Esta pesquisa foi suportada pelo National Institutes of Health (NIH) concessão R01 EB008268.

1. Fabricação de chips 12 <ol><li> Fazer duas placas de aglomerados Zeonex 690R: distribuir 8-9 gramas pelotas Zeonex uniformemente no centro de uma placa de metal, o pré-aquecimento na prensa aquecida a 198 ° C durante 5 min e, em seguida, aplicar a pressão lentamente até 2500 psi, durante mais 5 min. Para concluir esta etapa, foi utilizado um Carver imprensa quente. </li><li> Grava o canal microfluídico na placa com um molde de epóxi. Os detalhes sobre a fabricação do molde são descritos em outra parte 12 (canal de desenho na Figura 2b): colocar uma placa sobre o molde epóxi; preaquecimento los na prensa aquecida a 157 ° C durante 10 min e, em seguida, aplicar a pressão lentamente até 1000 psi por mais 10 min . Remova a placa do molde à mão ou usando uma pinça, antes que esfrie. (Nota: usar luvas térmicas durante esta etapa.) </li><li> Perfura-se o chip em relevo, na entrada, a porta de resíduos, e a saída do canal microfluídico. Usamos um diâmetro de 1,25 mmeter broca para fazer os três orifícios. </li><li> Ligação do chip em relevo com uma outra placa (em cima): Lavar as fichas com IPA, RNAse Fora água desionizada e em sequência; ar seco e pré-aquecer-los na prensa aquecida a 131 ° C durante 10 min e em seguida pressionar a 350 psi para uma outra 10 min. </li><li> Anexar Nanoports na entrada, de resíduos, e as portas de saída separadamente usando JB Weld Epoxy; curar durante 15 a 24 horas de acordo com as instruções do fabricante. </li><li> Lavar o canal SPE com 50 ul RNAse Away, seguiu com 100 ul de água nuclease livre. </li><li> Carregar o canal de SPE com 4 ul de solução de enxerto (metacrilato de metilo, de 3% w / v de benzofenona) e, em seguida, a ligação cruzada de metacrilato, por incubação durante 10 minutos num forno de UV de comprimento de onda 365 nm sob UV e 2.000 mJ / cm 2. Remover a solução residual com enxerto de vácuo. Utilizou-se o vácuo de parede. Para obter os melhores resultados, uma solução fresca deve ser usado. É geralmente aceitável, no entanto, para armazenar umsolução preparada a 4 ° C durante até uma semana. </li><li> Tornar a solução coluna SPE (todos% v / v): dimetacrilato de etileno de 16%, 24% de metacrilato de butilo, 42% de 1-dodecanol, ciclohexanol 18%, e adicionar 1% de 2-dimetilamino-4-(metil-f enilamino)-fenol como o foto-iniciador. </li><li> Retire o mesmo volume de solução de sílica microesferas como sendo a solução da coluna SPE (para fazer uma mistura a 1:1), e secá-la completamente, a 70 ° C durante a noite num forno de vácuo. Quebra-se o pellet tocando o tubo com a tampa fechada. Adicionar a solução da coluna SPE dentro do tubo de sílica seco de microsferas e vortex para misturar. </li><li> Carregar 4 ul de solução de SPE no mesmo canal e, em seguida, formar ligações cruzadas por radiação UV é de 2,5 min. Em seguida, vire o chip de uma e outra irradiar para 2,5 min. A coluna de SPE polimerizada deve ser um sólido opaco branco. </li><li> Lavar o canal com 500 ul de metanol a 100% para enxaguar o excesso de reagentes. Esta lavagem remove também intra-polímero posolventes rogenic, criando desse modo a estrutura de poros abertos da coluna SPE, após secagem em condições ambientes. </li><li> Anexar dois aquecedores de película fina para o fundo do chip com a fita condutora térmica. Os lados dos aquecedores têm de estar alinhadas com a borda dos canais largos para desnaturação e recozimento separadamente (ver Figura 1). </li><li> Coloque cinco termopares no chip através da morte terminou canais laterais abertas de cada chip (ver Figura 2b). Os termopares ficar nestes canais via ajuste de imprensa. Fita foi utilizada para continuar a fixar a localização dos termopares. </li></ol> 2. Método de Extracção em Fase Sólida <ol><li> Preparar 96 ul de etanol a 70%, com água tratada com DEPC e autoclavadas (desionizada ou Millipore-filtrada). </li><li> Adicionar 4 de ul de RNA 25X Fixe a 96 ul de etanol a 70% em um tubo, e duplicar a mesma mistura com 100% de etanol, num tubo separado. </li><li> Prepare 300ul de tampão de canal através da mistura do seguinte: 75 jul de tiocianato de guanidina 6 M (GuSCN), 150 ul de 2-propanol, 63 ul de água livre de nuclease, e 12 ul de RNA 25X seguros. </li><li> Preparar 312 ul de tampão de lise por mistura de: 100 uL de 6 M GuSCN, 200 ul de 2-propanol, e 12 ul de RNA 25X seguros. </li><li> Aquece-se a etanol a 70%, etanol a 100% de tampão de canal, e tampões de lise a 60 ° C durante 10-20 min para ativar o Secure RNA. </li><li> Para equilibrar o canal microfluídico, execute o buffer de canal através do canal de SPE com um caudal de 0,8 ml / hr. </li><li> Quick-descongelar a amostra de teste em VTM a 37 ° C em banho de água, e remover a amostra do banho de água imediatamente, logo que ele é descongelado. </li><li> Centrifugar amostra a 13.000 rpm durante 10 min, e transferir 100 ul do sobrenadante para 300 ul de tampão de lise. Completa a mistura por adição de 6 ul de 1 ug / ul de ARN transportador. </li><li> Vortex para misturar, e após centrifugação para5 seg, carregar o lisado inteiro para uma seringa Luer-lok cc 1. </li><li> Executar lisado através do canal de SPE com um caudal de 0,8 ml / hr. </li><li> Lava-se a canal de SPE com 100 ul de etanol a 70%, seguido de 100 ul de etanol of100% a 1 ml / hr. (Há um volume de 50 ul morto na ponta da seringa que não será empurrada para fora, então na verdade apenas 50 ul está a atravessar o canal.) </li><li> Posicionar uma seringa vazia na marca de 0,5 ml e empurrar o ar através do canal para secá-la, a 1 ml / hr. </li><li> Executar 67,5 ul de água livre de nuclease através do canal para eluir os ácidos nucleicos ligados a 0,5 ml / h, e recolher 13,5 uL da nanoport no porto de resíduos. (A perda de líquidos é devido ao volume de 54 ul morto na ponta da seringa e o canal de SPE.) </li></ol> 3. RT-PCR <ol><li> Prepare 36,5 uL da mistura principal de RT-PCR durante o passo de ar seco (2,12), utilizando os reagentes em um OneStep RT-PCR Qiagen kits. </li><li> Carregar RT-PCR reagente no nanoport no porto de resíduos, e mistura-se com o RNA para obter a seguinte reacção 50 uL de RT-PCR: 13,5 ul de RNA eluído em água livre de nuclease, 4 ul de mistura de RT-PCR de enzima, solução de 10 ul de Q, 10 μl1X um tampão passo, 2 mM de MgCl 2 25 uL, 1 ul de 50 mM de primer forward 5&#39;-GAC CRA TGT TCC CAC CTC TGA C-3 &#39;, 1 ul de 50 mM de iniciadores inverso 5&#39;-TGG AGG GCA TTY ACA AAK CGT CTA-3 &#39;, 2 ul de dNTPs, 0,75 ul de 1,0% w / v de BSA, 0,73 e 5,02 ul de água PEG8000 nuclease ul livre. </li><li> Coloque a mistura de RT-PCR para o canal de RT por vácuo suave (utilizou-se o vácuo de parede) e selar o Nanoport com um encaixe fechado. </li><li> Aplicar 35 V para o aquecedor 1. Manter os reagentes no canal de RT, durante 30 min, após o aquecedor 1 equilibra a 50 ° C. Manter os reagentes no canal de RT, durante 15 min, após o aquecedor uma equilibra a 95 ° C. </li><li> Aplicar 27 V para o aquecedor 1 e 50 V para o aquecedor 2, esperar cerca de três minutos ou até que um aquecedor equilibra a 60 e deg, C e aquecimento 2 equilibra a 95 ° C. </li><li> Empurrar os reagentes para dentro do canal de PCR com 0,5 ul / min. Deve ter cerca de 20 min para os reagentes fluir através do canal de serpentina. </li><li> Como a amostra torna-o no final do canal de serpentina, recolher os produtos de PCR na saída utilizando um pipetador de tamanho adequado e de ponta. </li></ol> 4. Detecção de Produtos PCR <ol><li> Usamos um Agilent teste de DNA de alta sensibilidade para detectar os produtos. Para executar este teste tirar a Agilent Kit DNA Alta Sensibilidade da geladeira 30 minutos antes do teste. </li><li> Ligue o Bioanalyzer e abra o software especialista 2100. </li><li> Carregar o 1 uL dos produtos de PCR para o ensaio e seguir o protocolo Agilent ( <a href="http://gcf.pbrc.edu/docs/Agilent/Agilent%20Manual.pdf" target="_blank">http://gcf.pbrc.edu/docs/Agilent/Agilent 20Manual.pdf%</a> ). </li><li> Analisar e registrar os dados. </li></ol>

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Os autores declararam não interesses concorrentes financeiros.

O método de diagnóstico aqui apresentados demonstram a capacidade de um chip microfluídico integrado plástico para amplificar influenza A RNA a partir de amostras de pacientes com elevada especificidade e um limite de detecção de baixo 13 Concebemos este chip para o ponto de teste de potencial cuidado:. (A) a temperatura do fluido e controle foram simplificados, (b) o chip é de baixo custo e conveniente para a fabricação de alto rendimento usando moldagem por injecção, e (c) o chip é descartável ...

Milhões de mortes foram relatadas durante os três pandemias de gripe do século 20 1. Além disso, a pandemia de gripe mais recente foi declarada pela Organização Mundial da Saúde (OMS) 2, em 2009, e em 1 de agosto de 2010, 18.449 mortes foram registradas pela OMS 3. Esta pandemia demonstrou mais uma vez a alta carga de doenças infecciosas, e da necessidade de rápida e precisa detecção de gripe para ativar a confirmação da doença rápida, resposta adequada de saúde pública e tratamento eficaz 4. Existem vários métodos utilizados para o diagnóstico da gripe, que incluem imunoensaios, ensaios rápidos antigénio directa fluorescente (DFA) e os métodos de cultura viral. Imunoensaios rápidos dramaticamente falta de sensibilidade 5-8, enquanto que os outros dois métodos são muito trabalhosa e demorada 9. Os testes moleculares oferecem múltiplas vantagens, incluindo um curto tempo de rotação SensIT, de altaivity, e maior especificidade. Várias entidades comerciais têm vindo a trabalhar no sentido rápidos testes moleculares (também chamados de testes de ácidos nucleicos ou NAT) para doenças infecciosas, e vários ensaios têm influenza em suas condutas. No entanto a maior parte deles necessitam off-chip de preparação da amostra. Nenhuma das alterações Clinical Laboratory Improvement (CLIA) renunciou testes moleculares incorporar a preparação da amostra para o cartucho de ensaio ou módulo. Lab-on-a-chip desempenha um papel importante no desenvolvimento de dispositivos de point-of-care de teste. Após a introdução do chip primeira PCR, em 1993, 10, numerosos esforços têm sido feito para desenvolver chips de teste de ácido nucleico. No entanto, apenas alguns deles têm a preparação da amostra integrado bruto com amplificação a jusante. Temos anteriormente demonstrada a miniaturização de uma coluna de extracção em fase sólida (SPE) em um chip microfluídico plástico 11 ea desenvolvermento e otimização de um contínuo fluxo de chip de PCR 12. Aqui, nós estendemos o trabalho anterior para integrar a SPE com RT e as etapas de PCR em um único chip para diagnósticos clínicos e demonstrar a sua capacidade para amplificar ácidos nucléicos de paciente nasofaringe (NP), esfregaços e aspirados.

<table border="1"><tbody><tr><td> Nome do reagente / Material </td><td> Companhia </td><td> Número de Catálogo </td></tr><tr><td> 1-dodecanol </td><td> Sigma-Aldrich, St. Louis, MO </td><td> 443816-500G </td></tr><tr><td> 2,2-dimetoxi-2-fenilacetofenona </td><td> Sigma-Aldrich, St. Louis, MO </td><td> 196118-50G </td></tr><tr><td> 2100 Bioanalyzer </td><td> Agilent Technologies, Santa Clara, CA </td><td> G2943CA </td></tr><tr><td> 2-Propanol </td><td> Sigma-Aldrich, St. Louis, MO </td><td> 19516 </td></tr><tr><td> Benzofenona </td><td> Sigma-Aldrich, St. Louis, MO </td><td> 239852-50G </td></tr><tr><td> BSA </td><td> Thermo Fisher Scientific, pittsburge, PA </td><td> A7979-50ML </td></tr><tr><td> Metacrilato de butilo </td><td> Sigma-Aldrich, St. Louis, MO </td><td> 235865-100 ml </td></tr><tr><td> RNA Transportador </td&gt; <td> Qiagen, Valencia, CA </td><td> 1017647 </td></tr><tr><td> Ciclohexanol </td><td> Sigma-Aldrich, St. Louis, MO </td><td> 105899-1L </td></tr><tr><td> Etanol </td><td> Sigma-Aldrich, St. Louis, MO </td><td> E7023 </td></tr><tr><td> Dimetacrilato de etileno </td><td> Sigma-Aldrich, St. Louis, MO </td><td> 335861 </td></tr><tr><td> Dimetacrilato de etilenoglicol </td><td> Sigma-Aldrich, St. Louis, MO </td><td> 335.681-100ML </td></tr><tr><td> Seringa de vidro de 250 jil </td><td> Hamilton, Reno, NV </td><td> 81127 </td></tr><tr><td> Tiocianato de guanidina </td><td> Sigma-Aldrich, St. Louis, MO </td><td> 50981 </td></tr><tr><td> Kit DNA Alta Sensibilidade </td><td> Agilent Technologies, Santa Clara, CA </td><td> 5067-4626 </td></tr><tr><td> Prensagem a quente </td><td> Carver, Wabash, EM </td><td> 4386 </td></tr><tr><td> JB Weld Epóxis </td><td> McMaster-Carr, Elmhurst, IL </td><td> 7605A11 </td></tr><tr><td> Luer-Lok seringas </td><td> BD-Medical, Franklin Lakes, NJ </td><td> 309628 </td></tr><tr><td> Cloreto de Magnésio </td><td> Thermo Fisher Scientific, pittsburge, PA </td><td> AB-0359 </td></tr><tr><td> Metanol </td><td> Sigma-Aldrich, St. Louis, MO </td><td> 494437 </td></tr><tr><td> Metacrilato de metilo </td><td> Sigma-Aldrich, St. Louis, MO </td><td> M55909 </td></tr><tr><td> Nanoport </td><td> Upchurch Científico </td><td> N-333-01 </td></tr><tr><td> Nanoport Fitting </td><td> Upchurch Científico </td><td> F-120x </td></tr><tr><td> Água livre de nuclease </td><td> Thermo Fisher Scientific, pittsburge, PA </td><td> PR-P1193 </td></tr><tr><td> OneStep RT-PCR Kit </td><td> Qiagen, Valencia, CA </td><td> 210210 </td></tr><tr><td> PEG8000 </td><td> Sigma-Aldrich, St. Louis, MO </td><td> 41009 </td></tr&gt; <tr><td> Fonte de alimentação </td><td> VWR, Radnor, PA </td><td> 300V </td></tr><tr><td> RNAse Fora </td><td> Sigma-Aldrich, St. Louis, MO </td><td> 83931-250ML </td></tr><tr><td> RNASecure </td><td> Applied Biosystems, Foster City, CA </td><td> AM7005 </td></tr><tr><td> Sílica microesferas </td><td> Polysciences, Warrington, PA </td><td> 24324-15 </td></tr><tr><td> Bomba de seringa </td><td> Harvard Apparatus, Holliston, MA </td><td> HA2000P/10 </td></tr><tr><td> Tape termicamente condutiva </td><td> McMaster-Carr, Elmhurst, IL </td><td> 6838A11 </td></tr><tr><td> Par termoelétrico </td><td> Omega Engenharia, Stamford, CT </td><td> 5SRTC-TT-J-40-36 </td></tr><tr><td> Película fina Aquecedores </td><td> Minco, Minneapolis, MN </td><td> HK5166R529L12A </td></tr><tr><td> Crosslinker ultravioleta </td><td> UPV, Upland, CA </td><td> CL-1000 </td></tr><tr><td> Zeonex </td> &lt;td&gt; Zeon Chemicals, Louisville, KY </td><td> 690R </td></tr></tbody></table>

microfluidic chip fabrication and method to detect influenza

Diagnóstico rápidos e eficazes têm um papel importante no controle de doenças infecciosas, permitindo que a gestão do paciente e tratamento eficazes. Aqui, apresentamos um chip microfluídico integrado termoplástico com a capacidade de amplificar vírus influenza A em paciente nasofaringe (NP), esfregaços e aspirados. Durante o carregamento da amostra do paciente, o dispositivo micro sequencialmente realiza on-chip a lise das células, purificação e concentração de RNA dentro da extracção em fase sólida (SPE), a transcrição reversa (RT) e de reacção em cadeia da polimerase (PCR), em câmaras de RT-PCR, respectivamente. Detecção do ponto final é realizada usando um Bioanalyzer off-chip (Agilent Technologies, Santa Clara, CA). Para os periféricos, utilizou-se uma bomba de seringa para dirigir reagentes e amostras, ao passo que dois aquecedores de película fina foram utilizadas como fontes de calor para a temperatura ambiente e as câmaras de PCR. O chip é desenhado para ser de camada única e adequado para o fabrico de elevado rendimento para reduzir o Fabricatiem tempo e custo. O chip microfluídico proporciona uma plataforma para analisar uma grande variedade de vírus e bactérias, limitado apenas pelas mudanças na concepção reagente necessários para detectar novos agentes patogénicos de interesse.

<html> Um resultado típico é mostrado na Figura 3, para uma amostra de influenza A lavagem nasofaríngeo infectado. Devido às diferentes quantidades de vírus influenza em cada amostra de paciente, a concentração final de produto de PCR pode variar. Um bom resultado deve ter baixo ruído, dois picos escada claras (35 e 10380 pb) e um pico único produto com o tamanho do produto concebido (107 pb) para a amostra positiva. Enquanto que o pico do produto deveria, teoricamente, estar ausente dos control...

Watch this Scientific Journal Video about Microfluidic Chip Fabrication and Method to Detect Influenza at JoVE.com

Microfluidic Chip Fabrication and Method to Detect Influenza

Um chip microfluídico integrado termoplástico tem sido desenvolvido para utilização como um diagnóstico molecular. O chip realiza a extracção dos ácidos nucleicos, a transcriptase reversa, e PCR. Os métodos para fabricar e executando o chip encontram-se descritos.

Fabricação de chips microfluídicos e método para detectar a gripe

Fast  and effective diagnostics play an important role in controlling infectious  disease by enabling effective patient management and treatment. Here, we ...

microfluidic-chip-fabrication-and-method-to-detect-influenza

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14.9K visualizações.  Boston University. Um chip microfluídico integrado termoplástico tem sido desenvolvido para utilização como um diagnóstico molecular. O chip realiza a extracção dos ácidos nucleicos, a transcriptase reversa, e PCR. Os métodos para fabricar e executando o chip encontram-se descritos.

Vídeo: Microfluidic Chip Fabrication and Method to Detect Influenza

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence of novel viral pathogens, such as SARS coronavirus and 2009 pandemic H1N1 influenza virus, that are not detectable by traditional methods. To address these challenges, we have previously developed and validated a pan-viral microarray platform called the Virochip with the capacity to detect all known viruses as well as novel variants on the basis of conserved sequence homology1. Using the Virochip, we have identified the full spectrum of viruses associated with respiratory infections, including cases of unexplained critical illness in hospitalized patients, with a sensitivity equivalent to or superior to conventional clinical testing2-5. The Virochip has also been used to identify novel viruses, including the SARS coronavirus6,7, a novel rhinovirus clade5, XMRV (a retrovirus linked to prostate cancer)8, avian bornavirus (the cause of a wasting disease in parrots)9, and a novel cardiovirus in children with respiratory and diarrheal illness10. The current version of the Virochip has been ported to an Agilent microarray platform and consists of ~36,000 probes derived from over ~1,500 viruses in GenBank as of December of 2009. Here we demonstrate the steps involved in processing a Virochip assay from start to finish (~24 hour turnaround time), including sample nucleic acid extraction, PCR amplification using random primers, fluorescent dye incorporation, and microarray hybridization, scanning, and analysis.

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

The Virochip is a pan-viral microarray designed to simultaneously detect all known viruses as well as novel viruses on the basis of conserved sequence homology. Here we demonstrate how to run a Virochip assay to analyze clinical samples for the presence of both known and unknown viruses.

Usando um ensaio Microarray Pan-Viral (Virochip) para Tela de amostras clínicas para patógenos virais

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

Imunologia e Infecção

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

Métodos de detecção de fluorescência para as plataformas de gotículas microfluídicos

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

Bioengenharia

ampliPHOX Colorimetric Detection on a DNA Microarray for Influenza

DNA microarrays have emerged as a powerful tool for pathogen detection.1-5 For instance, many examples of the ability to type and subtype influenza virus have been demonstrated.6-11 The identification and subtyping of influenza on DNA microarrays has applications in both public health and the clinic for early detection, rapid intervention, and minimizing the impact of an influenza pandemic. Traditional fluorescence is currently the most commonly used microarray detection method. However, as microarray technology progresses towards clinical use,1 replacing expensive instrumentation with low cost detection technology exhibiting similar performance characteristics to fluorescence will make microarray assays more attractive and cost-effective.
The ampliPHOX colorimetric detection technology is intended for research applications, and has a limit of detection within one order of magnitude of traditional fluorescence11, with a main advantage being an approximate ten-fold lower instrument cost compared to the confocal microarray scanners required for fluorescence microarray detection. Another advantage is the compact size of the instrument which allows for portability and flexibility, unlike traditional fluorescence instruments. Because the polymerization technology is not as inherently linear as fluorescence detection, however, it is best suited for lower density microarray applications in which a yes/no answer for the presence of a certain sequence is desired, such as for pathogen detection arrays. Currently the maximum spot density compatible with ampliPHOX detection is &tilde;1800 spots/array. Because of the spot density limitations, higher density microarrays are not suitable for ampliPHOX detection.
Here, we present ampliPHOX colorimetric detection technology as a method of signal amplification on a low density microarray developed for the detection and characterization of influenza viruses (FluChip). Although this protocol uses the FluChip (a DNA microarray) as one specific application of ampliPHOX detection, any microarray incorporating biotinylated target can be labeled and detected in a similar manner. The microarray design and biotinylation of the target to be captured are the responsibility of the user. Once the biotinylated target has been captured on the array, ampliPHOX detection can be performed by first tagging the array with a streptavidin-label conjugate (ampliTAG). Upon light exposure using the ampliPHOX Reader instrument, polymerization of a monomer solution (ampliPHY) occurs only in regions containing ampliTAG-labeled targets. The polymer formed can be subsequently stained with a non-toxic solution to improve visual contrast, followed by imaging and analysis using a simple software package (ampliVIEW). The entire FluChip assay from un-extracted sample to result can be performed in about 6 hours, and the ampliPHOX detection steps described above can be completed in about 30 min.

ampliPHOX colorimetric detection technology is presented as an inexpensive alternative to fluorescence detection for microarrays. Based on photopolymerization, ampliPHOX produces solid polymer spots visible to the naked eye in just a few minutes. Results are then imaged and automatically interpreted with a simple yet powerful software package.

ampliPHOX Detecção colorimétrica em um Microarray de DNA para Influenza

DNA microarrays have emerged as a powerful tool for pathogen detection.1-5  For instance, many examples of the ability to type and subtype influenza virus ...

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

Alta capacidade método de detecção de vírus Influenza

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this purpose, the cells are passively trapped in the middle of a microchamber. Upon activation of the control layer, the cell is isolated from the surrounding volume in a small chamber. The surrounding volume can then be exchanged without affecting the isolated cell. However, upon short opening and closing of the chamber, the solution in the chamber can be replaced within a few hundred milliseconds. Due to the reversibility of the chambers, the cells can be exposed to different solutions sequentially in a highly controllable fashion, e.g. for incubation, washing, and finally, cell lysis. The tightly sealed microchambers enable the retention of the lysate, minimize and control the dilution after cell lysis. Since lysis and analysis occur at the same location, high sensitivity is retained because no further dilution or loss of the analytes occurs during transport. The microchamber design therefore enables the reliable and reproducible analysis of very small copy numbers of intracellular molecules (attomoles, zeptomoles) released from individual cells. Furthermore, many microchambers can be arranged in an array format, allowing the analysis of many cells at once, given that suitable optical instruments are used for monitoring. We have already used the platform for proof-of-concept studies to analyze intracellular proteins, enzymes, cofactors and second messengers in either relative or absolute quantifiable manner.

In this article we present a microfluidic chip for single cell analysis. It allows the quantification of intracellular proteins, enzymes, cofactors, and second messengers by means of fluorescent assays or immunoassays.&#160;

Um chip microfluídico para a Versátil Análise Química de células individuais

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this ...

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 ...

A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the natural reservoir for IAV, but IAV can cross the species barrier to poultry, swine, horses and humans. Avian viruses recognize sialic acid attached to a penultimate galactose by a &#945;2-3 linkage (avian-type receptors) whereas human viruses preferentially recognize sialic acid with a &#945;2-6 linkage (human-type receptors). To monitor if avian viruses are adapting to human type receptors, several methods can be used. Glycan microarrays with diverse libraries of synthetic sialosides are increasingly used to evaluate receptor specificity. However, this technique is not used for measuring avidities. Measurement of avidity is typically achieved by evaluating the binding of serially diluted hemagglutinin or virus to glycans adsorbed to conventional polypropylene 96-well plates. In this assay, glycans with &#945;2-3 or &#945;2-6 sialic acids are coupled to biotin and adsorbed to streptavidin plates, or are coupled to polyacrylamide (PAA) which directly adsorb to the plastic. We have significantly miniaturized this assay by directly printing PAA-linked sialosides and their non PAA-linked counterparts on micro-well glass slides. This set-up, with 48 arrays on a single slide, enables simultaneous assays of 6 glycan binding proteins at 8 dilutions, interrogating 6 different glycans, including two non-sialylated controls. This is equivalent to 18x 96-well plates in the traditional plate assay. The glycan array format decreases consumption of compounds and biologicals and thus greatly enhances efficiency.

Using a printed glycan microarray strategy, a conventional 96-well plate assay was miniaturized for analysis of influenza A virus hemagglutinin avidity and specificity for sialic acid containing receptors.

A Miniaturized Glycan Microarray Ensaio para a Avaliação de avidez e especificidade do Hemaglutininas vírus da gripe A

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the ...

Computer Numerical Control Micromilling of a Microfluidic Acrylic Device with a Staggered Restriction for Magnetic Nanoparticle-Based Immunoassays

Microfluidic systems have greatly improved immunoassay techniques. However, many microfabrication techniques require specialized, expensive, or complicated equipment, making fabrication costly and incompatible with mass production, which is one of the most important preconditions for point-of-care tests (POCT) to be adopted in low-resource settings. This work describes the fabrication process of an acrylic (polymethylmethacrylate, PMMA) device for nanoparticle-conjugated enzymatic immunoassay testing using the computer numerical control (CNC) micromilling technique. The functioning of the microfluidic device is shown by performing an immunoassay to detect a commercial antibody using lysozyme as a model antigen conjugated to 100 nm magnetic nanoparticles. This device integrates a physical staggered restriction of only 5 &#181;m in height, used to capture magnetic microparticles that make up a magnetic trap by placing an external magnet. In this way, the magnetic force on the immunosupport of conjugated nanoparticles is enough to capture them and resist flow drag. This microfluidic device is particularly suitable for low-cost mass production without the loss of precision for immunoassay performance.

Microfluidics is a powerful tool for the development of diagnostic tests. However, expensive equipment and materials, as well as laborious fabrication and handling techniques, are often required. Here, we detail the fabrication protocol of an acrylic microfluidic device for magnetic micro- and nanoparticle-based immunoassays in a low-cost and simple-to-use setting.

Microfresagem de Controle Numérico Computacional de um Dispositivo Acrílico Microfluídico com uma Restrição Escalonada para Imunoensaios Magnéticos Baseados em Nanoppartículas

Microfluidic systems have greatly improved immunoassay techniques. However, many microfabrication techniques require specialized, expensive, or ...

Engenharia

Rapid Molecular Detection and Differentiation of Influenza Viruses A and B

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality every year. The Influenza A and B assay was the first CLIA-waived molecular rapid flu test available. The Influenza A and B test works by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. Here, the performance of the Influenza A and B assay on frozen, archived nasopharyngeal swab (NPS) specimens stored in viral transport medium (VTM) were compared to a respiratory panel assay.
The performance of the Influenza A and B assay was evaluated by comparing the results to the respiratory panel reference method. The sensitivity for total influenza virus A was 67.5% (95% CI (CI), 56.6-78.5) and the specificity was 86.9% (CI, 71.0-100). For influenza virus B testing, the sensitivity and specificity were 90.2% (CI, 68.5-100) and 98.8% (CI, 68.5-100), respectively.
This system has the advantage of a significantly shorter test time than any other currently available molecular assay and the simple, pipette-free procedure runs on a fully integrated, closed, small-footprint system. Overall, the Influenza A and B assay evaluated in this study has the potential to serve as a point-of-care rapid influenza diagnostic test.

We describe a rapid, molecular-based Influenza A and B assay. The Influenza assay detects each target within 15 min by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. The Influenza A and B assay is user-friendly and required minimal hands-on time to perform.

Rápida Detecção Molecular e diferenciação de Influenza vírus A e B

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality ...

Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An easy-to-use and low-cost sensor platform is highly desired to measure various types of analytes (e.g., biomarkers, hormones, and drugs) quantitatively and specifically. For this reason, dry film photoresist technology - enabling cheap, facile, and high-throughput fabrication - was used to manufacture the microfluidic biosensor presented here. Depending on the bioassay used afterwards, the versatile platform is capable of detecting various types of biomolecules. For the fabrication of the device, platinum electrodes are structured on a flexible polyimide (PI) foil in the only clean-room process step. The PI foil serves as a substrate for the electrodes, which are insulated with an epoxy-based photoresist. The microfluidic channel is subsequently generated by the development and lamination of dry film photoresist (DFR) foils onto the PI wafer. By using a hydrophobic stopping barrier in the channel, the channel is separated into two specific areas: an immobilization section for the enzyme-linked assay and an electrochemical measurement cell for the amperometric signal readout.
The on-chip bioassay immobilization is performed by the adsorption of the biomolecules to the channel surface. The glucose oxidase enzyme is used as a transducer for electrochemical signal generation. In the presence of the substrate, glucose, hydrogen peroxide is produced, which is detected at the platinum working electrode. The stop-flow technique is applied to obtain signal amplification along with rapid detection. Different biomolecules can quantitatively be measured by means of the introduced microfluidic system, giving an indication of different types of diseases, or, in regard to therapeutic drug monitoring, facilitating a personalized therapy.

A microfluidic biosensor platform was designed and fabricated using low-cost dry film photoresist technology for the rapid and sensitive quantification of various analytes. This single-use system allows for the electrochemical readout of on-chip-immobilized enzyme-linked assays by means of the stop-flow technique.

Película seca baseada em fotorresiste eletroquímica Microfluidic Biosensor plataforma: Fabricação de dispositivo, ensaio em-microplaqueta preparação e operação de sistema

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An ...