Finanziamento supporto fornito dal NIH concedere HL89764, borse di NSF 0546012, 0730503 e 0725528.

A. biotinilazione del DNA DNA plasmidico sono state covalentemente biotinilato con guanina-specifiche etichette biotina, come descritto dal produttore (Mirus Bio, Madison, WI), ma in scala di avere ~ 1-2 etichette biotina al DNA. DNA plasmidico (pEGFP-C1, 4,9 kb, Clontech, Mountain View, CA) è stato etichettato in questo protocollo. <ol><li> Sciogliere quantità desiderata di PDNA in TE buffer di DNasi e senza RNasi-free (biologia molecolare-grado di qualità) di acqua per ottenere una soluzione 1μg/μL DNA. </li><li> Condurre la reazione di marcatura usando le miscele di reazione seguente. Aggiungere l&#39;etichetta IT reagente scorso. </li></ol> Per reazione il DNA 100μg: <table border="1"><tbody><tr><td> DNasi RNasi-free e senza acqua </td><td> 75 microlitri </td></tr><tr><td> Etichettatura Buffer 10X A </td><td> 20 l </td></tr><tr><td> 1μg/μL DNA </td><td> 100 ul </td></tr><tr><td> Etichetta IT reagente </td><td> 5 microlitri </td></tr><tr><td> Volume totale </td><td> 200 l </td></tr></tbody></table><ol start="3"><li> Incubare la reazione a 37 ° C per 1 ora. </li><li> Purificare il campione etichettato da etanolo o isopropanolo precipitazione seguenti protocolli standard. </li></ol> Nota: le colonne di filtrazione gel a base può portare ad assorbimento UV ad alta o fluorescenza di fondo, che possono influenzare la quantificazione del DNA o la caratterizzazione fluorescenza. Nota: Il livello di biotinilazione può essere determinata da HABA a base di test. B. Etichettatura dei Cy5-polimeri cationici Chitosano (390 kDa, 83,5% deacetilata, Vanson, Redmond, WA) è stato utilizzato come modello di un polimero cationico in questo studio. Le ammine primarie libero sulla dorsale chitosano polimero sono stati etichettati con Cy5-NHS (Amersham Biosciences, Piscataway, NJ). <ol><li> Per facilitare la coniugazione completa di Cy5 colorante, calcolare in modo tale che il rapporto molare tra Cy5 la quantità richiesta di Cy5-SSN: ammine primarie è di 1: 200. </li><li> Regolare il pH della soluzione di chitosano (in tampone acetato 25mM) per ~ 6,5 con l&#39;aggiunta di NaOH. Si noti che la reazione NHS è più efficiente a pH basico, ma la solubilità del chitosano qui limita l&#39;intervallo di pH di lavoro. </li><li> Sotto agitazione, aggiungere lentamente la quantità calcolata di Cy5-NHS (1 mg / ml DMSO) alla soluzione chitosano in una goccia a goccia modo. </li><li> Agitare la miscela al buio a temperatura ambiente per una notte. </li><li> Per purificare, Dializzare con 10k MWCO Slide-a-Lyzer (Pierce) per 2 ore contro tampone acetato 1% a temperatura ambiente al buio. </li><li> Sostituire il tampone e Dializzare altre 2 ore a temperatura ambiente al buio. </li><li> Sostituire il tampone e Dializzare notte a 4 ° C al buio. </li><li> Conservare purificato etichettati polimero a -20 ° C. </li></ol> Nota: In questo studio, una curva standard è costruito misurando l&#39;intensità di emissione di Cy5-NHS estere a 670 nm. Caratterizzano la densità etichettatura misurando l&#39;emissione ottenuti a 670 nm da Cy5 marcato chitosano nella curva standard. Assorbanza può anche essere usato per determinare l&#39;efficienza etichettatura, ma non è stato eseguito qui. Preparazione C. QD marcato DNA e Cy5-Polymer Il rapporto molare di PDNA di QD è stato mantenuto in eccesso (PDNA: QD ≈ 1: 2) per garantire la coniugazione completa di QD di PDNA. Il numero di QD etichetta su ogni PDNA può essere stimata attraverso immagini TEM o altre strutture equivalenti. Nel nostro studio, il numero di QD per PDNA è stimato in ~ 1-3 da TEM e spettroscopia di singola molecola. 1 Utilizzare Millipore Milli-Q acqua pendenza (&gt; 18,0 MW, 0.2um filtrata) durante la preparazione. <ol><li> Calcolare la quantità di chitosano per 10μg PDNA secondo desiderato N / P, il rapporto teorico di ammine protonate nella soluzione di chitosano per i fosfati negativo nella soluzione del DNA. </li><li> Aggiungi streptavidina-funzionalizzati 605QDs (Qdot 605 ITK, Invitrogen, Carlsbad, CA) nella soluzione biotinilato PDNA. </li><li> Incubare la soluzione a temperatura ambiente al buio per 15 min. </li><li> Aggiungere il QD marcato DNA in 50 mM soluzione di solfato di sodio per rendere il volume finale 200μL. </li><li> Diluire Cy5-chitosano, secondo desiderato N / P, con acqua Milli-Q per rendere il volume finale 200μL. </li></ol> Nota: Tenere la reazione di scuro per impedire alla photobleaching possibile. Importante: Siate cauti di utilizzare il Qdot 605 ITK ™ streptavidina coniugato (la serie ITK), come Punti quantici in questo catalogo sono progettati per lo scopo di FRET. La serie Qdot regolari sono coniugati con uno strato PEG per evitare legame non specifico, in particolare per l&#39;etichettatura cellulare. Tuttavia, questa ulteriore rivestimento allarga il donatore-accettore distanza, con conseguente reduced trasferimento di efficienza energetica. D. Realizzazione della SU-8 Masters Utilizzando Fotolitografia standard <ol><li> Wafer di silicio è piranha pulito e cotto a 200 ° C per 5 min. </li><li> Per lo spessore di 25 micron padrone designato, cappotto girare il photoresist negativo (SU-8 2025, MicroChem, Newton, MA) su wafer di silicio a 2000 rpm per 30 sec. </li><li> Morbido cuocere la cialda su una piastra con una rampa di 65 ° C / hr a 95 ° C. </li><li> Esporre ai raggi UV (365nm) per 250mJ/cm 2 a un film maschera (CAD / Servizi Arte, Bandon, OR) contenente la progettazione di microcanali. </li><li> Post-esposizione cuocere la cialda su una piastra con una rampa di 65 ° C / hr a 95 ° C. </li><li> Sviluppare il wafer utilizzando SU-8 sviluppatore photoresist. </li><li> Il wafer fantasia è duro cotta su una piastra con una rampa di 65 ° C / hr a 200 ° C. Mantenere il wafer a 200 ° C per almeno 5 ore, poi gradualmente raffreddare la cialda a temperatura ambiente. </li></ol> Importante: rampa graduale durante il SU-8 processo di cottura master è necessario, altrimenti il SU-8, la struttura può staccato dal wafer di silicio o crepe sulla struttura di SU-8 può essere indotta da stress-release. E. Stampaggio Replica del PDMS dai maestri e l&#39;adesione al vetro di copertura <ol><li> Il SU-8 master è posto in una barca di pesatura. </li><li> Mix di silicone elastomero e catalizzatore (Poly (dimetilsiloxano), PDMS, Sylgard 184, Dow Corning, Midland, MI) in un 10: 1 rapporto. </li><li> Versare il composto PDMS sul SU-8 master e lasciare la barca pesa in un essiccatore a vuoto per rimuovere le bolle. </li><li> Curare il PDMS a 65 ° C per 1-2 ore. </li><li> Sbucciare la striscia PDMS dallo stampo maestro Si. </li><li> Punch insenature canale e uscite del dispositivo fluidica. </li><li> Pulire la striscia di PDMS e vetro di copertura con etanolo e poi asciugare all&#39;aria. </li><li> Trattare la pulizia PDMS striscia di vetro e coprire con plasma di ossigeno (20 W per 1 minuto). </li><li> Immediatamente la striscia PDMS legame con il vetro di copertura. </li><li> Lascia il chip incollato microfluidica in forno a 95 ° C per una notte. </li></ol> Importante: il trattamento al plasma e da forno durante la notte sono essenziali per la forza di legame maggiore. F. Monitorare la formazione di DNA Nanocomplexes Nel dispositivo a microfluidi <ol><li> Riempire il canale microfluidica con acqua (per assicurarsi che ci sia assenza di bolle all&#39;interno del canale microfluidica), prima di caricare i reagenti per assicurare un flusso regolare durante l&#39;esperimento. </li><li> Caricare il DNA QD-etichettati e marcati con Cy5 soluzioni di chitosano in due siringhe di vetro singolo, attraverso il tubo descritto nel video. </li><li> Collegare il tubo con le due insenature di dispositivi microfluidici. Essere cauti a non introdurre aria durante il processo. Impostare la velocità di flusso a 20nL/min (PHD-2000 pompa a siringa, Holliston, MA), in condizioni di flusso laminare. </li><li> Controllare la microcanali sotto il microscopio. </li><li> Quando il flusso è stabile (~ 15 a 20 minuti), QD-mediata FRET devono essere osservate al centro del canale. </li><li> Scatta foto di fluorescenza (CCD raffreddata, Qimaging, BC, Canada) in luoghi diversi lungo il canale. </li><li> Analizzare le immagini a fluorescenza con ImageJ e OriginLab. </li></ol><img src="/files/ftp_upload/1432/1432fig1.jpg" alt="Figura 1" /> Figura 1. QD-FRET fornisce un&#39;indicazione sensibile l&#39;insorgenza di Nanocomplexes DNA auto-assemblaggio <ol style="list-style-type:lower-alpha;"><li> Quantum dot-mediato trasferimento di energia di risonanza di fluorescenza (QD-FRET) in grado di fornire una indicazione quantitativa e altamente sensibile di stabilità Polyplex sia in ambienti extra o intra-cellulari, permettendo per la rilevazione inequivocabile l&#39;inizio delle interazioni tra il DNA e il vettore genico. Il FRET coppia, 605QD e Cy5, è stato scelto sulla base di massimizzare sovrapposizione spettrale tra il donatore e accettore e riducendo al minimo il potenziale cross-talk. Per questa coppia, la distanza di Förster è 69.4Å 3. </li><li> Auto-assemblaggio del QD-FRET nanocomplexes DNA. Anionici DNA plasmidico (PDNA) e il vettore gene cationici sono stati etichettati con QD (donatore di energia) e Cy5 (energia accettore), rispettivamente. QD-FRET nanocomplexes si sono formati attraverso elettrostatiche coacervazione complessi. Al momento di eccitazione a 488 nm, QD-FRET-mediata Cy5 emissione indicati formazione di un nanocomplex compatta e intatta. Il tempo di permanenza (t R) può essere calcolato in base alla distanza (x) che misura, da dove i due flussi si incontrano per la posizione di reazione sotto inchiesta, e la velocità di flusso medio (v). A causa della natura del flusso laminare, la miscelazione avviene esclusivamente a livello di interfaccia (centro di ogni immagine), che consente il calcolo preciso di trasporto di massa in funzione di t R. Risoluzione temporale può essere regolata variando la applied portate. (Interno) FRET-mediata segnale è stato osservato immediatamente a livello di interfaccia, quando i due corsi d&#39;acqua ha incontrato, a indicare che il legame è stato rapido, che si verificano in pochi millisecondi in base ai tassi di flusso applicati. Scala Bar: 100μm. </li></ol>

<ol>
	<li>Ho, Y. P., Chen, H. H., Leong, K. W., Wang, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+the+intracellular+stability+and+unpacking+of+DNA+nanocomplexes+by+quantum+dots-FRET.">Evaluating the intracellular stability and unpacking of DNA nanocomplexes by quantum dots-FRET.</a> J Control Release. 116, 83-89 (2006).</li><li>Chen, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+comparison+of+intracellular+unpacking+kinetics+of+polyplexes+by+a+model+constructed+from+quantum+dot-FRET.">Quantitative comparison of intracellular unpacking kinetics of polyplexes by a model constructed from quantum dot-FRET.</a> Mol. Ther. 16, 324-332 (2008).</li><li>Zhang, C. Y., Yeh, H. C., Kuroki, M., Wang, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-Quantum-Dot-Based+DNA+Nanosensor.">Single-Quantum-Dot-Based DNA Nanosensor.</a> Nat Mat. 4, 826-831 (2005).</li></ol>

The authors have nothing to disclose.

<ul><li> Significato del nostro lavoro: <ol><li> Questo è il primo tentativo di monitorare polimerici DNA nanocomplex auto-assemblaggio cinetica in tempo reale (millisecondi risoluzione) attraverso QD-FRET risposte all&#39;interno di un chip semplice microfluidica. </li><li> QD-mediata FRET fornisce un&#39;indicazione molto sensibile e quantitativa l&#39;insorgenza di interazioni molecolari e in tutto il processo di self-assembly, mentre microfluidica offre una ben controllata microambiente per analizzare spazialmente il processo durante la sintesi del DNA nanocomplex. </li><li> L&#39;integrazione di microfluidica e nanofotonica suggerisce un approccio nuovo e interessante per indagare su qualunque tipo di reazioni di complessazione. </li><li> La risultante QD-FRET nanocomplexes DNA polimerici potrebbe essere facilmente applicato per stabilire relazioni struttura-funzione. 1,2 </li></ol></li></ul>

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>SU-8</td>
<td>&nbsp;</td>
<td>MicroChem</td>
<td>SU-8 2025</td>
<td>&nbsp;</td>
<tr>
<td>PDMS</td>
<td>&nbsp;</td>
<td>Dow Corning</td>
<td>Sylgard 184</td>
<td>&nbsp;</td>
<tr>
<td>Plasmid DNA</td>
<td>&nbsp;</td>
<td>Clontech</td>
<td>pEGFP-C1</td>
<td>4.9 kb, MW = 3.3 x 106</td>
</tr>
<tr>
<td>LabelIT Biotin Labeling Kit</td>
<td>&nbsp;</td>
<td>Mirus Bio LLC</td>
<td>MIR 3400</td>
<td>Standard protocol yields labeling efficiency of approximately one label every 20-60 bp of double-stranded DNA, The density of labeling was adjusted in this work.</td>
</tr>
<tr>
<td>Streptavidin 605QD</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>Qdot&reg; 605 ITK&trade; Streptavidin Conjugate</td>
<td>&nbsp;</td>
<tr>
<td>Cy5-NHS Ester</td>
<td>&nbsp;</td>
<td>Amersham Biosciences</td>
<td>PA15101</td>
<td>&nbsp;</td>
<tr>
<td>Chitosan</td>
<td>&nbsp;</td>
<td>Vanson</td>
<td>390 kDa </td>
<td>83.5% deacetylated</td>
</tr>
<tr>
<td>Cover Glass</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>12-545C</td>
<td>No. 1; Size: 40 x 22mm</td>
</tr>
<tr>
<td>Gastight Glass Syringe</td>
<td>&nbsp;</td>
<td>Hamilton</td>
<td>TLL series</td>
<td>50&mu;L to 500&mu;L depending on sample volume</td>
</tr>
<tr>
<td>Tygon Tubing</td>
<td>&nbsp;</td>
<td>SmallParts</td>
<td>0.02 ID, 100ft</td>
<td>Tygon Tubes Microbore, 0.02 ID, 100ft</td>
</tr>
<tr>
<td>Connector</td>
<td>&nbsp;</td>
<td>SmallParts</td>
<td>HTX-23R</td>
<td>Customized in length of 0.750"</td>
</tr>
<tr>
<td>Syringe Pump</td>
<td>&nbsp;</td>
<td>Harvard Apparatus</td>
<td>PHD-2000</td>
<td>&nbsp;</td>
<tr>
<td>CCD</td>
<td>&nbsp;</td>
<td>Qimaging</td>
<td>Intensified Retiga Cooled</td>
<td>&nbsp;</td>
<tr>
<td>Microscope</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>BX-51</td>
<td>100W mercury arc lamp</td>
</tr>
<tr>
<td>ImageJ</td>
<td>&nbsp;</td>
<td>NIH</td>
<td>v1.36b</td>
<td>http://rsb.info.nih.gov/ij</td>
</tr>
<tr>
<td>Origin Pro8</td>
<td>&nbsp;</td>
<td>OriginLab</td>
<td>Student Version</td>
<td>&nbsp;</td>
<tr>
<td>Microscope Filter sets</td>
<td>&nbsp;</td>
<td>Omega Optical</td>
<td>475AF40</td>
<td>Excitation filter in both channels</td>
</tr>
<tr>
<td>Microscope Filter sets</td>
<td>&nbsp;</td>
<td>Omega Optical</td>
<td>595AF60</td>
<td>Emission filter in 605QD channel</td>
</tr>
<tr>
<td>Microscope Filter sets</td>
<td>&nbsp;</td>
<td>Omega Optical</td>
<td>670DF40</td>
<td>Emission filter in QD-FRET channel</td>
</tr>
<tr>
<td>Microscope Filter sets</td>
<td>&nbsp;</td>
<td>Omega Optical</td>
<td>500 DRLP</td>
<td>Long pass dichroic in 605QD channel</td>
</tr>
<tr>
<td>Microscope Filter sets</td>
<td>&nbsp;</td>
<td>Omega Optical</td>
<td>595DRLP</td>
<td>Long pass dichroic in QD-FRET channel</td>
</tr>		</tbody>
		</table>
		</div>

combining qd-fret and microfluidics to monitor dna nanocomplex self-assembly in real-time

I progressi nella genomica continuano ad alimentare lo sviluppo di terapie che possono bersaglio patogenesi a livello cellulare e molecolare. Tipicamente funzionale all&#39;interno della cellula, acidi nucleici terapie a base richiedono un efficiente sistema di distribuzione intracellulare. Un metodo ampiamente adottato è quello del DNA complesso con un vettore gene per formare nanocomplexes tramite auto-assemblaggio elettrostatico, facilitando l&#39;assorbimento cellulare di DNA, mentre la protezione contro la degradazione. La sfida consiste nella progettazione razionale dei vettori gene efficiente, dal momento prematuro dissociazione o eccessivamente vincolante stabile sarebbe dannoso per la captazione cellulare e l&#39;efficacia terapeutica. Nanocomplexes sintetizzato mescolando di massa ha mostrato una vasta gamma di comportamenti intracellulari disimballaggio e il traffico, che è stata attribuita alla eterogeneità di dimensioni e la stabilità del nanocomplexes. Tale eterogeneità ostacola la corretta valutazione del self-assembly cinetica e aggiunge alla difficoltà di correlare le loro proprietà fisiche di efficienza di trasfezione o bioactivities. Vi presentiamo una convergenza romanzo di nanofotonica (cioè QD-FRET) e microfluidica per caratterizzare in tempo reale cinetica del nanocomplex self-assembly sotto flusso laminare. QD-FRET fornisce un&#39;indicazione molto sensibile della insorgenza di interazioni molecolari e misura quantitativa per tutto il processo di sintesi, mentre microfluidica offre una ben controllata microambiente per analizzare spazialmente il processo con alta risoluzione temporale (~ millisecondi). Per il sistema modello di nanocomplexes polimerici, due fasi distinte nel processo di auto-assemblaggio sono stati catturati da questa piattaforma analitica. L&#39;aspetto cinetico del processo di auto-assemblaggio presso la microscala sarebbe particolarmente utile per microreattore basato reazioni che sono rilevanti per molte applicazioni micro-e nano-scala. Inoltre, nanocomplexes può essere personalizzato attraverso la corretta progettazione di dispositivi microfludic, e la conseguente QD-FRET nanocomplexes DNA polimerici può essere facilmente applicato per stabilire relazioni struttura-funzione.

Watch this Scientific Journal Video about Combining QD-FRET and Microfluidics to Monitor DNA Nanocomplex Self-Assembly in Real-Time at JoVE.com

Combining QD-FRET and Microfluidics to Monitor DNA Nanocomplex Self-Assembly in Real-Time

Vi presentiamo un nuovo e potente integrazione di nanofotonica (QD-FRET) e microfluidica per studiare la formazione di polyplexes polielettrolita, che si prevede di fornire un migliore controllo e la sintesi di polyplexes uniforme e personalizzabili per i futuri nucleici terapie a base di acido.

Combinando QD-FRET e microfluidica per il monitoraggio Nanocomplex DNA di auto-assemblaggio in tempo reale

Advances in genomics continue to fuel the development of therapeutics that can target pathogenesis at the cellular and molecular level. Typically ...

combining-qd-fret-microfluidics-to-monitor-dna-nanocomplex-self

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Biology

11.0K Views.  Johns Hopkins University. Vi presentiamo un nuovo e potente integrazione di nanofotonica (QD-FRET) e microfluidica per studiare la formazione di polyplexes polielettrolita, che si prevede di fornire un migliore controllo e la sintesi di polyplexes uniforme e personalizzabili per i futuri nucleici terapie a base di acido.

Video: Combining QD-FRET and Microfluidics to Monitor DNA Nanocomplex Self-Assembly in Real-Time

Chemotactic Response of Marine Micro-Organisms to Micro-Scale Nutrient Layers

The degree to which planktonic microbes can exploit microscale resource patches will have considerable implications for oceanic trophodynamics and biogeochemical flux. However, to take advantage of nutrient patches in the ocean, swimming microbes must overcome the influences of physical forces including molecular diffusion and turbulent shear, which will limit the availability of patches and the ability of bacteria to locate them. Until recently, methodological limitations have precluded direct examinations of microbial behaviour within patchy habitats and realistic small-scale flow conditions. Hence, much of our current knowledge regarding microbial behaviour in the ocean has been procured from theoretical predictions. To obtain new information on microbial foraging behaviour in the ocean we have applied soft lithographic fabrication techniques to develop 2 microfluidic devices, which we have used to create (i) microscale nutrient patches with dimensions and diffusive characteristics relevant to oceanic processes and (ii) microscale vortices, with shear rates corresponding to those expected in the ocean. These microfluidic devices have permitted a first direct examination of microbial swimming and chemotactic behaviour within a heterogeneous and dynamic seascape. The combined use of epifluorescence and phase contrast microscopy allow direct examinations of the physical dimensions and diffusive characteristics of nutrient patches, while observing the population-level aggregative response, in addition to the swimming behaviour of individual microbes. These experiments have revealed that some species of phytoplankton, heterotrophic bacteria and phagotrophic protists are adept at locating and exploiting diffusing microscale resource patches within very short time frames. We have also shown that up to moderate shear rates, marine bacteria are able to  fight the flow  and swim through their environment at their own accord. However, beyond a threshold high shear level, bacteria are aligned in the shear flow and are less capable of swimming without disturbance from the flow. Microfluidics represents a novel and inexpensive approach for studying aquatic microbial ecology, and due to its suitability for accurately creating realistic flow fields and substrate gradients at the microscale, is ideally applicable to examinations of microbial behaviour at the smallest scales of interaction.  We therefore suggest that microfluidics represents a valuable tool for obtaining a better understanding of the ecology of microorganisms in the ocean.

The fabrication of microfluidic channels and their implementation in experiments for studying the chemotactic foraging behaviour of marine microbes within a patchy nutrient seascape and the swimming behaviour of bacteria within shear flow are described.

Risposta chemiotattica di microrganismi marini a livelli di nutrienti Micro-Scale

The degree to which planktonic microbes can exploit microscale resource patches will have considerable implications for oceanic trophodynamics and ...

Ricerca

Biologia

Studies of Bacterial Chemotaxis Using Microfluidics - Interview

Gli studi di chemiotassi batterica Utilizzando Microfluidica - Intervista

Applying Microfluidics to Electrophysiology

Microfluidics can be integrated with standard electrophysiology techniques to allow new experimental modalities.  Specifically, the motivation for the microfluidic brain slice device is discussed including how the device docks to standard perfusion chambers and the technique of passive pumping which is used to deliver boluses of neuromodulators to the brain slice.  By simplifying the device design, we are able to achieve a practical solution to the current unmet electrophysiology need of applying multiple neuromodulators across multiple regions of the brain slice.  This is achieved by substituting the standard coverglass substrate of the perfusion chamber with a thin microfluidic device bonded to the coverglass substrate.  This was then attached to the perfusion chamber and small holes connect the open-well of the perfusion chamber to the microfluidic channels buried within the microfluidic substrate.  These microfluidic channels are interfaced with ports drilled into the edge of the perfusion chamber to access and deliver stimulants.  This project represents how the field of microfluidics is transitioning away from proof-of concept device demonstrations and into practical solutions for unmet experimental and clinical needs.

Applicando Microfluidica di Elettrofisiologia

Microfluidics can be integrated with standard electrophysiology techniques to allow new experimental modalities.  Specifically, the motivation for the ...

Digital Microfluidics for Automated Proteomic Processing

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and prognosis of disease. While clinical proteomic methods vary widely, a common characteristic is the need for (i) extraction of proteins from extremely heterogeneous fluids (i.e. serum, whole blood, etc.) and (ii) extensive biochemical processing prior to analysis. Here, we report a new digital microfluidics (DMF) based method integrating several processing steps used in clinical proteomics. This includes protein extraction, resolubilization, reduction, alkylation and enzymatic digestion. Digital microfluidics is a microscale fluid-handling technique in which nanoliter-microliter sized droplets are manipulated on an open surface. Droplets are positioned on top of an array of electrodes that are coated by a dielectric layer - when an electrical potential is applied to the droplet, charges accumulate on either side of the dielectric. The charges serve as electrostatic handles that can be used to control droplet position, and by biasing a sequence of electrodes in series, droplets can be made to dispense, move, merge, mix, and split on the surface. Therefore, DMF is a natural fit for carrying rapid, sequential, multistep, miniaturized automated biochemical assays. This represents a significant advance over conventional methods (relying on manual pipetting or robots), and has the potential to be a useful new tool in clinical proteomics.
Mais J. Jebrail, Vivienne N. Luk, and Steve C. C. Shih contributed equally to this work.
Sergio L. S. Freire's current address is at the University of the Sciences in Philadelphia located at 600 South 43rd Street, Philadelphia, PA 19104.

Digital Microfluidics is a technique characterized by the manipulation of discrete droplets (~nL - mL) on an array of electrodes by the application of electrical fields. It is well-suited for carrying out rapid, sequential, miniaturized automated biochemical assays. Here, we report a platform capable of automating several proteomic processing steps.

Microfluidica digitale per trattamento automatizzato di proteomica

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and ...

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, messages are carried by neurotransmitters released from presynaptic terminals and received by receptors on postsynaptic cells. Our previous research in Caenorhabditis elegans has shown that VSM-1 negatively regulates exocytosis. Additionally, analysis of synapses in vsm-1 mutants showed that animals lacking a fully functional VSM-1 have increased synaptic connectivity. Based on these preliminary findings, we hypothesized that C. elegans VSM-1 may play a crucial role in synaptogenesis. To test this hypothesis, double-labeled microarray analysis was performed, and gene expression profiles were determined. First, total RNA was isolated, reversely transcribed to cDNA, and hybridized to the DNA microarrays. Then, in-silico analysis of fluorescent probe hybridization revealed significant induction of many genes coding for members of the major sperm protein family (MSP) in mutants with enhanced synaptogenesis. MSPs are the major component of sperm in C. elegans and appear to signal nematode oocyte maturation and ovulation . In fruit flies, Chai and colleagues 1 demonstrated that MSP-like molecules regulate presynaptic bouton number and size at the neuromuscular junction. Moreover, analysis performed by Tsuda and coworkers 2 suggested that MSPs may act as ligands for Eph receptors and trigger receptor tyrosine kinase signaling cascades. Lastly, real time PCR analysis corroborated that the gene coding for MSP-32 is induced in vsm-1(ok1468) mutants. Taken together, research performed by our laboratory has shown that vsm-1 mutants have a significant increase in synaptic density, which could be mediated by MSP-32 signaling.

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Microarray analysis was conducted to determine genetic expression profiles in C. elegans, and real-time PCR was used to validate and quantify microarray data.

Determinare profili di espressione genetica in C. elegans Utilizzo Microarray e Real-time PCR

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, ...

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.

Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres.

Combinando singola molecola di manipolazione e di imaging per lo studio delle interazioni proteina-DNA

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

In tempo reale Imaging del singolo Engineered RNA trascritti in cellule viventi Utilizzando Ratiometric bimolecolari Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix containing polysaccharides that include cellulose microfibrils that form both crystalline structures and cellulose chains of amorphous organization. The orientation of the cellulose fibers and their concentrations dictate the mechanical properties of the cell. Several methods are used to determine the levels of crystalline cellulose, each bringing both advantages and limitations. Some can distinguish the proportion of crystalline regions within the total cellulose. However, they are limited to whole-organ analyses that are deficient in spatiotemporal information. Others relying on live imaging, are limited by the use of imprecise dyes. Here, we report a sensitive polarized light-based system for specific quantification of relative light retardance, representing crystalline cellulose accumulation in cross sections of Arabidopsis thaliana roots. In this method, the cellular resolution and anatomical data are maintained, enabling direct comparisons between the different tissues composing the growing root. This approach opens a new analytical dimension, shedding light on the link between cell wall composition, cellular behavior and whole-organ growth.

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging. Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution.

Risoluzione Quantificazione alta del cristallino cellulosa accumulo in<em&gt; Arabidopsis</em&gt; Roots a Monitor tessuto-specifica delle cellule parete Modifiche

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix ...

Detecting and Characterizing Protein Self-Assembly In Vivo by Flow Cytometry

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from low-sensitivity, indirect read-outs, limited throughput, and/or population-level rather than single-cell resolution. We designed a flow cytometry-based single methodology that addresses all of these limitations: Distributed Amphifluoric FRET or DAmFRET. DAmFRET detects and quantifies protein self-assemblies by sensitized emission FRET in vivo, enables deployment across model systems-from yeast to human cells-and achieves sensitive, single-cell, high-throughput read-outs irrespective of protein localization or solubility.

This article describes a FRET-based flow cytometry protocol to quantify protein self-assembly in both S. cerevisiae and HEK293T cells.

Rilevamento e caratterizzazione dell'autoassemblaggio proteico in vivo da parte della Cytometria di flusso

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from ...

Wide-Field, Real-Time Imaging of Local and Systemic Wound Signals in Arabidopsis

Plants respond to mechanical stresses such as wounding and herbivory by inducing defense responses both in the damaged and in the distal undamaged parts. Upon wounding of a leaf, an increase in cytosolic calcium ion concentration (Ca2+ signal) occurs at the wound site. This signal is rapidly transmitted to undamaged leaves, where defense responses are activated. Our recent research revealed that glutamate leaking from the wounded cells of the leaf into the apoplast around them serves as a wound signal. This glutamate activates glutamate receptor-like Ca2+ permeable channels, which then leads to long-distance Ca2+ signal propagation throughout the plant. The spatial and temporal characteristics of these events can be captured with real-time imaging of living plants expressing genetically encoded fluorescent biosensors. Here we introduce a plant-wide, real-time imaging method to monitor the dynamics of both the Ca2+ signals and changes in apoplastic glutamate that occur in response to wounding. This approach uses a wide-field fluorescence microscope and transgenic Arabidopsis plants expressing Green Fluorescent Protein (GFP)-based Ca2+ and glutamate biosensors. In addition, we present methodology to easily elicit wound-induced, glutamate-triggered rapid and long-distance Ca2+ signal propagation. This protocol can also be applied to studies on other plant stresses to help investigate how plant systemic signaling might be involved in their signaling and response networks.

Wide-Field, Real-Time Imaging of Local and Systemic Wound Signals in Arabidopsis

Extracellular glutamate-triggered systemic calcium signaling is critical for the induction of plant defense responses to mechanical wounding and herbivore attack in plants. This article describes a method to visualize the spatial and temporal dynamics of both these factors using Arabidopsis thaliana plants expressing calcium- and glutamate-sensitive fluorescent biosensors.

Imaging in tempo reale a ampio campo dei segnali di ferita locali e sistemici in Arabidopsis

Plants respond to mechanical stresses such as wounding and herbivory by inducing defense responses both in the damaged and in the distal undamaged parts. ...