YG è sostenuto da una borsa di studio IDB. IN è un tipo Alon e membro della facoltà del J Edmond ‭. ‬ Safra Centro di Bioinformatica dell&#39;Università di Tel Aviv ‭. ‬ Questa ricerca è stata sostenuta da ISF sovvenzione 1499-1410.

1. Creazione di un nastro Scotch-Master 5 <ol><li> Disegna o stampare il layout desiderato microcanali, in scala, su carta. Nel nostro caso il disegno consiste di due canali a forma di Y, larghe 3 mm ciascuna (Figura 2). </li><li> Coprire un vetro scorrevole con strati di nastro adesivo. Il numero di strati determinerà l&#39;altezza del canale (circa 60 micron per strato). Abbiamo usato 3 strati di nastro scotch. </li><li> Posizionare la progettazione del layout su una superficie piana. Allineare il cursore sopra il modello di progettazione. Tagliare con cautela il nastro sul vetrino con un bisturi in base al layout. </li><li> Rimuovere il nastro Scotch da tutte le regioni del vetrino eccetto quelle del layout del microcanale. </li><li> Posizionare il vetrino in un forno di riscaldamento a 65 ° C per 2-3 min. Pulire delicatamente con etanolo. </li></ol> 2. Realizzazione di un dispositivo PDMS Microfluidic <ol><li> Miscelare la base ed indurimento componenti di PDMS come raccomandato dal produttore. Usiamo un 10:01rapporto della base e catalizzatore. Versare circa 30 ml di miscela PDMS in una piastra di Petri ad un&#39;altezza di ~ 0,5 cm. Degassarla PDMS nel vuoto, se necessario. Immergere il vetrino nel PDMS con il nastro adesivo fantasia rivolto verso l&#39;alto. Posizionare il modello dopo il PDMS impedisce la formazione di bolle d&#39;aria tra la slitta e il fondo piatto. Cure per 48 ore a 65 ° C, assicurandosi che il piatto sia orizzontale con una livella a bolla. </li><li> In un piatto nuovo 90 millimetri Petri pour 3 ml di miscela PDMS, risultante in uno strato di circa 0,5 mm di profondità. (Se un rivestitore a rotazione è disponibile, può essere utilizzata per questa fase). </li><li> Degas e cura come in 2.1. </li><li> Delicatamente tagliare il dispositivo microfluidico alla dimensione desiderata con un bisturi. Questo strato viene definito il &quot;livello di flusso&quot;. </li><li> Tagliare un pezzo di dimensioni simili di sottile strato di PDMS dal secondo piatto Petri. Questo sarà definito il &quot;livello pozzi&quot;. </li><li> Utilizzare un puncher di dimensioni adeguate biopsia (Usiamo 1.2 ID mm) a fori di perforazione per le entrate e le uscite nel livello di flusso. </li&gt; <li> Posizionare il livello pozzetti su un vetrino di spessore sul layout design e punch out pozzetti, utilizzando 2 mm ID perforatore biopsia, in allineamento con i microcanali sul formato. </li><li> Pulire entrambi gli strati PDMS nonché coprioggetto da 24 mm x 60 mm con etanolo. Asciugare e pulire. </li><li> Plasma trattare sia il coprioggetto di vetro e dei pozzi strato PDMS utilizzando incisore standard di plasma (per non reversibile legame) o un portatile corona treater 6 per incollaggio reversibile. Appoggiare delicatamente lo strato pozzetti sopra il vetrino per provocare adesione (Figura 3). Strofinare delicatamente le eventuali bolle d&#39;aria (se necessario). </li></ol> 3. Cella Imaging Experiment <ol><li> Assicurarsi di avere il supporto desiderato pronto in siringhe adeguate collegati a tubi flessibili di plastica con diametro interno da 0,02 &quot;(Tygon) nella pompa a siringa. </li><li> Grow S. cerevisiae alla fase desiderata in YPD liquido. Vortex accuratamente e posto 300 μl di ciascun ceppo in una provetta da microcentrifuga. </li><li> Posizionare delicatamente 1 ml di Concanavalina A (Sigma) 2 mg / ml in ciascun pozzetto. Lavare Concanavalina eccesso A di ogni ben due volte pipettando gentilmente acqua. </li><li> Mentre il Concanavalina A sta asciugando, lavare i ceppi volte con 300 ml di mezzo privo di glucosio SC. Lavaggio glucosio residuo dalle pareti cellulari aiuta l&#39;adesione adeguata alla Concanavalina A. </li><li> Cellule Vortex accuratamente. Pipettare 0,75 microlitri o meno di ciascuna sospensione cellulare in proprio pozzo (Figura 3). Lavare delicatamente le cellule residue con terreno ricco. I due passaggi successivi devono essere eseguiti rapidamente per impedire alle cellule di essiccazione. </li><li> Plasma trattare il chip e i pozzi accuratamente con l&#39;hand-held trattamento corona, facendo attenzione a non colpire i pozzi direttamente. Cura il chip sulla pozzi, prestando particolare attenzione all&#39;allineamento tra i due (questo passaggio può essere fatto sotto uno stereoscopio). Premere delicatamente per aderire i due strati di PDMS. Lentamente riempire completamente il dispositivo con circa 50 microlitri media ricca, prestando attenzione a non bolle d&#39;aria rimangono all&#39;interno del canale. </li><li> Collegare il dispositivo, inserire i tubi negli ingressi appropriati e avviare il flusso dei media. Evitare che le bolle d&#39;aria vengono introdotti nel dispositivo come questi possono essere difficili da rimuovere, che può essere compiuta da scollegare un ingresso o di uscita e picchiettando leggermente sul dispositivo in cui le bolle sono, facendo attenzione a non rompere il vetrino coprioggetti. </li><li> Mettere al microscopio e trovare punti di imaging appropriate in ciascun pozzetto. </li><li> Conservare le cellule sotto ricco flusso medio per 1 ora o più a lungo per consentire il ripristino in caso di necessità. </li><li> Iniziare l&#39;esperimento: usiamo flusso costante da ciascuna delle due pompe a siringa per impedire il riflusso dei media, cambiando portate relativi come desiderato. Impostazione pompa A e 0,8 ml / hr e B pompa a 0,2 ml / hr risultati in medium A scorre oltre l&#39;80% della larghezza del canale (Figura 4). Questo può essere dinamicamente cimpiccato modificando le due portate. Le nuove condizioni stabilizzare pochi secondi lungo l&#39;intera lunghezza del canale 2. </li></ol>

<ol>
	<li>Dertinger, S. K., Jiang, X., Li, Z., Murthy, V. N., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gradients+of+substrate-bound+laminin+orient+axonal+specification+of+neurons.">Gradients of substrate-bound laminin orient axonal specification of neurons.</a> Proceedings of the National Academy of Sciences of the United States of America. 99, 12542-12547 (2002).</li><li>Hersen, P., McClean, M. N., Mahadevan, L., Ramanathan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+processing+by+the+HOG+MAP+kinase+pathway.">Signal processing by the HOG MAP kinase pathway.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 7165-7170 (2008).</li><li>Paliwal, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MAPK-mediated+bimodal+gene+expression+and+adaptive+gradient+sensing+in+yeast.">MAPK-mediated bimodal gene expression and adaptive gradient sensing in yeast.</a> Nature. 446, 46-51 (2007).</li><li>Bennett, M. R., Hasty, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+devices+for+measuring+gene+network+dynamics+in+single+cells.">Microfluidic devices for measuring gene network dynamics in single cells.</a> Nat. Rev. Genet. 10, 628-638 (2009).</li><li>Shrirao, A. B., Perez-Castillejos, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+Fabrication+of+Microfluidic+Devices+by+Replicating+Scotch-tape+Masters.">Simple Fabrication of Microfluidic Devices by Replicating Scotch-tape Masters.</a> Chips &#38; Tips. , (2010).</li><li>Haubert, K., Drier, T., Beebe, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PDMS+bonding+by+means+of+a+portable,+low-cost+corona+system.">PDMS bonding by means of a portable, low-cost corona system.</a> Lab Chip. 6, 1548-1549 (2006).</li><li>Gerber, D., Maerkl, S. J., Quake, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+microfluidic+approach+to+generating+protein-interaction+networks.">An in vitro microfluidic approach to generating protein-interaction networks.</a> Nat. Methods. 6, 71-74 (2009).</li></ol>

Nessun conflitto di interessi dichiarati.

In questo articolo presentiamo un metodo semplice da banco per la creazione di un dispositivo a microfluidi che consente i seguenti ceppi di lievito contemporaneamente in condizioni dinamiche. Seguendo diversi ceppi in un canale fornisce uno strumento affidabile per il confronto dinamica di risposte di cellule singole in più ceppi. Un vantaggio del nostro approccio è la capacità di fabbricare il dispositivo con tecniche semplici senza la necessità di una camera pulita. In realtà, abbiamo anche svolto il protocollo ...

Le cellule sono costantemente reagire ad un ambiente in perpetuo cambiamento, cambiando il loro metabolismo, profilo trascrizionale e funzioni cellulari. Per studiare questi fenomeni, metodi che possono quantificare tali cambiamenti sono necessari. Un tipo di lettura di queste risposte è il cambiamento di localizzazione di fattori di trascrizione riferiti sforzo in risposta a stress nutrizionale. Dispositivi microfluidici 1 sono stati usati per manipolare dinamicamente condizioni ambientali alle cellule 2-4. Essi presentano diversi vantaggi per l&#39;imaging cellulare dal vivo: l&#39;ambiente delle cellule può essere controllato con precisione e dinamicamente cambiato, le cellule possono essere vivo con immagini in qualsiasi momento, anche durante la manipolazione delle condizioni esterne, e volumi di reagente minimi sono obbligatori. Un disegno molto semplice per un dispositivo microfluidico, consentendo alternanza tra due condizioni, è un dispositivo a forma di Y 2. Noi abitualmente uso dispositivi microfluidici a Y che consentonoapplicazione di variazioni dinamiche delle cellule nel canale. Usiamo questo dispositivo per seguire i cambiamenti di localizzazione di etichettati con colori fluorescenti fattori di trascrizione di stress correlati in cellule di lievito. Seguiamo questi cambiamenti nutrizionali in condizioni di instabilità. Per esempio, si può fornire un impulso (o una serie di impulsi) di glucosio contenente terreno entro un periodo di non-glucosio medio, ad una buona risoluzione temporale. Spesso in tali esperimenti, si è interessato a confrontare le risposte dei ceppi cellulari diversi (ad esempio, mutanti diversi, o diversi isolati selvatici). Il canale a forma di Y è limitata ad un ceppo in ogni canale - se più di un ceppo viene utilizzato, non esiste un modo semplice per distinguere tra i ceppi. Per ovviare a questo, i canali possono essere usati diversi. Se vogliamo applicare le stesse condizioni dinamiche a più ceppi, vorremmo combinare la Y-canale concetto con più canali. Ci sono due sfide di implementazione di questa soluzione: Può essere difficile da applicare tegli stesse condizioni contemporaneamente a tutti i canali, e vi è una limitazione geometrica al numero di Y canali che possono essere montati in un unico dispositivo. Abbiamo quindi cercando un modo per visualizzare diversi ceppi in un canale in condizioni dinamiche. Qui si descrive un doppio strato dispositivo microfluidico destinato ceppi di imaging diversi canali alle stesse condizioni dinamiche. Lo strato inferiore è costituito da sottili PDMS con una fila di fori. Questo strato è attaccato al coprioggetti creando pozzi in cui si collocano i diversi ceppi, aderendo con concanavalina A al vetro. Il secondo strato è una forma di Y dispositivo microfluidico creato utilizzando il metodo scotch 5. Dopo aver posizionato i ceppi nei pozzetti secondo strato è rapidamente allineato e collegato alla prima, creando un canale con diversi pozzi che contengono i diversi ceppi. Questo dispositivo permette finale dopo diversi ceppi in un canale, dove tutticeppi sono sottoposti alle stesse condizioni al tempo stesso esatto. Tutta la progettazione e processo di produzione può essere fatto sul piano del banco, con mezzi semplici, senza litografia morbida.

<table border="1"><tbody><tr><td> Reagente / attrezzature </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> PDMS-SYLGARD 184 </td><td> Dow Corning USA </td><td></td></tr><tr><td> Vuoto essiccatore </td><td> Nalgene </td><td> 5310-0250 </td></tr><tr><td> Biopsia punzoni </td><td> Ted Pella Inc. </td><td> Harris Uni-Core 15.076 (2 mm), 15.074 (1,2 mm) </td></tr><tr><td> Pompe a siringa </td><td> Chemyx </td><td> Fusion 200 </td></tr><tr><td> Corona che cura </td><td> Electro-technic prodotti </td><td> BD-20 </td></tr><tr><td> Tygon tubo </td><td> Tygon </td><td> S-54-HL </td></tr><tr><td> Concanavalina A- </td><td> Sigma </td><td> C7275 </td></tr><tr><td> Nastro adesivo </td><td> 3M Scotch </td><td> Nastro trasparente 1/2 &quot; </td></tr></tbody></table>

a microfluidic device for studying multiple distinct strains

Lo studio delle risposte delle cellule ai cambiamenti ambientali pone molte sfide sperimentali: cellule hanno bisogno di essere ripreso in condizioni mutevoli, spesso in modo comparativo. Piastre multipozzetto sono abitualmente utilizzati per confrontare molti diversi ceppi o linee cellulari, ma permettono un controllo limitato sulle dinamiche ambiente. Dispositivi microfluidici, invece, consentono squisito controllo dinamico delle condizioni ambientali, ma è difficile da immagine e distinguere più di pochi ceppi in loro. Qui si descrive un metodo per produrre facilmente e rapidamente un dispositivo microfluidico in grado di applicare condizioni mutevoli a più ceppi distinti in un canale. Il dispositivo è progettato e costruito in modo semplice senza la necessità di litografia soft. Esso è composto da un canale a forma di Y flusso attaccato ad un secondo strato harboring micropozzetti. I ceppi sono posti in pozzetti separati, e ripreso nelle esatte condizioni dinamiche. Abbiamo demonstrate l&#39;uso del dispositivo per misurare le risposte di localizzazione della proteina di impulsi di cambiamenti nutrienti in differenti ceppi di lievito.

Per dimostrare la separazione tra i vari ceppi abbiamo ripreso due ceppi di lievito distinguibili in pozzi alternativi. Imaging pieno pozzetti mostra alcuna perdita cellulare tra pozzetti (Figura 5a, b). Entrambi i ceppi hanno il fattore di trascrizione MSN2 etichettato con YFP. Per verificare l&#39;effetto simultaneo di modificare dinamicamente condizioni, abbiamo cambiato le portate nei due canali di ingresso, creando una fase di mezzo di no-glucosio, che ha portato localizzazione di MSN2-YFP al nucle...

Watch this Scientific Journal Video about A Microfluidic Device for Studying Multiple Distinct Strains at JoVE.com

A Microfluidic Device for Studying Multiple Distinct Strains

Presentiamo un metodo semplice per produrre dispositivi microfluidici capaci di applicare simili condizioni dinamiche a più ceppi distinti, senza la necessità di una stanza pulita o litografia morbida.

Un dispositivo a microfluidi per lo studio più ceppi distinti

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a ...

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Research

JoVE Journal

Bioengineering

8.7K Views.  Tel Aviv University. Presentiamo un metodo semplice per produrre dispositivi microfluidici capaci di applicare simili condizioni dinamiche a più ceppi distinti, senza la necessità di una stanza pulita o litografia morbida.

Video: A Microfluidic Device for Studying Multiple Distinct Strains

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in vitro. Nutrients delivered by vasculature fail to reach all parts of tumors, giving rise to heterogeneous microenvironments consisting of viable, quiescent and necrotic cell types. Many cancer drugs fail to effectively penetrate and treat all types of cells because of this heterogeneity. Monolayers of cancer cells do not mimic this heterogeneity, making it difficult to test cancer drugs with a suitable in vitro model. Our microfluidic devices were fabricated out of PDMS using soft lithography. Multicellular tumor spheroids, formed by the hanging drop method, were inserted and constrained into rectangular chambers on the device and maintained with continuous medium perfusion on one side. The rectangular shape of chambers on the device created linear gradients within tissue. Fluorescent stains were used to quantify the variability in apoptosis within tissue. Tumors on the device were treated with the fluorescent chemotherapeutic drug doxorubicin, time-lapse microscopy was used to monitor its diffusion into tissue, and the effective diffusion coefficient was estimated. The hanging drop method allowed quick formation of uniform spheroids from several cancer cell lines. The device enabled growth of spheroids for up to 3 days. Cells in proximity of flowing medium were minimally apoptotic and those far from the channel were more apoptotic, thereby accurately mimicking regions in tumors adjacent to blood vessels. The estimated value of the doxorubicin diffusion coefficient agreed with a previously reported value in human breast cancer. Because the penetration and retention of drugs in solid tumors affects their efficacy, we believe that this device is an important tool in understanding the behavior of drugs, and developing new cancer therapeutics.

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We present the procedure for fabrication and operation of a microfluidic device that recreates heterogeneous tumor microenvironments in vitro. The variability in apoptosis within tumor tissue was quantified using fluorescent stains and the effective diffusion coefficient of the chemotherapeutic drug doxorubicin into tumor tissue was evaluated.

Dispositivo Microfluidic per ricreare un microambiente tumorale<em&gt; In Vitro</em

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in ...

Ricerca

Bioingegneria

Microfluidic Mixers for Studying Protein Folding

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason for this is that folding takes place over a wide range of timescales, from nanoseconds to seconds or longer, depending on the protein1. Conventional stopped-flow mixers have allowed measurement of folding kinetics starting at about 1 ms. We have recently developed a microfluidic mixer that dilutes denaturant ~100-fold in ~8 &mu;s2. Unlike a stopped-flow mixer, this mixer operates in the laminar flow regime in which turbulence does not occur. The absence of turbulence allows precise numeric simulation of all flows within the mixer with excellent agreement to experiment3-4.
Laminar flow is achieved for Reynolds numbers Re &le;100. For aqueous solutions, this requires micron scale geometries. We use a hard substrate, such as silicon or fused silica, to make channels 5-10 &mu;m wide and 10 &mu;m deep (See Figure 1). The smallest dimensions, at the entrance to the mixing region, are on the order of 1 &mu;m in size. The chip is sealed with a thin glass or fused silica coverslip for optical access. Typical total linear flow rates are ~1 m/s, yielding Re~10, but the protein consumption is only ~0.5 nL/s or 1.8 &mu;L/hr. Protein concentration depends on the detection method: For tryptophan fluorescence the typical concentration is 100 &mu;M (for 1 Trp/protein) and for FRET the typical concentration is ~100 nM.
The folding process is initiated by rapid dilution of denaturant from 6 M to 0.06 M guanidine hydrochloride. The protein in high denaturant flows down a central channel and is met on either side at the mixing region by buffer without denaturant moving ~100 times faster (see Figure 2). This geometry causes rapid constriction of the protein flow into a narrow jet ~100 nm wide. Diffusion of the light denaturant molecules is very rapid, while diffusion of the heavy protein molecules is much slower, diffusing less than 1 &mu;m in 1 ms. The difference in diffusion constant of the denaturant and the protein results in rapid dilution of the denaturant from the protein stream, reducing the effective concentration of the denaturant around the protein. The protein jet flows at a constant rate down the observation channel and fluorescence of the protein during folding can be observed using a scanning confocal microscope5.

In this work we explain the fabrication and use of a microfluidic mixer capable of mixing two solutions in ~8 &mu;s. We also demonstrate the use of these mixers with spectroscopic detection using UV fluorescence and fluorescence resonance energy transfer (FRET).

Miscelatori per lo studio microfluidici Protein Folding

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason ...

Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in porous media are relevant to several industrial and environmental processes such as wastewater treatment and CO2 sequestration. We used Pseudomonas fluorescens, a Gram-negative aerobic bacterium, to investigate biofilm formation in a microfluidic device that mimics porous media. The microfluidic device consists of an array of micro-posts, which were fabricated using soft-lithography. Subsequently, biofilm formation in these devices with flow was investigated and we demonstrate the formation of filamentous biofilms known as streamers in our device. The detailed protocols for fabrication and assembly of microfluidic device are provided here along with the bacterial culture protocols. Detailed procedures for experimentation with the microfluidic device are also presented along with representative results.

Protocols for the study of biofilm formation in a microfluidic device that mimics porous media are discussed. The microfluidic device consists of an array of micro-pillars and biofilm formation by Pseudomonas fluorescens in this device is investigated.

Protocollo per il Biofilm Streamer formazione in un dispositivo a microfluidi con Micro-pilastri

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in ...

Analyzing Mixing Inhomogeneity in a Microfluidic Device by Microscale Schlieren Technique

In this paper, we introduce the use of microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. The microscale schlieren system is constructed from a Hoffman modulation contrast microscope, which provides easy access to the rear focal plane of the objective lens, by removing the slit plate and replacing the modulator with a knife-edge. The working principle of microscale schlieren technique relies on detecting light deflection caused by variation of refractive index1-3. The deflected light either escapes or is obstructed by the knife-edge to produce a bright or a dark band, respectively. If the refractive index of the mixture varies linearly with its composition, the local change in light intensity in the image plane is proportional to the concentration gradient normal to the optical axis. The micro-schlieren image gives a two-dimensional projection of the disturbed light produced by three-dimensional inhomogeneity.
To accomplish quantitative analysis, we describe a calibration procedure that mixes two fluids in a T-microchannel. We carry out a numerical simulation to obtain the concentration gradient in the T-microchannel that correlates closely with the corresponding micro-schlieren image. By comparison, a relationship between the grayscale readouts of the micro-schlieren image and the concentration gradients presented in a microfluidic device is established. Using this relationship, we are able to analyze the mixing inhomogeneity from associate micro-schlieren image and demonstrate the capability of microscale schlieren technique with measurements in a microfluidic oscillator4. For optically transparent fluids, microscale schlieren technique is an attractive diagnostic tool to provide instantaneous full-field information that retains the three-dimensional features of the mixing process.

Herein, we describe a procedure that employs microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. Through calibration, distribution of concentration gradient can be derived from the micro-schlieren image.

Analizzando disomogeneità Mescolando in un dispositivo a microfluidi da Tecnica microscala Schlieren

In this paper, we introduce the use of microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. The microscale schlieren ...

Fully Automated Centrifugal Microfluidic Device for Ultrasensitive Protein Detection from Whole Blood

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform for reduced sample volume and assay time as well as full automation are required for potential use in point-of-care-diagnostics. Recently, we have demonstrated ultrasensitive detection of serum proteins, C-reactive protein (CRP) and cardiac troponin I (cTnI), utilizing a lab-on-a-disc composed of TiO2 nanofibrous (NF) mats. It showed a large dynamic range with femto molar (fM) detection sensitivity, from a small volume of whole blood in 30 min. The device consists of several components for blood separation, metering, mixing, and washing that are automated for improved sensitivity from low sample volumes. Here, in the video demonstration, we show the experimental protocols and know-how for the fabrication of NFs as well as the disc, their integration and the operation in the following order: processes for preparing TiO2 NF mat; transfer-printing of TiO2 NF mat onto the disc; surface modification for immune-reactions, disc assembly and operation; on-disc detection and representative results for immunoassay. Use of this device enables multiplexed analysis with minimal consumption of samples and reagents. Given the advantages, the device should find use in a wide variety of applications, and prove beneficial in facilitating the analysis of low abundant proteins.

This protocol demonstrates how to achieve femto molar detection sensitivity of proteins in 10 &#181;L of whole blood within 30 min. This can be achieved by using electrospun nanofibrous mats integrated in a lab-on-a-disc, which offers high surface area as well as effective mixing and washing for enhanced signal-to-noise ratio.

Completamente dispositivo automatizzato centrifuga Microfluidic per il rilevamento di proteine ​​Ultrasensitive da sangue intero

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform ...

Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human gut-on-a-chip microphysiological device. We recapitulate the intestinal lumen-capillary tissue interface in a microfluidic device, where physiological mechanical deformations and fluid shear flow are constantly applied to mimic peristalsis. In the lumen microchannel, human intestinal epithelial Caco-2 cells are cultured to form a &#39;germ-free&#39; villus epithelium and regenerate small intestinal villi. Pre-cultured microbial cells are inoculated into the lumen side to establish a host-microbe ecosystem. After microbial cells adhere to the apical surface of the villi, fluid flow and mechanical deformations are resumed to produce a steady-state microenvironment in which fresh culture medium is constantly supplied and unbound bacteria (as well as bacterial wastes) are continuously removed. After extended co-culture from days to weeks, multiple microcolonies are found to be randomly located between the villi, and both microbial and epithelial cells remain viable and functional for at least one week in culture. Our co-culture protocol can be adapted to provide a versatile platform for other host-microbiome ecosystems that can be found in various human organs, which may facilitate in vitro study of the role of human microbiome in orchestrating health and disease.

We describe an in vitro protocol to co-culture gut microbiome and intestinal villi for an extended period using a human gut-on-a-chip microphysiological system.

Co-cultura del vivere Microbiome con Microengineered umana intestinale Villi in un Gut-on-a-chip microfluidico dispositivo

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human ...

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 ...

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the spheroid cultures, a perfusable vascular network in the spheroids remains a critical challenge for long-term culture required to maintain and develop their functions, such as protein expressions and morphogenesis. The protocol presents a novel method to integrate a perfusable vascular network within the spheroid in a microfluidic device. To induce a perfusable vascular network in the spheroid, angiogenic sprouts connected to microchannels were guided to the spheroid by utilizing angiogenic factors from human lung fibroblasts cultured in the spheroid. The angiogenic sprouts reached the spheroid, merged with the endothelial cells co-cultured in the spheroid, and formed a continuous vascular network. The vascular network could perfuse the interior of the spheroid without any leakage. The constructed vascular network may be further used as a route for supply of nutrients and removal of waste products, mimicking blood circulation in vivo. The method provides a new platform in spheroid culture toward better recapitulation of living tissues.

The protocol describes how to engineer a perfusable vascular network in a spheroid. The spheroid's surrounding microenvironment is devised to induce angiogenesis and connect the spheroid to the microchannels in a microfluidic device. The method allows the perfusion of the spheroid, which is a long-awaited technique in three-dimensional cultures.

Perfusable rete vascolare con un modello di tessuto in un dispositivo microfluidico

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

Generazione di condizioni ambientali dinamiche utilizzando un dispositivo microfluidico ad alta produttività

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...

Configurazione di un dispositivo microfluidico per la produzione di tappi combinatoriali

Bilayer Microfluidic Device for Combinatorial Plug Production

Droplet microfluidics is a versatile tool that allows the execution of a large number of reactions in chemically distinct nanoliter compartments. Such systems have been used to encapsulate a variety of biochemical reactions - from incubation of single cells to implementation of PCR reactions, from genomics to chemical synthesis. Coupling the microfluidic channels with regulatory valves allows control over their opening and closing, thereby enabling the rapid production of large-scale combinatorial libraries consisting of a population of droplets with unique compositions. In this paper, protocols for the fabrication and operation of a pressure-driven, PDMS-based bilayer microfluidic device that can be utilized to generate combinatorial libraries of water-in-oil emulsions called plugs are presented. By incorporating software programs and microfluidic hardware, the flow of desired fluids in the device can be controlled and manipulated to generate combinatorial plug libraries and to control the composition and quantity of constituent plug populations. These protocols will expedite the process of generating combinatorial screens, particularly to study drug response in cells from cancer patient biopsies.

Autore in primo piano: Integrazione di approcci computazionali e sperimentali nell'oncologia di precisione

The fabrication of a polydimethylsiloxane (PDMS)-based bilayer device for the production of combinatorial libraries in water-in-oil emulsions (plugs) is presented here. The necessary hardware and software required to automate plug production are detailed in the protocol, and the production of a quantitative library of fluorescent plugs is also demonstrated.

Dispositivo microfluidico a doppio strato per la produzione di tappi combinatori

Droplet microfluidics is a versatile tool that allows the execution of a large number of reactions in chemically distinct nanoliter compartments. Such ...