blue sal de diazônio PR. Enxágüe com água deionizada e armazenar em buffer ou água deionizada quando não em uso. </li><li> A pasta de carbono lactato sensível é baseado fora de uma receita anterior, e é obtida por mistura de 2,5 mg de ródio sobre carbono em pó e 2,5 mg de lactato-oxidase, alternando entre 5 minutos de sonicação e 5 minutos de agitação em vórtice durante cinco rotações. 18 </li><li> Embalagem das pastas modificados no cabo plano preparada flexível é realizado através da aplicação das pastas respectivas sobre as cavidades dos eléctrodos. Usando uma parte fina de plástico (por exemplo, uma aresta de um barco de pesar de plástico) como uma espátula e t pacoteele colar até uma superfície lisa é alcançada. Repita com um barco limpo segunda pesagem até colar em excesso é removido. Lavar com água desionizada. Um esquema que mostra a ablação de laser para criar cavidades, a embalagem de pastas de carbono, e de microagulhas integração (descrito no ponto 2 e 3) é apresentada na Figura 3. </li></ol> 4. Detecção e calibração de sensores <ol><li> Detecção de lactato é conseguido através da medição da resposta cronoamperométrica do sensor a -0,15 V e de registo da corrente após 15 segundos em tampão fosfato 0,1 M (pH = 7,5). Figura 4 (a) contém um diagrama esquemático da reacção eletrocatalítica para a detecção de lactato . </li><li> Detecção de glicose é realizada de uma maneira semelhante, medindo a resposta cronoamperométrica do sensor a -0,05 V e de registo da corrente após 15 segundos em tampão fosfato 0,1 M (pH 7,0). Figura 4 (b) contém um diagrama esquemático da reacção eletrocatalítica para detection de glicose. </li><li> pH é monitorizado por execução cíclicos varreduras voltamétricas de -0,7 V a 0,8 V a 100 mV / s, e gravando a posição do pico potencial oxidativo. Um esquemática das reacções redox para a detecção de pH é mostrado na Figura 5. </li><li> As curvas de calibração para a glicose e os sensores de lactato pode ser criado por adições sucessivas de substância a analisar respectivo; medições cronoamperométricos estão realizando depois de cada adição do analito, como descrito nas secções 5.1 e 5.2. Alternativamente, fixos potenciais medições cronoamperométricos pode ser feita sob agitação enquanto permite que o tempo suficiente (~ de 10-100 segundos) entre cada adição analito para a estabilização de corrente. </li><li> as curvas de calibração de pH pode ser criado através da medição da posição do potencial de pico oxidativo longo de uma série de valores de pH conhecidos 5-8 em incrementos de 1,0 unidades de pH e gravando voltamogramas cíclicos como descrito na secção 5.3. </li></ol> 5. Re Representantesultados Quando a obtenção de curvas de cronoamperometria (por exemplo, para a detecção de glicose ou de detecção de lactato) em soluções quiescentes de carbono modificado com cole-cheias microagulhas, a corrente irá diminuir de imediato a pedido do respectivo potencial de detecção. Será, eventualmente decair para um valor de estado estacionário. Um resultado representativo é mostrado na Figura 6, sendo este resultado foi obtido a partir de 2 adições mM de lactato e gravação na microagulhas lactato. A solução deve ser brevemente agitada após cada adição de lactato. A corrente após 15 segundos sobe sobre o aumento da concentração de lactato; a resposta de corrente pode então ser usado para determinar a concentração de lactato em uma solução desconhecida. Alternativamente, a monitorização contínua pode ser usado em uma solução agitada (ou em uma solução de fluxo) tal como demonstrado por uma solução com uma concentração de glicose crescente (Figura 5). Novamente, o aumento na corrente mediante o aumento tele concentração de glucose pode ser usada para padronizar a resposta da glucose a uma solução desconhecida. Tempo suficiente deve ser permitido após cada espiga, a fim de permitir que a solução para estabilizar. Os voltamogramas cíclicos na microagulhas sensível ao pH em 0,1 M de tampão fosfato são mostrados ao longo de quatro soluções de pH diferentes, de 5 a 8 em incrementos de 1 unidade de pH na Figura 6. Os turnos oxidativas de pico potenciais com o aumento do pH; este fenómeno é usado como um indicador do valor do pH. <img alt="A Figura 1" src="/files/ftp_upload/4067/4067fig1.jpg" /> Figura 1. Imagens do arquivo STL da matriz de microagulhas criado em Solidworks (A) e da tela de impressão, que mostra a estrutura de suporte (B). <img alt="A Figura 2" src="/files/ftp_upload/4067/4067fig2.jpg" /> Figura 2. Scanning micrografias electrónicas da matriz de microagulhas (A) e uma única microagulhas dentro deste matriz (B). Figura 3. Esquema de montagem de cabo plano flexível. Os passos envolvidos incluem modificando o cabo plano flexível (A), a ablação dos círculos estampados (B), adicionando o inicialmente ablated camada Melinex, que é enchido com pasta de carbono (C), bem como a adição do segundo camada Melinex ablacionada e acasalamento o microneedle array (D). <a href="http://www.jove.com/files/ftp_upload/4067/4067fig3large.jpg" target="_blank">Clique aqui para ver maior figura</a> . <img alt="A Figura 4" src="/files/ftp_upload/4067/4067fig4.jpg" /> Figura 4. Calibração de lactato-sensível pasta com 15 segundos scans cronoamperométricos -0,15 V em tampão fosfato 0,1 M (pH = 7,5). Cada aumento na corrente corresponde a uma adição de 2 mM de lactato. <img alt="A Figura 5" src="/files/ftp_upload/4067/4067fig5.jpg" /> Figura 5. </sTrong&gt; Calibração de pasta de glucose-sensível executando scans cronoamperométricos em -0,05 V. Cada diminuição da resposta corresponde a uma adição de 2 mM de glucose. O 0,1 M de solução de tampão fosfato (pH = 7,0) foi agitada durante a calibragem. Externo de Ag / AgCl e Pt de referência e eléctrodos de contador foram utilizados neste estudo. <img alt="A Figura 6" src="/files/ftp_upload/4067/4067fig6.jpg" /> Figura 6. Voltamograma cíclico (CV) de carbono sensível ao pH colar em tampão fosfato 0,1 M ao longo de pH 5-8 em incrementos de 1 unidade de pH (pH 8,0 = verde azulado, verde = pH 7,0, púrpura = pH 6,0, vermelho = pH 5,0). Um quinto CV foi utilizada para análise contra Ag / AgCl referência e os eléctrodos do contador de Pt de arame.

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Múltiplos aspectos da concepção deste sensor microneedle baseado foram considerados antes da fabricação do dispositivo. A fim de utilizar este sensor para detecção em tempo real, o tempo de resposta do sensor deve ser baixa; neste protocolo, cada sensor testados exibiram um tempo de resposta inferior a quinze segundos. Pastas utilizadas neste protocolo foram também escolhidos por sua seletividade dentro em ambientes in vivo, que contêm biomoléculas eletroativos que podem interferir na respost...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td></tr><tr><td> Cabo flexível plano </td><td> Molex </td><td> 3302/10SF </td></tr><tr><td> 0,003 fita Side &quot;face </td><td> Melinex </td><td></td></tr><tr><td> 0,004 &quot;fita dupla face </td><td> Melinex </td><td></td></tr><tr><td> Lactato-oxidase </td><td> Sigma </td><td> L0638 </td></tr><tr><td> Glucose oxidase </td><td> Sigma </td><td> G7141 </td></tr><tr><td> Ródio sobre carbono </td><td> Sigma </td><td> 206164 </td></tr><tr><td> Pó de grafite </td><td> Sigma </td><td> 385031000 </td></tr><tr><td> poli (etilenimina) </td><td> Acros </td><td> 178570010 </td></tr><tr><td> Óleo mineral </td><td> Sigma </td><td> M5904 </td></tr><tr><td> Glicose </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> Shell e-300 </td><td> EnvisionTEC </td><td></td></tr><tr><td> Shell e-200 </td><td> EnvisionTEC </td><td></td></tr><tr><td> Ag / AgCl eletrodo de referência </td><td> Basi </td><td> MF-2052 </td></tr><tr><td> Fio de Pt </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 sistema Postcuring </td><td> EnvisionTEC </td><td></td></tr><tr><td> O ácido fosfórico </td><td> Pescador </td><td> A366-4 </td></tr><tr><td> 60W Modelo 6,75 CO 2 raster / vector sistema laser </td><td> Universidadesal 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

O desenvolvimento de um sistema de monitoramento minimamente invasivo multiplexados para análise rápida de moléculas biologicamente relevantes poderia oferecer aos indivíduos que sofrem de doenças crônicas avaliação simplista de seu estado fisiológico imediato. Além disso, poderia servir como uma ferramenta de pesquisa para a análise de complexos, multifatoriais condições médicas. A fim de que um tal sensor de multianalyte a ser realizado, ele deve ser minimamente invasiva, a amostragem de fluido intersticial deve ocorrer sem dor ou danos para o utilizador, e análise deve ser rápida, bem como selectiva. Inicialmente desenvolvido para a dor livre de entrega da droga, microagulhas têm sido utilizados para fornecer vacinas e agentes farmacológicos (por exemplo, insulina) através da pele. 1-2 Uma vez que estes dispositivos de acesso ao espaço intersticial, microagulhas que são integrados com microeletrodos pode ser usado como transdérmica sensores eletroquímicos. Detecção selectiva da glucose, o glutamato, lactato, hácido ydrogen peróxido, e ascórbico foi demonstrada utilizando integrados microagulhas de eléctrodos de dispositivos com fibras de carbono, pastas de carbono modificados e microagulhas platina revestidos de polímero que servem como elementos de transdução. 3-7,8 Esta tecnologia de sensor de microagulhas permitiu uma nova e sofisticada abordagem analítica para detecção in situ e simultânea de múltiplos analitos. Multiplexing oferece a possibilidade de controlar microambientes complexos, que são de outro modo difícil de caracterizar de forma rápida e minimamente invasiva. Por exemplo, esta tecnologia pode ser utilizado para monitorização simultânea dos níveis extracelulares de, lactato, glicose e pH 9, que são importantes indicadores metabólicos de estados de doença 7,10-14 (por exemplo, cancro da proliferação) e induzida por exercício acidose. 15

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Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing

Este artigo detalha a construção de um sensor de microagulhas multiplexados baseado. O dispositivo está a ser desenvolvido para a amostragem in situ ea análise electroquímica de analitos múltiplos de uma maneira rápida e selectiva. Nós encaramos a medicina clínica e pesquisa biomédica usa para esses sensores baseados microagulhas.

Sensor de microagulhas baseado oco para Multiplexed Transdermal Eletroquímica Sensing

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 visualizações.  University of North Carolina and North Carolina State University. Este artigo detalha a construção de um sensor de microagulhas multiplexados baseado. O dispositivo está a ser desenvolvido para a amostragem in situ ea análise electroquímica de analitos múltiplos de uma maneira rápida e selectiva. Nós encaramos a medicina clínica e pesquisa biomédica usa para esses sensores baseados microagulhas.

Vídeo: 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.

Detecção de Biomolecular empregando o sensor de imagem Interferometric Reflectância (IRIS)

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

Bioengenharia

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.

Multiplexados molécula única Força Medidas proteólise, usando uma pinça magnética

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.

Bacteriana Detecção e Identificação Utilizando sensores eletroquímicos

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.

Sensoriamento de Barreira rompimento do tecido com um transistor Eletroquímica Orgânica

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.

A Biochip eletroquímica microfluídica à base de Análise de hibridação DNA livre-Label

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.

Biomembrane Fabrication pelo Solvent-assistida Lipid bicamada (SALB) Método

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 fibras ocas para<em&gt; In Vivo</em&gt; -como Cultura tecidos 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.

Cápsulas à base de plantas de extração de para aplicações de microencapsulação

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.

Snap o Chip para imunoensaios livre de Cross-reactivity e Spotter sanduíche multiplexado

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.

Desenvolvimento de um Biosensor eletroquímico do DNA para detectar um patógeno de origem alimentar

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