Les auteurs tiennent à remercier les sciences naturelles et en génie de recherches du Canada, Programme des chaires de recherche du Canada, Instituts de recherche en santé du Canada et le ministère de la Recherche et de l&#39;Innovation de l&#39;Ontario par l&#39;entremise de leur Programme de bourses des chercheurs pour un soutien financier.

1. Photolithographie pour Mold Maître Fabrication <ol><li> Baigner un 3 pouces plaquette de silicium dans de l&#39;acétone pendant 30 sec, puis le méthanol pendant 30 sec. Rincer avec dH 2 0 eau pendant 5 min. </li><li> Sécher la surface de la plaquette avec un pistolet à air de N2. Chauffer la tranche sur une plaque chauffante à 140 ° C pendant 2 min. </li><li> Plasma oxyder la surface de la tranche de silicium (1 min, 50 W). </li><li> Spin-coat surface de la plaquette avec 3 ml SU-8 100 photosensible (40 sec; 1,750 rpm). </li><li> Pré-cuire la plaquette revêtue sur une plaque chauffante à 65 ° C pendant 10 min, puis la rampe la température jusqu&#39;à 95 ° C en 2 minutes. Maintenir ce réglage pour un montant supplémentaire de 1 h. </li><li> Aligner un masque photographique contenant la conception du canal désiré. Exposer la résister à 550-600 mJ / cm 2 de lumière UV (350-400 nm). Masques peuvent être conçus dans AutoCAD et imprimés sur un transparent avec impression haute résolution. </li><li> Post-cuire la galette sur une plaque chauffante à 65 ° C pendant 1 min et 95 ° C pendant 10 min, rampe til température comme avant. </li><li> Plonger la plaquette dans la solution de révélateur SU-8 pendant 10-15 min. Vérifiez achèvement du développement d&#39;un rinçage avec de l&#39;isopropanol. Si un précipité blanc apparaît, continuer à développer. Le moule maître est représenté sur la figure 2A. </li></ol> 2. Lithographie douce pour Fabrication Microchannel <ol><li> Mélanger 35 ml de polydiméthylsiloxane (PDMS) de la base d&#39;élastomère avec 3,5 ml PDMS agent de durcissement. </li><li> Placez le moule maître fabriqué (modèle vers le haut) et une plaquette de silicium vierge dans des boîtes de Pétri recouverte de papier d&#39;aluminium. </li><li> Verser 20 ml prépolymère PDMS dans le plat du moule maître et de 15 ml dans le second plat. Éliminer les poches d&#39;air sous les plaquettes en appuyant doucement sur eux avec un applicateur jetable en bois. </li><li> Couvrir les deux plats et mettre de côté pour un jour à guérir. Sinon, pour un durcissement plus rapide, éliminer les bulles d&#39;air du PDMS en utilisant un dégazeur à vide et ensuite laisser les plats sur une plaque chauffante à 80 ° C pendant 2hr. </li><li> Retirez le film et le zeste du PDMS à partir des plaquettes. </li><li> Utilisez le Harris Uni-Core (2,5 mm) pour percer des orifices d&#39;accès de fluide aux deux extrémités de la chaîne. Couper le canal et blanc PDMS en bandes de taille similaire. </li><li> Charger le canal, la bande ébauche PDMS et d&#39;une lame de verre (75 x 25 mm 2) dans un dispositif d&#39;oxydation par plasma, probablement situé dans une salle blanche. Exposer à un plasma d&#39;oxygène pendant 40 secondes à 40 W de puissance. </li><li> Coller le morceau de canal et lame de verre à faces opposées de la bande ébauche. Mettez de côté pendant 2 heures pour achever le collage. </li><li> Placez l&#39;ensemble sur une plaque chauffante à 120 ° C. Fixer le tube en plastique (diamètre intérieur 1/32 &quot;, diamètre extérieur de 3/32&quot;), chacun au moins 6 po de long, les réservoirs percés à l&#39;aide de PDMS prépolymère. Fixer un raccord en plastique fluidique à un ou deux tubes pour permettre la fixation de la seringue, ou utiliser des raccords disponibles dans le commerce. </li><li> Laisser le PDMS fixer le tube à guérir. Insérez 3 &quot;longueurs de calibre 22 fil de cuivre isolé dans each réservoir, entre le tube d&#39;admission et le canal, et sûr PDMS prépolymère. Le produit fini est illustré à la figure 2B. </li></ol> 3. Électrotaxie Experiment <ol><li> Placer le microcanal sur la scène (de préférence XY mobile) d&#39;un microscope avec une caméra montée reliée à un moniteur (Figure 1). </li><li> Connectez l&#39;alimentation ou les fils de sortie de l&#39;amplificateur à des électrodes de la microcanaux. Une alimentation CC simple est suffisant, si seulement un signal de courant continu est souhaité, mais un amplificateur connecté à un générateur de fonction permet à l&#39;application de courant continu à impulsions et des signaux alternatifs de même. </li><li> Fixer le tuyau de sortie du micro-canal à une seringue jetable. Immerger l&#39;embouchure du tube d&#39;entrée dans un tampon physiologique, M9 et doucement aspirer du liquide dans le canal par application d&#39;une pression négative à l&#39;intérieur de la seringue (soit manuellement, soit en utilisant une pompe à seringue). Lorsque les tubes d&#39;entrée et de sortie sont tous deux remplis de M9, déconnecter la seringue from le tube. Niveler les deux tubes à la même hauteur pour empêcher l&#39;écoulement hydrostatique entraînée. </li><li> Appliquer une tension de courant continu sur le canal et faire en sorte que la résistance (R = U / I) est d&#39;environ 0,6 MQ (pour une longueur de 50 mm, 0,3 mm de largeur et d&#39;environ 0,1 mm de profondeur à microcanaux). </li><li> S&#39;il est satisfait de l&#39;intégrité de la chaîne, suivez les étapes ci-dessus pour charger les vers d&#39;une suspension diluée dans le canal. </li><li> Déconnecter la seringue et hydrostatique manipuler le flux de réglage de la hauteur par rapport aux tubes d&#39;. Utilisez cette méthode pour mettre un ver dans le centre du chenal et puis posez les deux tubes plats à la même altitude. </li><li> Régler l&#39;alimentation sur la tension appropriée: 4-12 V / cm pour les animaux de scène L3, 4-10 V / cm pour L4s et 2-4 V / cm pour les jeunes adultes. Activez le signal électrique et permettre 1 min de pré-exposition pour le ver de s&#39;acclimater au terrain. Le ver devrait commencer à se déplacer vers la cathode. Lorsque la minute est passée, utiliser l&#39;appareil photo pour commencer l&#39;enregistrement. </li><li> Pour AC et le poulsd expériences DC, le champ électrique en réponse au maximum peut être adoptée de cycle du signal ci-dessus et de la fréquence et de service peuvent être modulées à volonté 7, 8. </li><li> Quand l&#39;expérience est terminée, retirer tout le liquide (et vers) du canal, le rincer avec dH 2 0, et laisser l&#39;appareil sur une plaque chauffante à 125 ° C pour sécher. </li><li> Extraire des données locomotrices de vidéos enregistrées manuellement à l&#39;aide NIH ImageJ ( <a href="http://rsbweb.nih.gov/ij/" target="_blank">http://rsbweb.nih.gov/ij/</a> ) ou un logiciel de suivi de ver MATLAB basée personnalisé. </li></ol>

<ol>
	<li>Kuwahara, T., Koyama, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Familial+Parkinson+mutant+&#945;-synuclein+causes+dopamine+neuron+dysfunction+in+transgenic+Caenorhabditis+elegans.">Familial Parkinson mutant &#945;-synuclein causes dopamine neuron dysfunction in transgenic Caenorhabditis elegans.</a> J. Biol. Chem. 281 (1), 334-340 (2006).</li><li>Kuwahara, T., Koyama, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+RNAi+screen+reveals+involvement+of+endocytic+pathway+in+neuronal+dysfunction+in+a-synuclein+transgenic.">A systematic RNAi screen reveals involvement of endocytic pathway in neuronal dysfunction in a-synuclein transgenic.</a> 17 (19), 2997-3009 (2008).</li><li>Su, L. J., Auluck, P. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compounds+from+an+unbiased+chemical+screen+reverse+both+ER-to-Golgi+trafficking+defects+and+mitochondrial+dysfunction+in+Parkinson's+disease+models.">Compounds from an unbiased chemical screen reverse both ER-to-Golgi trafficking defects and mitochondrial dysfunction in Parkinson's disease models.</a> Dis. Model Mech. 3 (3-4), 194-208 (2010).</li><li>Rezai, P., Siddiqui, A., Selvaganapathy, P. R., Gupta, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrotaxis+of+Caenorhabditis+elegans+in+a+microfluidic+environment.">Electrotaxis of Caenorhabditis elegans in a microfluidic environment.</a> Lab Chip. 10 (2), 220-226 (2010).</li><li>Salam, S., Ansari, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microfluidics+set+up+to+study+neuronal+degeneration+and+identification+of+neuroprotective+compounds+in+C.+elegans.">A microfluidics set up to study neuronal degeneration and identification of neuroprotective compounds in C. elegans.</a> , (2013).</li><li>Rezai, P., Salam, S., Selvaganapathy, P. R., Gupta, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+sorting+of+Caenorhabditis+elegans.">Electrical sorting of Caenorhabditis elegans.</a> Lab Chip. 12 (10), 1831-1840 (2012).</li><li>Rezai, P., Salam, S., Selvaganapathy, P. R., Gupta, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+pulse+direct+current+signals+on+electrotactic+movement+of+nematodes+Caenorhabditis+elegans+and+Caenorhabditis+briggsae.">Effect of pulse direct current signals on electrotactic movement of nematodes Caenorhabditis elegans and Caenorhabditis briggsae.</a> Biomicrofluidics. 5 (4), 044116 (2011).</li><li>Rezai, P., Siddiqui, A., Selvaganapathy, P. R., Gupta, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavior+of+Caenorhabditis+elegans+in+alternating+electric+field+and+its+application+to+their+localization+and+control.">Behavior of Caenorhabditis elegans in alternating electric field and its application to their localization and control.</a> Appl. Phys. Lett. 96 (15), 153702 (2010).</li><li>van Ham, T. J., Thijssen, K. L., Breitling, R., Hofstra, R. M., Plasterk, R. H., Nollen, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C.+elegans+model+identifies+genetic+modifiers+of+alpha-synuclein+inclusion+formation+during+aging.">C. elegans model identifies genetic modifiers of alpha-synuclein inclusion formation during aging.</a> PLoS Genet. 4, e1000027 (2008).</li><li>Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetics+of+Caenorhabditis+elegans.">The genetics of Caenorhabditis elegans.</a> Genetics. 77 (1), 71-94 (1974).</li><li>Gabel, C. V., Gabel, H., Pavlichin, D., Kao, A., Clark, D. A., Samuel, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+circuits+mediate+electrosensory+behavior+in+Caenorhabditis+elegans.">Neural circuits mediate electrosensory behavior in Caenorhabditis elegans.</a> J. Neurosci. 27 (28), 7586-7596 (2007).</li><li>Chuang, H. -. S., Raizen, D. M., Lamb, A., Dabbish, N., Bau, 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=Dielectrophoresis+of+Caenorhabditis+elegans.">Dielectrophoresis of Caenorhabditis elegans.</a> Lab Chip. 11 (4), 599-604 (2011).</li><li>Cronin, C. J., Mendel, J. E., Mukhtar, S., Kim, Y. -. M., Stirbl, R. C., Bruck, J., Sternberg, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+automated+system+for+measuring+parameters+of+nematode+sinusoidal+movement.">An automated system for measuring parameters of nematode sinusoidal movement.</a> BMC Genet. 6, 5 (2005).</li><li>Mani&#232;re, X., Lebois, F., Matic, I., Ladoux, B., Meglio, J. -. M. D. i., Hersen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Running+worms:+C.+elegans+self-sorting+by+electrotaxis.">Running worms: C. elegans self-sorting by electrotaxis.</a> PLoS One. 6 (2), e16637 (2011).</li></ol>

La technologie d&#39;analyse électrotaxie microfluidique a été déposé de brevet aux États-Unis d&#39;Amérique et le Canada.

Profitant du phénomène comportemental d&#39;abord décrit par Gabel et ses collègues et en s&#39;appuyant sur ​​le travail de manipulation diélectrophorétique de Chuang et ses collègues 11,12, notre test de électrotaxie microfluidique à base offre une méthode simple, robuste et sensible pour sonder l&#39;activité neuronale dans les vers utilisant le mouvement comme une sortie. L&#39;analyse des paramètres de mouvement permet une comparaison quantitative entre les différents génotypes. La pré...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>CALEDON Labs</td>
			<td>1200-1-30</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>CALEDON Labs</td>
			<td>6700-1-30</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>CALEDON Labs</td>
			<td>8600-1-40</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SU-8</td>
			<td>Microchem Corp.</td>
			<td>Y131273</td>
			<td>SU-8 100</td>
		</tr>
		<tr>
			<td>SU-8 Developer</td>
			<td>Microchem Corp.</td>
			<td>Y020100</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>92x16 mm Petri dish</td>
			<td>Sarstedt</td>
			<td>82.1473.001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sylgard 184 Silicone Elastomer Kit</td>
			<td>Dow Corning</td>
			<td>&nbsp;</td>
			<td>Contains elastomer base and curing agent</td>
		</tr>
		<tr>
			<td>Function generator</td>
			<td>Tektronix Inc.</td>
			<td>&nbsp;</td>
			<td>Model AFG3022B</td>
		</tr>
		<tr>
			<td>Amplifier</td>
			<td>Trek Inc.</td>
			<td>&nbsp;</td>
			<td>Model 2210-CE</td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>Harvard Apparatus</td>
			<td>70-4506</td>
			<td>Model 11 ELITE</td>
		</tr>
		<tr>
			<td>Hot plate</td>
			<td>Fisher Scientific</td>
			<td>11675916Q</td>
			<td>Model HP131725Q</td>
		</tr>
	</tbody>
</table>

microfluidic-based electrotaxis for on-demand quantitative analysis of caenorhabditis elegans' locomotion

Les Caenorhabditis elegans nématode est un modèle polyvalent organisme pour la recherche biomédicale en raison de sa conservation de gènes et les voies liées à la maladie ainsi que sa facilité de culture. Plusieurs C. modèles de maladies elegans ont été signalés, y compris les maladies neurodégénératives telles que la maladie de Parkinson (PD), ce qui implique la dégénérescence des dopaminergiques (DA) des neurones 1. Les deux transgènes et des produits chimiques neurotoxiques ont été utilisés pour induire DA neurodégénérescence et les anomalies de mouvement conséquente vers, permettant des enquêtes sur la base de la neurodégénérescence et les écrans des gènes et des composés 2,3 neuroprotecteurs. Écrans chez les eucaryotes inférieurs comme C. elegans fournir un moyen efficace et économique pour identifier les composés et les gènes affectant la signalisation neuronale. Écrans classiques sont généralement effectuées manuellement et marqués par inspection visuelle, par conséquent, ils sont contre-tempsuming et sujettes à des erreurs humaines. En outre, la plupart se concentrent sur l&#39;analyse du niveau cellulaire tout en ignorant locomotion, qui est un paramètre particulièrement important pour les troubles du mouvement. Nous avons développé un système de criblage microfluidique roman (Figure 1) qui contrôle et quantifie C. La locomotion elegans de l&#39;aide stimuli de champ électrique à l&#39;intérieur de microcanaux. Nous avons montré qu&#39;un champ courant continu (CC) peut induire robuste sur demande locomotion vers la cathode (&quot;électrotaxie&quot;) 4. Inversion de polarité du champ entraîne le ver à inverser rapidement la direction ainsi. Nous avons également montré que les défauts dans les neurones sensoriels et d&#39;autres dopaminergiques modifier la réponse de natation 5. Par conséquent, des anomalies dans la signalisation neuronale peuvent être déterminées en utilisant locomotion comme une lecture. La réponse du mouvement peut être quantifiée avec précision en utilisant une gamme de paramètres tels que la vitesse de nage, le corps fréquence de flexion et temps d&#39;inversion. <p class = &quot;jove_content&quot;&gt; Notre travail a révélé que la réponse electrotactic varie avec l&#39;âge. Plus précisément, les jeunes adultes répondent à une gamme inférieure des champs électriques et se déplacent plus rapidement par rapport aux larves 4. Ces résultats nous ont amenés à concevoir un nouveau dispositif microfluidique pour trier passivement vers l&#39;âge et le phénotype 6. Nous avons également testé la réponse des vers à impulsions DC et courant alternatif champs électriques (AC). Champs continus pulsés de différents cycles efficacement électrotaxie générés à la fois C. elegans et sa cousine C. briggsae 7. Dans une autre expérience, symétriques champs AC avec des fréquences allant de 1 Hz à 3 kHz immobilisés vers l&#39;intérieur du canal 8. Mise en oeuvre du champ électrique dans un environnement microfluidique permet une exécution rapide et automatique du dosage électrotaxie. Cette approche promet de faciliter les écrans génétiques et chimiques à haut débit pour les facteursaffectant la fonction neuronale et la viabilité.

Une vidéo représentant des électrotaxie d&#39;un type sauvage jeune adulte nématodes et de sa position et sorties de la vitesse du logiciel de suivi du ver sont présentés dans la vidéo supplémentaire 1 et la Figure 3. Le logiciel d&#39;analyse de mouvement lui-même ne reconnaît pas le sens de la polarité du champ et le moment de l&#39;inversion de polarité, mais plutôt, cette information doit être obtenue à partir de la source vidéo. Cela pourrait être fait en utilisant...

Watch this Scientific Journal Video about Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion at JoVE.com

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans&#039; Locomotion

Procédé de micro-électro-fluidique semi-automatisé pour induire à la demande dans la locomotion<em&gt; Caenorhabditis elegans</em&gt; Est décrite. Cette méthode est basée sur le phénomène neurophysiologique de vers répondant à des champs électriques légers («électrotaxie&quot;) à l&#39;intérieur des canaux microfluidiques. Microfluidique électrotaxie sert d&#39;une technique rapide, sensible, faible coût, et évolutive pour dépister les facteurs affectant la santé neuronale.

Électrotaxie microfluidique à base d&#39;analyse quantitative à la demande de<em&gt; Caenorhabditis elegans</em&gt; &#39;Locomotion

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and ...

microfluidic-based-electrotaxis-for-on-demand-quantitative-analysis

Research

JoVE Journal

Bioengineering

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

9.7K vues.  McMaster University. Procédé de micro-électro-fluidique semi-automatisé pour induire à la demande dans la locomotion Caenorhabditis elegans Est décrite. Cette méthode est basée sur le phénomène neurophysiologique de vers répondant à des champs électriques légers («électrotaxie") à l'intérieur des canaux microfluidiques. Microfluidique électrotaxie sert d'une technique rapide, sensible, faible coût, et évolutive pour dépister les facteurs affec...

Vidéo: Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

Improved Visualization and Quantitative Analysis of Drug Effects Using Micropatterned Cells

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated micro-plates. Under these conditions, cells spread and divide in all directions resulting in an inherent variability in cell shape, morphology and behavior. The high cell-to-cell variance of the overall population impedes the success of HCA, especially for drug development. The ability of micropatterns to normalize the shape and internal polarity of every individual cell provides a tremendous opportunity for solving this critical bottleneck 1-2.
To facilitate access and use of the micropatterning technology, CYTOO has developed a range of ready to use micropatterns, available in coverslip and microwell formats. In this video article, we provide detailed protocols of all the procedures from cell seeding on CYTOOchip micropatterns, drug treatment, fixation and staining to automated acquisition, automated image processing and final data analysis. With this example, we illustrate how micropatterns can facilitate cell-based assays. Alterations of the cell cytoskeleton are difficult to quantify in cells cultured on homogenous substrates, but culturing cells on micropatterns results in a reproducible organization of the actin meshwork due to systematic positioning of the cell adhesion contacts in every cell. Such normalization of the intracellular architecture allows quantification of even small effects on the actin cytoskeleton as demonstrated in these set of protocols using blebbistatin, an inhibitor of the actin-myosin interaction.

Adhesive micropatterns that normalize cellular architecture can be used to increase sensitivity in the detection of drug effects, improve reproducibility and simplify automated image acquisition and analysis. Such technology will benefit drug/siRNA screening assays, performed on conventional cell culture supports and consequently suffering from excessive cell-to-cell variability.

Amélioration de la visualisation et l&#39;analyse quantitative des effets des drogues utilisant des cellules Micropatterned

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated ...

Recherche

Bio-ingénierie

A Microfluidic-based Hydrodynamic Trap for Single Particles

The ability to confine and manipulate single particles in free solution is a key enabling technology for fundamental and applied science. Methods for particle trapping based on optical, magnetic, electrokinetic, and acoustic techniques have led to major advancements in physics and biology ranging from the molecular to cellular level. In this article, we introduce a new microfluidic-based technique for particle trapping and manipulation based solely on hydrodynamic fluid flow. Using this method, we demonstrate trapping of micro- and nano-scale particles in aqueous solutions for long time scales. The hydrodynamic trap consists of an integrated microfluidic device with a cross-slot channel geometry where two opposing laminar streams converge, thereby generating a planar extensional flow with a fluid stagnation point (zero-velocity point). In this device, particles are confined at the trap center by active control of the flow field to maintain particle position at the fluid stagnation point. In this manner, particles are effectively trapped in free solution using a feedback control algorithm implemented with a custom-built LabVIEW code. The control algorithm consists of image acquisition for a particle in the microfluidic device, followed by particle tracking, determination of particle centroid position, and active adjustment of fluid flow by regulating the pressure applied to an on-chip pneumatic valve using a pressure regulator. In this way, the on-chip dynamic metering valve functions to regulate the relative flow rates in the outlet channels, thereby enabling fine-scale control of stagnation point position and particle trapping. The microfluidic-based hydrodynamic trap exhibits several advantages as a method for particle trapping. Hydrodynamic trapping is possible for any arbitrary particle without specific requirements on the physical or chemical properties of the trapped object. In addition, hydrodynamic trapping enables confinement of a "single" target object in concentrated or crowded particle suspensions, which is difficult using alternative force field-based trapping methods. The hydrodynamic trap is user-friendly, straightforward to implement and may be added to existing microfluidic devices to facilitate trapping and long-time analysis of particles. Overall, the hydrodynamic trap is a new platform for confinement, micromanipulation, and observation of particles without surface immobilization and eliminates the need for potentially perturbative optical, magnetic, and electric fields in the free-solution trapping of small particles.

In this article, we present a microfluidic-based method for particle confinement based on hydrodynamic flow. We demonstrate stable particle trapping at a fluid stagnation point using a feedback control mechanism, thereby enabling confinement and micromanipulation of arbitrary particles in an integrated microdevice.

Un piège hydrodynamique microfluidique à base de particules uniques

The ability to confine and manipulate single particles in free solution is a key enabling technology for fundamental and applied science.  Methods for ...

Quantitative Locomotion Study of Freely Swimming Micro-organisms Using Laser Diffraction

Soil and aquatic microscopic organisms live and behave in a complex three-dimensional environment. Most studies of microscopic organism behavior, in contrast, have been conducted using microscope-based approaches, which limit the movement and behavior to a narrow, nearly two-dimensional focal field.1 We present a novel analytical approach that provides real-time analysis of freely swimming C. elegans in a cuvette without dependence on microscope-based equipment. This approach consists of tracking the temporal periodicity of diffraction patterns generated by directing laser light through the cuvette. We measure oscillation frequencies for freely swimming nematodes.
Analysis of the far-field diffraction patterns reveals clues about the waveforms of the nematodes. Diffraction is the process of light bending around an object. In this case light is diffracted by the organisms. The light waves interfere and can form a diffraction pattern. A far-field, or Fraunhofer, diffraction pattern is formed if the screen-to-object distance is much larger than the diffracting object. In this case, the diffraction pattern can be calculated (modeled) using a Fourier transform.2
C. elegans are free-living soil-dwelling nematodes that navigate in three dimensions. They move both on a solid matrix like soil or agar in a sinusoidal locomotory pattern called crawling and in liquid in a different pattern called swimming.3 The roles played by sensory information provided by mechanosensory, chemosensory, and thermosensory cells that govern plastic changes in locomotory patterns and switches in patterns are only beginning to be elucidated.4 We describe an optical approach to measuring nematode locomotion in three dimensions that does not require a microscope and will enable us to begin to explore the complexities of nematode locomotion under different conditions.

Microscopic organisms like the free-swimming nematode C. elegans, live and behave in a complex three-dimensional environment. We report on a novel approach that provides analysis of C. elegans using diffraction patterns. This approach consists of tracking the temporal periodicity of diffraction patterns generated by directing laser light through a cuvette.

Étude quantitative de la Locomotion librement Piscine Micro-organismes utilisant la diffraction laser

Soil and aquatic microscopic organisms live and behave in a complex three-dimensional environment. Most studies of microscopic organism behavior, in ...

Quantitative FRET (F&#246;rster Resonance Energy Transfer) Analysis for SENP1 Protease Kinetics Determination

Reversible posttranslational modifications of proteins with ubiquitin or ubiquitin-like proteins (Ubls) are widely used to dynamically regulate protein activity and have diverse roles in many biological processes. For example, SUMO covalently modifies a large number or proteins with important roles in many cellular processes, including cell-cycle regulation, cell survival and death, DNA damage response, and stress response 1-5. SENP, as SUMO-specific protease, functions as an endopeptidase in the maturation of SUMO precursors or as an isopeptidase to remove SUMO from its target proteins and refresh the SUMOylation cycle 1,3,6,7.
The catalytic efficiency or specificity of an enzyme is best characterized by the ratio of the kinetic constants, kcat/KM. In several studies, the kinetic parameters of SUMO-SENP pairs have been determined by various methods, including polyacrylamide gel-based western-blot, radioactive-labeled substrate, fluorescent compound or protein labeled substrate 8-13. However, the polyacrylamide-gel-based techniques, which used the "native" proteins but are laborious and technically demanding, that do not readily lend themselves to detailed quantitative analysis. The obtained kcat/KM from studies using tetrapeptides or proteins with an ACC (7-amino-4-carbamoylmetylcoumarin) or AMC (7-amino-4-methylcoumarin) fluorophore were either up to two orders of magnitude lower than the natural substrates or cannot clearly differentiate the iso- and endopeptidase activities of SENPs.
Recently, FRET-based protease assays were used to study the deubiquitinating enzymes (DUBs) or SENPs with the FRET pair of cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) 9,10,14,15. The ratio of acceptor emission to donor emission was used as the quantitative parameter for FRET signal monitor for protease activity determination. However, this method ignored signal cross-contaminations at the acceptor and donor emission wavelengths by acceptor and donor self-fluorescence and thus was not accurate.
We developed a novel highly sensitive and quantitative FRET-based protease assay for determining the kinetic parameters of pre-SUMO1 maturation by SENP1. An engineered FRET pair CyPet and YPet with significantly improved FRET efficiency and fluorescence quantum yield, were used to generate the CyPet-(pre-SUMO1)-YPet substrate 16. We differentiated and quantified absolute fluorescence signals contributed by the donor and acceptor and FRET at the acceptor and emission wavelengths, respectively. The value of kcat/KM was obtained as (3.2 &plusmn; 0.55) x107 M-1s-1 of SENP1 toward pre-SUMO1, which is in agreement with general enzymatic kinetic parameters. Therefore, this methodology is valid and can be used as a general approach to characterize other proteases as well.

Quantitative FRET F&amp;#246;rster Resonance Energy Transfer Analysis for SENP1 Protease Kinetics Determination

A novel method involving quantitative analysis of FRET (F&ouml;rster Resonance Energy Transfer) signals is described for studying enzyme kinetics. KM and kcat were obtained for the hydrolysis of the catalytic domain of SENP1 (SUMO/Sentrin specific protease 1) to pre-SUMO1 (Small Ubiquitin-like MOdifier). The general principles of this quantitative-FRET-based protease kinetic study can be applied to other proteases.

Quantitative FRET (transfert de Förster Resonance Energy) Analyse de la protéase SENP1 Kinetics Détermination

Reversible posttranslational modifications of proteins with ubiquitin or ubiquitin-like proteins (Ubls) are widely used to dynamically regulate protein ...

Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse and Parkinson&#39;s disease. Acute activation of G&#945;i/o-linked receptors inhibits AC activity, whereas persistent activation of these receptors results in heterologous sensitization of AC and increased levels of intracellular cAMP. Previous studies have demonstrated that this enhancement of AC responsiveness is observed both in vitro and in vivo following the chronic activation of several types of G&#945;i/o-linked receptors including D2 dopamine and &#956; opioid receptors. Although heterologous sensitization of AC was first reported four decades ago, the mechanism(s) that underlie this phenomenon remain largely unknown. The lack of mechanistic data presumably reflects the complexity involved with this adaptive response, suggesting that nonbiased approaches could aid in identifying the molecular pathways involved in heterologous sensitization of AC. Previous studies have implicated kinase and Gb&#947; signaling as overlapping components that regulate the heterologous sensitization of AC. To identify unique and additional overlapping targets associated with sensitization of AC, the development and validation of a scalable cAMP sensitization assay is required for greater throughput. Previous approaches to study sensitization are generally cumbersome involving continuous cell culture maintenance as well as a complex methodology for measuring cAMP accumulation that involves multiple wash steps. Thus, the development of a robust cell-based assay that can be used for high throughput screening (HTS) in a 384&#160;well format would facilitate future studies. Using two D2 dopamine receptor cellular models (i.e.&#160;CHO-D2L and HEK-AC6/D2L), we have converted our 48-well sensitization assay (&#62;20 steps 4-5 days) to a five-step, single day assay in 384-well format. This new format is amenable to small molecule screening, and we demonstrate that this assay design can also be readily used for reverse transfection of siRNA in anticipation of targeted siRNA library screening.

Persistent activation of inhibitory G protein-coupled receptors results in sensitization of adenylyl cyclase signaling. To identify the essential molecular pathways, nonbiased approaches are necessary; however, this strategy requires the development of a scalable cell-based cAMP sensitization assay. Herein, we describe a sensitization assay for small molecule and siRNA screening.

Sensibilisation à la drogue induit des adénylylcyclase: Rationalisation de dosage et la miniaturisation des petites molécules et siARN préalable Applications

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse ...

A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration of pre-processing steps. Utilizing these devices towards the analysis of DNA hybridization events is important because it offers a technology for real time assessment of biomarkers at the point-of-care for various diseases. However, when the device footprint decreases the dominance of various physical phenomena increases. These phenomena influence the fabrication precision and operation reliability of the device. Therefore, there is a great need to accurately fabricate and operate these devices in a reproducible manner in order to improve the overall performance. Here, we describe the protocols and the methods used for the fabrication and the operation of a microfluidic-based electrochemical biochip for accurate analysis of DNA hybridization events. The biochip is composed of two parts: a microfluidic chip with three parallel micro-channels made of polydimethylsiloxane (PDMS), and a 3 x 3 arrayed electrochemical micro-chip. The DNA hybridization events are detected using electrochemical impedance spectroscopy (EIS) analysis. The EIS analysis enables monitoring variations of the properties of the electrochemical system that are dominant at these length scales. With the ability to monitor changes of both charge transfer and diffusional resistance with the biosensor, we demonstrate the selectivity to complementary ssDNA targets, a calculated detection limit of 3.8 nM, and a 13% cross-reactivity with other non-complementary ssDNA following 20 min&#160;of incubation. This methodology can improve the performance of miniaturized devices by elucidating on the behavior of diffusion at the micro-scale regime and by enabling the study of DNA hybridization events.

We present a microfluidic-based electrochemical biochip for DNA hybridization detection. Following ssDNA probe functionalization, the specificity, sensitivity, and detection limit are studied with complementary and non-complementary ssDNA targets. Results illustrate the influence of the DNA hybridization events on the electrochemical system, with a detection limit of 3.8 nM.

Une biopuce microfluidique électrochimique à base d&#39;analyse ADN hybridation Sans étiquette

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration ...

Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of physiological dcEF in guiding cell movement during embryo development, cell differentiation, and wound healing has been demonstrated in many studies. By applying microfluidic chips to an electrotaxis assay, the investigation process is shortened and experimental errors are minimized. In recent years, microfluidic devices made of polymeric substances (e.g., polymethylmethacrylate, PMMA, or acrylic) or polydimethylsiloxane (PDMS) have been widely used in studying the responses of cells to electrical stimulation. However, unlike the numerous steps required to fabricate a PDMS device, the simple and rapid construction of the acrylic micro&#64258;uidic chip makes it suitable for both device prototyping and production. Yet none of the reported devices facilitate the efficient study of the simultaneous chemical and dcEF effects on cells. In this report, we describe our design and fabrication of an acrylic-based multichannel dual-electric-field (MDF) chip to investigate the concurrent effect of chemical and electrical stimulation on lung cancer cells. The MDF chip provides eight combinations of electrical/chemical stimulations in a single test. The chip not only greatly shortens the required experimental time but also increases accuracy in electrotaxis studies.

Many microfluidic devices have been developed for use in the study of electrotaxis. Yet, none of these chips allows the efficient study of the simultaneous chemical and electric-field (EF) effects on cells. We developed a polymethylmethacrylate-based device that offers better-controlled coexisting EF and chemical stimulation for use in electrotaxis research.

Électrotaxie l&#39;étude des cellules du cancer du poumon à l&#39;aide d&#39;un double-Multichannel champ électrique microfluidique Chip

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of ...

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as multiplex assays, enzyme evolution, and molecular biology enhanced cell sorting require the detection of two or more colors of fluorescence. Standard multicolor detection systems that couple free space lasers to epifluorescence microscopes are bulky, expensive, and difficult to maintain. In this paper, we describe a scheme to perform multicolor detection by exciting discrete regions of a microfluidic channel with lasers coupled to optical fibers. Emitted light is collected by an optical fiber coupled to a single photodetector. Because the excitation occurs at different spatial locations, the identity of emitted light can be encoded as a temporal shift, eliminating the need for more complicated light filtering schemes. The system has been used to detect droplet populations containing four unique combinations of dyes and to detect sub-nanomolar concentrations of fluorescein.

Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

Multicolor détection Fluorescence pour Droplet microfluidique utilisant des fibres optiques

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as ...

An Efficient and Reproducible Protocol for Distraction Osteogenesis in a Rat Model Leading to a Functional Regenerated Femur

This protocol describes the use of a newly developed external fixator for distraction osteogenesis in a rat femoral model. Distraction osteogenesis (DO) is a surgical technique leading to bone regeneration after an osteotomy. The osteotomized extremities are moved away from each other by gradual distraction to reach the desired elongation. This procedure is widely used in humans for lower and upper limb lengthening, treatment after a bone nonunion, or the regeneration of a bone defect following surgery for bone tumor excision, as well as in maxillofacial reconstruction. Only a few studies clearly demonstrate the efficiency of their protocol in obtaining a functional regenerated bone, i.e., bone that will support physiological weight-bearing without fracture after removal of the external fixator. Moreover, protocols for DO vary and reproducibility is limited by lack of information, making comparison between studies difficult. The aim of this study was to develop a reproducible protocol comprising an appropriate external fixator design for rat limb lengthening, with a detailed surgical technique that permits physiological weight-bearing by the animal after removal of the external fixator.

This study describes a reproducible and detailed protocol using a newly developed external fixator for distraction osteogenesis (DO) in a femoral rat model which permits physiological weight-bearing by the animal after removal of the external fixator.

Un protocole efficace et reproductible pour Distraction ostéogénèse dans un modèle de Rat conduisant à un fémur régénéré fonctionnel

This protocol describes the use of a newly developed external fixator for distraction osteogenesis in a rat femoral model. Distraction osteogenesis (DO) ...

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved useful as a convenient live imaging platform providing capabilities for precise control of experimental conditions in real time. In this article, we demonstrate live imaging of individual worms employing WormSpa, a previously-published custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes. To illustrate the capability, we performed proof-of-principle experiments in which worms were infected in the device with pathogenic bacteria, and the dynamics of expression of immune response genes and egg laying were monitored continuously in individual animals. The simple design and operation of this device make it suitable for users with no previous experience with microfluidic-based experiments. We propose that this approach will be useful for many researchers interested in longitudinal observations of biological processes under well-defined conditions.

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In this article, we demonstrate live imaging of individual worms employing a custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes.

Une plate-forme microfluidique d’imagerie longitudinales chez Caenorhabditis elegans

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved ...