Questa ricerca è stata sostenuta dal National Institutes of Health (NIH) concessione R01 EB008268.

1. Chip Fabrication 12 <ol><li> Fare due placche da Zeonex pellets 690R: distribuire 8-9 grammi pellets Zeonex uniformemente nel centro di una piastra metallica, preriscaldare il pressa riscaldata a 198 ° C per 5 min, e quindi applicare pressione lentamente a 2500 psi per altri 5 minuti. Per completare questo passaggio, abbiamo usato una pressa a caldo Carver. </li><li> Rilievo il canale microfluidica nella placca con uno stampo epossidica. Dettagli sulla fabbricazione dello stampo sono descritte altrove 12 (disegno del canale in Figura 2b): posizionare una placca sullo stampo epossidico; preriscaldare loro sulla pressa riscaldata a 157 ° C per 10 minuti e quindi applicare pressione lentamente a 1000 psi per 10 min . Rimuovere la placca dallo stampo a mano o con una pinza prima che si raffreddi. (Nota: indossare guanti termici durante questa fase.) </li><li> Forare il chip goffrato in ingresso, porta rifiuti, e l&#39;uscita del canale microfluidico. Abbiamo usato a 1.25 mm diameter punta da trapano per fare i tre fori. </li><li> Legame del chip goffrato con un&#39;altra placca (in alto): Lavare i chip con IPA, RNAse via e acqua deionizzata in sequenza; aria secca e preriscaldare loro sulla pressa riscaldata a 131 ° C per 10 min e poi premere a 350 psi per un&#39;altra 10 min. </li><li> Fissare Nanoports all&#39;ingresso, i rifiuti, e le porte di uscita separatamente utilizzando JB Weld epossidico; cura per 15 a 24 ore secondo le istruzioni del produttore. </li><li> Sciacquare il canale SPE con 50 microlitri RNasi Away, seguita con 100 microlitri di acqua priva di nucleasi. </li><li> Caricare il canale SPE con 4 ml di soluzione di innesto (metilmetacrilato con 3% w / v benzofenone) e quindi reticolare il metacrilato incubando per 10 minuti in un forno sotto UV di lunghezza d&#39;onda UV 365 nm e 2000 mJ / cm 2. Rimuovere la soluzione residua innesto con vuoto. Abbiamo utilizzato il vuoto muro. Per i migliori risultati, una soluzione fresca dovrebbe essere usato. Di solito è accettabile, tuttavia, per memorizzare unsoluzione preparata a 4 ° C per un massimo di una settimana. </li><li> Rendono la soluzione SPE colonna (tutti v / v%): dimetacrilato di etilene 16%, 24% di metacrilato di butile, 42% 1-dodecanolo, 18% cicloesanolo, e aggiungere 1% 2-dimetilammino-4-(metil-fenilammino)-fenolo come la foto-iniziatore. </li><li> Estrarre lo stesso volume di soluzione di microsfere di silice come la soluzione colonna SPE (per fare una miscela 1:1), ed asciugare completamente a 70 ° C per una notte in un forno sotto vuoto. Suddividere il pellet toccando il tubo con il coperchio chiuso. Aggiungere la soluzione di SPE colonna nel tubo di microsfere di silice essiccata e agitare per miscelare. </li><li> Caricare 4 microlitri della soluzione SPE nello stesso canale e quindi reticolare tramite irradiazione UV per 2,5 min. Quindi, capovolgere il chip più e irradiare per un altro min 2.5. La colonna SPE polimerizzato deve essere un solido bianco opaco. </li><li> Lavare il canale con 500 ml di metanolo 100% per risciacquare via reagenti in eccesso. Questo lavaggio rimuove anche intra-polimero posolventi rogenic, creando così la struttura a pori aperti della colonna SPE quando essiccato in condizioni ambientali. </li><li> Fissare due film sottile riscaldatori al fondo del chip con nastro termoconduttivo. I lati dei riscaldatori devono essere allineati con il bordo dei canali larghi per la denaturazione e annealing separatamente (vedi Figura 1). </li><li> Inserire cinque termocoppie nel chip tramite i morti finito canali laterali aperti di ogni chip (vedi figura 2b). Le termocoppie restare in questi canali tramite press-fit. Nastro è stato utilizzato per fissare ulteriormente la posizione delle termocoppie. </li></ol> 2. Metodo di estrazione in fase solida <ol><li> Preparare 96 ml di etanolo al 70% con acqua trattata con DEPC e autoclavato (deionizzata o Millipore filtrata). </li><li> Aggiungere 4 pl di RNA 25X Fissare a 96 pl di etanolo al 70% in una provetta, e duplicare la stessa miscela di etanolo al 100% in un tubo separato. </li><li> Preparare 300pl di tampone canale miscelando il seguente: 75 pl di 6 M guanidina tiocianato (GuSCN), 150 ml di 2-propanolo, 63 microlitri di acqua priva di nucleasi, e 12 pl di RNA 25X sicuri. </li><li> Preparare 312 ml di tampone di lisi mescolando: 100 pl di GuSCN 6 M, 200 ml di 2-propanolo, e 12 pl di RNA 25X sicuri. </li><li> Riscaldare l&#39;etanolo 70%, 100% etanolo, tampone canale e tamponi di lisi a 60 ° C per 10-20 min per attivare la sicura RNA. </li><li> Per equilibrare il canale microfluidico, eseguire il buffer canale attraverso il canale SPE ad una portata di 0,8 ml / hr. </li><li> Quick-scongelare il campione in VTM a 37 ° C in bagno d&#39;acqua, e rimuovere il campione immediatamente dal bagnomaria appena lo scongelamento. </li><li> Centrifugare campione a 13.000 rpm per 10 min, e trasferire 100 pl del supernatante in 300 pl di tampone di lisi. Completare l&#39;impasto aggiungendo 6 microlitri di 1 mg / RNA carrier pl. </li><li> Vortex per mescolare, e dopo la centrifuga per5 secondi, caricare il lisato intero in un Luer-Lok 1 siringa cc. </li><li> Eseguire il lisato attraverso il canale SPE ad una portata di 0,8 ml / hr. </li><li> Lavare il canale SPE con 100 ml di etanolo al 70%, seguito da etanolo al 100 microlitri of100% a 1 ml / hr. (C&#39;è un volume di 50 microlitri morto nella punta della siringa che non verrà spinto fuori, quindi in realtà solo 50 pl sta attraversando il canale.) </li><li> Posizionare una siringa vuota alla tacca 0,5 ml e spingere aria attraverso il canale di asciugare a 1 ml / hr. </li><li> Run 67,5 ml di acqua priva di nucleasi attraverso il canale per eluire gli acidi nucleici legati a 0,5 ml / h, e raccogliere 13,5 microlitri dal nanoport al porto rifiuti. (La perdita di fluido è dovuto al volume di 54 microlitri morto nella siringa e il canale SPE.) </li></ol> 3. RT-PCR <ol><li> Preparare 36,5 microlitri della RT-PCR Master Mix durante la fase di aria secca (2.12) utilizzando i reagenti in un Qiagen OneStep RT-PCR kit. </li><li> Caricare RT-PCR reagente nel nanoport al porto dei rifiuti, e mescolare con l&#39;RNA per ottenere il seguente 50 microlitri RT-PCR reazione: 13,5 pl RNA eluiti in acqua priva di nucleasi, 4 microlitri RT-PCR mix enzima, 10 ul di soluzione Q, 10 μl1X uno passo tampone, 2 microlitri 25 mM MgCl2, 1 pl 50 pm in avanti fondo 5&#39;-GAC CRA TCC TGT CAC CTC TGA C-3 &#39;, 1 ml 50 microns di primer inverso 5&#39;-AGG GCA TTY TGG ACA AAK CGT CTA-3 &#39;, 2 microlitri dNTP, 0,75 microlitri 1,0% w / v BSA, 0,73 microlitri PEG8000 e 5.02 microlitri di acqua priva di nucleasi. </li><li> Caricare il RT-PCR miscela nel canale RT da vuoto gentile (abbiamo usato il vuoto parete) e sigillare il Nanoport con un raccordo chiuso. </li><li> Applicare 35 V al riscaldatore 1. Mantenere i reagenti nel canale RT per 30 minuti dopo il riscaldatore 1 equilibra a 50 ° C. Mantenere i reagenti nel canale RT per 15 minuti dopo il 1 equilibra riscaldamento a 95 ° C. </li><li> Applicare 27 V, le resistenze del 1 e 50 V a riscaldatore 2, attendere circa tre minuti o fino a riscaldatore 1 equilibra a 60 &amp; deg; C e riscaldatore 2 equilibra a 95 ° C. </li><li> Spingere i reagenti nel canale PCR a 0,5 microlitri / min. Dovrebbe prendere circa 20 min per i reagenti di fluire attraverso il canale a serpentina. </li><li> Quando il campione rende alla fine del canale a serpentina, raccogliere i prodotti di PCR a presa utilizzando un pipettatore opportunamente dimensionato e punta. </li></ol> 4. Prodotti PCR Detection <ol><li> Usiamo un test del DNA ad alta Agilent sensibilità per individuare i prodotti. Per eseguire questo test vengono eseguiti fuori l&#39;Alto Agilent Kit DNA Sensibilità dal frigorifero 30 minuti prima del test. </li><li> Accendere il Bioanalyzer e aprire il 2100 del software Expert. </li><li> Caricare il 1 pl di prodotti di PCR nella sperimentazione e seguire il protocollo Agilent ( <a href="http://gcf.pbrc.edu/docs/Agilent/Agilent%20Manual.pdf" target="_blank">20Manual.pdf http://gcf.pbrc.edu/docs/Agilent/Agilent%</a> ). </li><li> Analizzare e registrare i dati. </li></ol>

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Gli autori hanno dichiarato alcun conflitto di interessi finanziari.

Il metodo diagnostico presentato qui dimostrato la capacità di un sistema integrato chip microfluidica plastica per amplificare influenzali RNA da campioni di pazienti con alta specificità e un limite di rilevabilità basso 13 Abbiamo progettato questo chip per potenziale punto di testing cura:. (A) la temperatura e fluidici controllo sono state semplificate, (b) il chip è a basso costo e adatto per fabbricazione ad alta con stampaggio ad iniezione, e (c), il chip è monouso e previsto per un solo uso, rid...

Milioni di morti sono stati segnalati nel corso delle tre pandemie influenzali del 20 ° secolo 1. Inoltre, la pandemia influenzale più recente è stato dichiarato dall&#39;Organizzazione Mondiale della Sanità (OMS) 2 nel 2009, e dal 1 ° agosto 2010, 18.449 decessi sono stati segnalati dall&#39;OMS 3. Questa pandemia dimostrato ancora una volta l&#39;elevato onere delle malattie infettive, e la necessità di una rapida ed accurata di influenza per la conferma rapida della malattia, la risposta del caso la salute pubblica e un trattamento efficace 4. Ci sono diversi metodi ampiamente utilizzati per la diagnosi di influenza, questi includono test immunologici rapidi, test antigene diretto (DFA) e metodi di coltura virale. Immunologici rapidi drammaticamente mancano sensibilità 5-8, mentre gli altri due metodi sono di manodopera e richiede tempo 9. Test molecolari offrono molteplici vantaggi tra cui un breve tempo di ritorno, SENsit altaivity e una maggiore specificità. Diversi enti commerciali hanno lavorato per veloci test molecolari (chiamato anche test di acidi nucleici o NAT) per le malattie infettive, e molti saggi hanno influenza nelle loro condotte. Tuttavia la maggior parte di essi richiedono off-chip preparazione del campione. Nessuno degli emendamenti Clinical Laboratory Improvement (CLIA) hanno rinunciato test molecolari integrare la preparazione del campione nella cartuccia dosaggio o il modulo. Lab-on-a-chip svolge un ruolo importante nello sviluppo di point-of-care test. Dopo l&#39;introduzione del primo chip PCR nel 1993 10, numerosi sforzi sono stati messi in sviluppo chip di test di acidi nucleici. Tuttavia, solo pochi di questi hanno integrato preparazione grezza campione con amplificazione a valle. Abbiamo precedentemente dimostrato la miniaturizzazione di una colonna di estrazione in fase solida (SPE) in un chip di plastica microfluidico 11 e lo svilupposviluppo e ottimizzazione di un chip a flusso continuo PCR 12. Qui, si estende la precedente lavoro di integrare la SPE con RT e PCR passi in un unico chip per la diagnostica clinica e dimostrare la sua capacità di amplificare gli acidi nucleici da paziente nasofaringei (NP) tamponi e aspirati.

<table border="1"><tbody><tr><td> Nome del reagente / materiale </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> 1-dodecanol </td><td> Sigma-Aldrich, St. Louis, MO </td><td> 443816-500G </td></tr><tr><td> 2,2-dimetossi-2-fenilacetofenone </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-propanolo </td><td> Sigma-Aldrich, St. Louis, MO </td><td> 19516 </td></tr><tr><td> Benzophenone </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 di butile </td><td> Sigma-Aldrich, St. Louis, MO </td><td> 235865-100 ml </td></tr><tr><td> Carrier RNA </td&gt; <td> Qiagen, Valencia, CA </td><td> 1017647 </td></tr><tr><td> Cicloesanolo </td><td> Sigma-Aldrich, St. Louis, MO </td><td> 105.899-1L </td></tr><tr><td> Etanolo </td><td> Sigma-Aldrich, St. Louis, MO </td><td> E7023 </td></tr><tr><td> Etilene dimetacrilato </td><td> Sigma-Aldrich, St. Louis, MO </td><td> 335861 </td></tr><tr><td> Glicole etilenico dimetacrilato </td><td> Sigma-Aldrich, St. Louis, MO </td><td> 335681-100ML </td></tr><tr><td> Vetro siringa 250 microlitri </td><td> Hamilton, Reno, NV </td><td> 81127 </td></tr><tr><td> Guanidina tiocianato </td><td> Sigma-Aldrich, St. Louis, MO </td><td> 50981 </td></tr><tr><td> High Sensitivity Kit DNA </td><td> Agilent Technologies, Santa Clara, CA </td><td> 5067-4626 </td></tr><tr><td> Stampa a caldo </td><td> Carver, Wabash, IN </td><td> 4386 </td></tr><tr><td> JB Weld Epossidiche </td><td> McMaster-Carr, Elmhurst, IL </td><td> 7605A11 </td></tr><tr><td> Luer-Lok siringhe </td><td> BD-Medical, Franklin Lakes, NJ </td><td> 309628 </td></tr><tr><td> Cloruro di magnesio </td><td> Thermo Fisher Scientific, pittsburge, PA </td><td> AB-0359 </td></tr><tr><td> Metanolo </td><td> Sigma-Aldrich, St. Louis, MO </td><td> 494437 </td></tr><tr><td> Metacrilato di metile </td><td> Sigma-Aldrich, St. Louis, MO </td><td> M55909 </td></tr><tr><td> Nanoport </td><td> Upchurch scientifico </td><td> N-333-01 </td></tr><tr><td> Nanoport Raccordo </td><td> Upchurch scientifico </td><td> F-120x </td></tr><tr><td> Acqua priva di nucleasi </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> Alimentazione </td><td> VWR, Radnor, PA </td><td> 300V </td></tr><tr><td> RNAsi in trasferta </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> Microsfere di silice </td><td> Polysciences, Warrington, PA </td><td> 24324-15 </td></tr><tr><td> Pompa a siringa </td><td> Harvard Apparatus, Holliston, MA </td><td> HA2000P/10 </td></tr><tr><td> Nastro termoconduttivo </td><td> McMaster-Carr, Elmhurst, IL </td><td> 6838A11 </td></tr><tr><td> Termocoppia </td><td> Omega Engineering, Stamford, CT </td><td> 5SRTC-TT-J-40-36 </td></tr><tr><td> A film sottile Riscaldatori </td><td> Minco, Minneapolis, MN </td><td> HK5166R529L12A </td></tr><tr><td> Crosslinker Ultraviolet </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

Diagnostica rapida ed efficace svolgono un ruolo importante nel controllo delle malattie infettive, consentendo la gestione efficace del paziente e del trattamento. Qui, presentiamo integrato chip microfluidica termoplastico con la capacità di amplificare virus in pazienti nasofaringei (NP) tamponi e aspirati. Una volta caricato il campione del paziente, il dispositivo microfluidico esegue sequenzialmente su-chip lisi cellulare, purificazione dell&#39;RNA e concentrazione all&#39;interno estrazione in fase solida (SPE), la trascrizione inversa (RT) e la reazione a catena della polimerasi (PCR) in RT-PCR camere, rispettivamente. End-point di rilevamento è effettuato con un off-chip Bioanalyzer (Agilent Technologies, Santa Clara, CA). Per le periferiche, abbiamo utilizzato una sola pompa siringa per guidare reagenti e campioni, mentre due riscaldatori a pellicola sottile sono stati utilizzati come sorgenti di calore per la PCR e RT camere. Il chip è progettato per essere a singolo strato e adatta per la produzione ad alto throughput per ridurre il fabricatiil tempo e il costo. Il chip microfluidico fornisce una piattaforma per analizzare una varietà di virus e batteri, limitato solo dalle variazioni di progettazione reagenti necessari per individuare nuovi agenti patogeni di interesse.

<html> Un tipico risultato è mostrato in Figura 3 per un influenza A esemplare infetto lavaggio nasofaringeo. A causa delle diverse quantità di virus dell&#39;influenza in ogni campione, la concentrazione finale del prodotto di PCR varierà. Un buon risultato deve avere basso rumore, due picchi scaletta chiari (35 e 10.380 bp) e un picco singolo prodotto con le dimensioni prodotto progettato (107 bp) per il campione positivo. Mentre il picco prodotto teoricamente dovrebbe essere assente per i controlli...

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

Integrato chip microfluidica termoplastico è stato sviluppato per l&#39;utilizzo come diagnostica molecolare. Il chip esegue l&#39;estrazione di acido nucleico, la trascrittasi inversa e PCR. Metodi per la fabbricazione e l&#39;esecuzione del chip sono descritti.

Chip Fabrication Microfluidic e metodo per rilevare l&#39;influenza

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

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14.8K Views.  Boston University. Integrato chip microfluidica termoplastico è stato sviluppato per l'utilizzo come diagnostica molecolare. Il chip esegue l'estrazione di acido nucleico, la trascrittasi inversa e PCR. Metodi per la fabbricazione e l'esecuzione del chip sono descritti.

Video: Microfluidic Chip Fabrication and Method to Detect Influenza

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.

Metodi di rilevamento a fluorescenza per piattaforme goccia microfluidica

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

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Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described in detail. The PLBR can be utilized for the analysis of single bacteria and microcolonies to investigate biotechnological and microbiological related questions concerning, e.g.&#160;cell growth, morphology, stress response, and metabolite or protein production on single-cell level. The device features continuous media flow enabling constant environmental conditions for perturbation studies, but in addition allows fast medium changes as well as oscillating conditions to mimic any desired environmental situation. To fabricate the single use devices, a silicon wafer containing sub micrometer sized SU-8 structures served as the replication mold for rapid polydimethylsiloxane casting. Chips were cut, assembled, connected, and set up onto a high resolution and fully automated microscope suited for time-lapse imaging, a powerful tool for spatio-temporal cell analysis. Here, the biotechnological platform organism Corynebacterium glutamicum&#160;was seeded into the PLBR and cell growth and intracellular fluorescence were followed over several hours unraveling time dependent population heterogeneity on single-cell level, not possible with conventional analysis methods such as flow cytometry. Besides insights into device fabrication, furthermore, the preparation of the preculture, loading, trapping of bacteria, and the PLBR cultivation of single cells and colonies is demonstrated. These devices will add a new dimension in microbiological research to analyze time dependent phenomena of single bacteria under tight environmental control. Due to the simple and relatively short fabrication process the technology can be easily adapted at any microfluidics lab and simply tailored towards specific needs.

In this protocol the fabrication, setup and basic operation of a microfluidic picoliter bioreactor (PLBR) for single-cell analysis of prokaryotic microorganisms is introduced. Industrially relevant microorganisms were analyzed as proof of principle allowing insights into growth rate, morphology, and phenotypic heterogeneity over certain time periods, hardly possible with conventional methods.

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described ...

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;

Un chip microfluidica per la Versatile analisi chimica di singole cellule

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

Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned Size and Shape

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and used to dictate the shape as well as the diameter of a core stream.&#160;Grooves in the top and bottom of a microfluidic channel were designed to direct the sheath fluid and shape the core fluid.&#160;By matching the viscosity and hydrophilicity of the sheath and core fluids, the interfacial effects are minimized and complex fluid shapes can be formed.&#160;Controlling the relative flow rates of the sheath and core fluids determines the cross-sectional area of the core fluid.&#160;Fibers have been produced with sizes ranging from 300 nm to ~1 mm, and fiber cross-sections can be round, flat, square, or complex as in the case with double anchor fibers. Polymerization of the core fluid downstream from the shaping region solidifies the fibers.&#160;Photoinitiated click chemistries are well suited for rapid polymerization of the core fluid by irradiation with ultraviolet light.&#160;Fibers with a wide variety of shapes have been produced from a list of polymers including liquid crystals, poly(methylmethacrylate), thiol-ene and thiol-yne resins, polyethylene glycol, and hydrogel derivatives. Minimal shear during the shaping process and mild polymerization conditions also makes the fabrication process well suited for encapsulation of cells and other biological components.

Two adjacent fluids passing through a grooved microfluidic channel can be directed to form a sheath around a prepolymer core; thereby&#160;determining both shape and cross-section. Photoinitiated polymerization, such as thiol click chemistry, is well suited for rapidly solidifying the core fluid into a microfiber with predetermined size and shape.

Fabbricazione microfluidica di fibre polimeriche e bioibride con dimensioni e forma predesignate

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and ...

A Microfluidic Chip for ICPMS Sample Introduction

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). The chip produces monodisperse aqueous sample droplets in perfluorohexane (PFH). Size and frequency of the aqueous droplets can be varied in the range of 40 to 60 &#181;m and from 90 to 7,000&#160;Hz, respectively. The droplets are ejected from the chip with a second flow of PFH and remain intact during the ejection. A custom-built desolvation system removes the PFH and transports the droplets into the ICPMS. Here, very stable signals with a narrow intensity distribution can be measured, showing the monodispersity of the droplets. We show that the introduction system can be used to quantitatively determine iron in single bovine red blood cells. In the future, the capabilities of the introduction device can easily be extended by the integration of additional microfluidic modules.

We present a discrete droplet sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). It is based on a cheap and disposable microfluidic chip that generates highly monodisperse droplets in a size range of 40&#8722;60 &#181;m at frequencies from 90 to 7,000 Hz.

Un chip microfluidico per ICPMS introduzione del campione

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma ...

Image-guided, Laser-based Fabrication of Vascular-derived Microfluidic Networks

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic networks embedded in PEGDA hydrogels. Here, we describe the creation of virtual masks that allow for image-guided laser control; the photopolymerization of a micromolded PEGDA hydrogel, suitable for microfluidic network fabrication and pressure head-driven flow; the setup and use of a commercially available laser scanning confocal microscope paired with a femtosecond pulsed Ti:S laser to induce hydrogel degradation; and the imaging of fabricated microfluidic networks using fluorescent species and confocal microscopy. Much of the protocol is focused on the proper setup and implementation of the microscope software and microscope macro, as these are crucial steps in using a commercial microscope for microfluidic fabrication purposes that contain a number of intricacies. The image-guided component of this technique allows for the implementation of 3D image stacks or user-generated 3D models, thereby allowing for creative microfluidic design and for the fabrication of complex microfluidic systems of virtually any configuration. With an expected impact in tissue engineering, the methods outlined in this protocol could aid in the fabrication of advanced biomimetic microtissue constructs for organ- and human-on-a-chip devices. By mimicking the complex architecture, tortuosity, size, and density of in vivo vasculature, essential biological transport processes can be replicated in these constructs, leading to more accurate in vitro modeling of drug pharmacokinetics and disease.

This protocol outlines the implementation of image-guided, laser-based hydrogel degradation to fabricate vascular-derived, biomimetic microfluidic networks embedded in poly(ethylene glycol) diacrylate (PEGDA) hydrogels. These biomimetic microfluidic systems may be useful for tissue engineering applications, generation of in vitro disease models, and fabrication of advanced "on-a-chip" devices.

Immagine-guidate, Fabrication laser a base di reti microfluidici vascolare-derivati

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic ...

Fabrication of Three-dimensional Paper-based Microfluidic Devices for Immunoassays

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and directed within a layer of paper. Moreover, stacking multiple layers of patterned paper creates sophisticated three-dimensional microfluidic networks that can support the development of analytical and bioanalytical assays. Paper-based microfluidic devices are inexpensive, portable, easy to use, and require no external equipment to operate. As a result, they hold great promise as a platform for point-of-care diagnostics. In order to properly evaluate the utility and analytical performance of paper-based devices, suitable methods must be developed to ensure their manufacture is reproducible and at a scale that is appropriate for laboratory settings. In this manuscript, a method to fabricate a general device architecture that can be used for paper-based immunoassays is described. We use a form of additive manufacturing (multi-layer lamination) to prepare devices that comprise multiple layers of patterned paper and patterned adhesive. In addition to demonstrating the proper use of these three-dimensional paper-based microfluidic devices with an immunoassay for human chorionic gonadotropin (hCG), errors in the manufacturing process that may result in device failures are discussed. We expect this approach to manufacturing paper-based devices will find broad utility in the development of analytical applications designed specifically for limited-resource settings.

We detail a method to fabricate three-dimensional paper-based microfluidic devices for use in the development of immunoassays. Our approach to device assembly is a type of multilayer, additive manufacturing. We demonstrate a sandwich immunoassay to provide representative results for these types of paper-based devices.

Fabbricazione di dispositivi microfluidici basati su carta tridimensionale per gli immunodosaggi

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and ...

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.

Film secco basati su Photoresist biosensore elettrochimico Microfluidic Platform: Fabbricazione di dispositivi, saggio su chip preparazione e funzionamento del sistema

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

Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful research tools for studying human health and disease. To characterize the barrier function of cell layers cultured inside organ-on-chip devices, often transendothelial or transepithelial electrical resistance (TEER) is measured. To this end, electrodes are usually integrated into the chip by micromachining methods to provide more stable measurements than is achieved with manual insertion of electrodes into the inlets of the chip. However, these electrodes frequently hamper visual inspection of the studied cell layer or require expensive cleanroom processes for fabrication. To overcome these limitations, the device described here contains four easily integrated electrodes that are placed and fixed outside of the culture area, making visual inspection possible. Using these four electrodes the resistance of six measurement paths can be quantified, from which the TEER can be directly isolated, independent of the resistance of culture medium-filled microchannels. The blood-brain barrier was replicated in this device and its TEER was monitored to show the device applicability. This chip, the integrated electrodes and the TEER determination method are generally applicable in organs-on-chips, both to mimic other organs or to be incorporated into existing organ-on-chip systems.

This publication describes the fabrication of an organ-on-chip device with integrated electrodes for direct quantification of transendothelial electrical resistance (TEER). For validation, the blood-brain barrier was mimicked inside this microfluidic device and its barrier function was monitored. The presented methods for electrode integration and direct TEER quantification are generally applicable.

Fabbricazione e validazione di un sistema di organo-on-chip con elettrodi integrati direttamente quantificare transendoteliale resistenza elettrica

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful ...

Scalable Fabrication of Stretchable, Dual Channel, Microfluidic Organ Chips

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human patients. Human Organ-on-a-Chip (Organ Chip) microfluidic cell culture devices, which provide an experimental in vitro platform to assess efficacy, toxicity, and pharmacokinetic (PK) profiles in humans, may be better predictors of therapeutic efficacy and safety in the clinic compared to animal studies. These devices may be used to model the function of virtually any organ type and can be fluidically linked through common endothelium-lined microchannels to perform in vitro studies on human organ-level and whole body-level physiology without having to conduct experiments on people. These Organ Chips consist of two perfused microfluidic channels separated by a permeable elastomeric membrane with organ-specific parenchymal cells on one side and microvascular endothelium on the other, which can be cyclically stretched to provide organ-specific mechanical cues (e.g., breathing motions in lung). This protocol details the fabrication of flexible, dual channel, Organ Chips through casting of parts using 3D printed molds, enabling combination of multiple casting and post-processing steps. Porous poly (dimethyl siloxane) (PDMS) membranes are cast with micrometer sized through-holes using silicon pillar arrays under compression. Fabrication and assembly of Organ Chips involves equipment and steps that can be implemented outside of a traditional cleanroom. This protocol provides researchers with access to Organ Chip technology for in vitro organ- and body-level studies in drug discovery, safety and efficacy testing, as well as mechanistic studies of fundamental biological processes.

Here, we present a protocol that describes the fabrication of stretchable, dual channel, organ chip microfluidic cell culture devices for recapitulating organ-level functionality in vitro.

Fabbricazione scalabile di Stretchable, Dual Channel, organo di Microfluidic chip

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human ...