Ce travail est soutenu par la National Science Fibr Foundation (NSF EF-0623664) et IDBR (NSF DBI-0754570). Ce travail a été partiellement financé par des fonds de la National Science Foundation Grant Fibr 0623664 administré par le Centre pour la biophotonique, une science NSF et de la technologie Center, géré par l&#39;Université de Californie, Davis, en vertu de l&#39;accord de coopération PHY 0120999. La recherche de Lisa Lapidus, Ph.D. est soutenu en partie par une bourse de carrière à l&#39;interface scientifique du Fonds Burroughs Welcome.

<ol>
	<li>Eaton, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+kinetics+and+mechanisms+in+protein+folding.">Fast kinetics and mechanisms in protein folding.</a> Annual Review of Biophysics and Biomolecular Structure. 29, 327-359 (2000).</li><li>Hertzog, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Femtomole+mixer+for+microsecond+kinetic+studies+of+protein+folding.">Femtomole mixer for microsecond kinetic studies of protein folding.</a> Analytical Chemistry. 76, 7169-7178 (2004).</li><li>Hertzog, D. E., Ivorra, B., Mohammadi, B., Bakajin, O., Santiago, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+a+microfluidic+mixer+for+studying+protein+folding+kinetics.">Optimization of a microfluidic mixer for studying protein folding kinetics.</a> Analytical Chemistry. 78, 4299-4306 (2006).</li><li>Yao, S., Bakajin, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improvements+in+Mixing+Time+and+Mixing+Uniformity+in+Devices+Designed+for+Studies+of+Protein+Folding+Kinetics.">Improvements in Mixing Time and Mixing Uniformity in Devices Designed for Studies of Protein Folding Kinetics.</a> Analytical Chemistry. 79, 5753-5759 (2007).</li><li>Lapidus, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+Hydrophobic+Collapse+and+Early+Folding+Steps+Observed+in+a+Microfluidic+Mixer.">Protein Hydrophobic Collapse and Early Folding Steps Observed in a Microfluidic Mixer.</a> Biophysical Journal. 93, 218-224 (2007).</li><li>Bilsel, O., Kayatekin, C., Wallace, L. A., Matthews, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microchannel+solution+mixer+for+studying+microsecond+protein+folding+reactions.">A microchannel solution mixer for studying microsecond protein folding reactions.</a> Review of Scientific Instruments. 76, (2005).</li><li>Chan, C. -. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Submillisecond+protein+folding+kinetics+studied+by+ultrarapid+mixing.">Submillisecond protein folding kinetics studied by ultrarapid mixing.</a> PNAS. 94, 1779-1784 (1997).</li><li>Shastry, M. C. R., Luck, S. D., Roder, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+continuous-flow+capillary+mixing+method+to+monitor+reactions+on+the+microsecond+time+scale.">A continuous-flow capillary mixing method to monitor reactions on the microsecond time scale.</a> Biophysical Journal. 74, 2714-2721 (1998).</li><li>Pollack, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compactness+of+the+denatured+state+of+a+fast-folding+protein+measured+by+submillisecond+small-angle+x-ray+scattering.">Compactness of the denatured state of a fast-folding protein measured by submillisecond small-angle x-ray scattering.</a> Proceedings of the National Academy of Sciences of the United States of America. 96, 10115-10117 (1999).</li><li>Takahashi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Folding+of+cytochrome+c+initiated+by+submillisecond+mixing.">Folding of cytochrome c initiated by submillisecond mixing.</a> Nature Structural Molecular Biology. 4, 44-50 (1997).</li><li>Knight, J. B., Vishwanath, A., Brody, J. P., Austin, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrodynamic+focusing+on+a+silicon+chip:+Mixing+nanoliters+in+microseconds.">Hydrodynamic focusing on a silicon chip: Mixing nanoliters in microseconds.</a> Physical Review Letters. 80, 3863-3866 (1998).</li><li>DeCamp, S. J., Naganathan, A. N., Waldauer, S. A., Bakajin, O., Lapidus, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+Observation+of+Downhill+Folding+of+lambda-Repressor+in+a+Microfluidic+Mixer.">Direct Observation of Downhill Folding of lambda-Repressor in a Microfluidic Mixer.</a> Biophysical Journal. 97, 1772-1777 (2009).</li><li>Waldauer, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ruggedness+in+the+folding+landscape+of+protein+L.">Ruggedness in the folding landscape of protein L.</a> Hfsp Journal. 2, 388-395 (2008).</li><li>Shastry, M. C. R., Roder, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+barrier-limited+protein+folding+kinetics+on+the+microsecond+time+scale.">Evidence for barrier-limited protein folding kinetics on the microsecond time scale.</a> Nature Structural Biology. 5, 385-392 (1998).</li><li>Uzawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collapse+and+search+dynamics+of+apomyoglobin+folding+revealed+by+submillisecond+observations+of+alpha-helical+content+and+compactness.">Collapse and search dynamics of apomyoglobin folding revealed by submillisecond observations of alpha-helical content and compactness.</a> Proceedings of the National Academy of Sciences of the United States of America. 101, 1171-1176 (2004).</li><li>Zhu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+of+Multiple+Folding+Pathways+for+the+Villin+Headpiece+Subdomain.">Evidence of Multiple Folding Pathways for the Villin Headpiece Subdomain.</a> The Journal of Physical Chemistry B. 115, 12632-12637 (2011).</li></ol>

Nous n&#39;avons rien à communiquer.

Mélange rapide a été un objectif de développement pour le champ du repliement des protéines pour de nombreuses années, car il est reconnu depuis longtemps que les processus moléculaires de biomolécules se produire sur des échelles de temps allant de picosecondes à quelques secondes. Classiques stopped-flow mélangeurs ont des temps morts de 1-5 ms, ce qui est principalement limitée par les turbulences. Turbulent mélangeurs à flux continu avec un temps de mélange de 30-300 ms ont été développés au cours des 15 dernières années par un petit nombre de groupes mais exigent généralement des débits élevés pour atteindre ces temps de mélange et donc d&#39;utiliser un grand nombre de l&#39;échantillon 6-8. Ce protocole décrit l&#39;utilisation de puces microfluidiques mélange pour obtenir une dilution rapide dans le régime d&#39;écoulement laminaire. Il ya de multiples avantages à travailler au sein de ce régime, mais un primaire est la possibilité de simuler l&#39;ensemble du processus de mélange avec une grande précision qui permet de modifier la réponse de mélange. T-mélangeurs développés par d&#39;autres groupes avec de simples geometries ont démontré un temps de mélange de 100-200 ms qui ont été principalement limités par la taille des canaux 9-10. Mettant à l&#39;échelle de la taille des canaux à 10 um chevalier de réduire le temps de mélange d&#39;environ 10 ms 11. Yao et Bakajin a montré que de petits changements dans la géométrie pourrait conduire à des améliorations significatives dans le temps de mélange 4. Toutefois, ce travail a montré que l&#39;accélération du mélange peut aussi conduire à un ralentissement des phases ainsi. La conception utilisée dans ce travail 12-13 est un compromis entre les deux meilleures designs de référence de 4 à minimiser le plus lent de décroissance exponentielle de la concentration en dénaturant et aussi pour rendre la fonction plus reproductible dans gravure DRIE. Il a eu une certaine controverse dans le domaine de la ultrarapide mélange sur la définition du temps de mélange. Il est important de reconnaître la différence entre &quot;temps de mélange» et «temps mort». Le temps mort est définie dans un mélangeur turbulente que le temps pendant lequel une MESUREt ne peut être faite et peut en fait être beaucoup plus long que le temps réel de mélange. Il est généralement déterminée en faisant plusieurs mesures d&#39;une réaction pseudo-premier ordre bimoléculaire (tels que trempe de fluorescence du tryptophane par N bromosuccinate) à différentes concentrations. Des caries observées exponentielles sont montés à converger à une valeur unique supposé être t = 0 s et le temps entre ce point et le premier point de mesure est le temps mort. Pour les mélangeurs à flux laminaire, des mesures peuvent être prises durant le processus de mélange de sorte qu&#39;il n&#39;ya pas de temps mort, seulement un temps de mélange. Le temps de mélange est tout simplement le temps de combiner deux solutions jusqu&#39;à ce que l&#39;uniformité suffisant est atteint. Nous avons défini précédemment le temps de mélange d&#39;un temps de 90/10, le temps de la concentration de dénaturant pour diminuer de 90% à 10% de la valeur sans mélange. Toutefois, la forme de la courbe de mélange n&#39;est pas parfaitement symétrique avec la queue de la désintégration ayant un petit composant exponentielle. En outre, le repliement des protéines a unefortement non-linéaire de dépendance sur la concentration de dénaturant de telle sorte que pliage peut souvent commencer même si le dénaturant est seulement réduit par deux fois. C&#39;est pourquoi nous avons plus récemment défini le temps de mélange que le temps de réduire la concentration de dénaturant de 80%. Certes, d&#39;autres définitions peuvent être utilisées en fonction des exigences de l&#39;expérience. L&#39;utilisation de cette table de mixage pour étudier le repliement des protéines a toujours révélé des résultats surprenants. Pliage de cytochrome c et apomyoglobine ont longtemps été étudiée pour avoir de multiples étapes de pliage et les premiers résultats avec des mélangeurs à flux continu ont montré l&#39;effondrement de se produire sur l&#39;échelle de temps ~ 100 ms 10,14-15. Nos mesures ont montré avec cette table de mixage qu&#39;il y ait au moins 2 étapes sur cette échelle de temps, un très rapide, probablement non spécifique, l&#39;effondrement (tel que mesuré par fluorescence Trp décalage spectral) dans le temps de mélange du mélangeur suivie par une phase plus lente (comme mesurée par la trempe du total des émissions de Trp) qui est probablement le fla formation de la structure native IRST 5. Nous avons également observé un processus 50 ms dans le domaine B1 de la protéine L, renversant l&#39;interprétation conventionnelle que cette protéine est un dossier 2-Etat 13. Un avantage de cette table de mixage rapide, c&#39;est qu&#39;il permet de sonder le repliement de protéines les plus rapides pliantes qui ont habituellement seulement été mesurable par ordre de la nanoseconde T-saut instruments. T-saut nécessite généralement l&#39;observation de pliage / dépliage détente à proximité de la température de fusion de la protéine et donc ne suit jamais le pliage de la population dans son ensemble. Avec cette table de mixage, nous avons examiné le pliage de la γ-répresseur (λ 6-86) à la fois les émissions totales Trp et décalage spectral et ont trouvé des preuves que la protéine n&#39;a pas de barrière à plier sous de fortes conditions de pliage qui n&#39;étaient pas accessibles à T précédentes -sauter des mesures 12. Enfin, nous avons mesuré le repliement de la coiffe villine HP-35, l&#39;un des til le plus rapide des dossiers encore mesuré et a constaté que le taux de pliage mesuré après le mélange est de ~ 5 fois plus lent que mesurée par T-bond dans les mêmes conditions 16. Ceci suggère que la cinétique de pliage dépend des conditions de départ, le renversement d&#39;une hypothèse majeure dans le domaine.

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td> AZ P4110 Photoresist </td><td> AZ Electronic Materials </td><td></td><td></td></tr><tr><td> AZ 400 K Developer </td><td> AZ Electronic Materials </td><td></td><td></td></tr><tr><td> PRS Baker, 2000 décapant résine photosensible </td><td> Performance Materials Avantor </td><td></td><td></td></tr><tr><td> AquaBond </td><td> Technologies AquaBond </td><td> AquaBond 55 </td><td></td></tr><tr><td> 500 plaquette de recouvrement pm </td><td> CAPTEUR services de préparation </td><td></td><td> Ø: 100 mm ± 0,5 mm Épaisseur: 0,5 mm ± 0,025 mm Tolérances standard (sauf indication contraire) Finition de la surface: SI: 4-8a; SII: 8-12A Appartements: Une primaire SEMI-STD; un scribe Propre et poli, des deux côtés </td></tr><tr><td> 170 plaquette de recouvrement pm </td><td> CAPTEUR services de préparation </td><td> 7980 Gaufrettes 2G </td><td> Ø: 100 mm ± 0,5 mm Epaisseur: 0,17 mm ± 0,025 mm Tolérances standard (sauf indication contraire) Finition de la surface: SI: 4-8a; SII: 8-12A Appartements: Une primaire SEMI-STD; un scribe Propre et poli, des deux côtés </td></tr><tr><td> Profonde graveur ionique réactive pour les oxydes </td><td> ULVAC </td><td> ULVAC NL-6000 </td><td></td></tr><tr><td> Laser à ions argon </td><td> Cambridge Laboratories laser </td><td> Lexel 95-SHG </td><td> X = 258 nm </td></tr><tr><td> Laser à ions argon </td><td> Melles Griot </td><td></td><td> X = 488 nm </td></tr><tr><td> Microscope </td><td> Olympe </td><td> IX-51</td><td></td></tr><tr><td> Objectif de microscope </td><td> Thor Labs </td><td> OFR LMU-40X-UVB 0,5 NA </td><td> X = 258 nm </td></tr><tr><td> Objectif de microscope </td><td> Olympe </td><td> UPLSAPO 60XW 1,2 NA </td><td> X = 488 nm </td></tr><tr><td> Nanopositioner </td><td> Mad Labs Ville </td><td> Nano-LP100 </td><td></td></tr><tr><td> Stade Traduction motorisé </td><td> Semprex Corp </td><td> KL-Série 12-6436 </td><td></td></tr><tr><td> GaAsP Photon contre </td><td> Hamamatsu </td><td> H7421-40 </td><td></td></tr><tr><td> Monochromateur </td><td> Horiba Instruments Inc </td><td> MicroHR </td><td></td></tr><tr><td> La caméra CCD </td><td> Andor Technology </td><td> idus 420A-BU </td><td></td></tr><tr><td> Chlorhydrate de guanidine (GuHCl) </td><td> Sigma-Aldrich </td><td&gt; G4505 </td><td></td></tr></tbody></table>

microfluidic mixers for studying protein folding

Le processus par lequel une protéine se replie dans sa conformation native est très pertinente pour la biologie et la santé humaine mais encore mal compris. Une raison à cela est que le pliage a lieu sur une large gamme d&#39;échelles temporelles, de la nanoseconde à quelques secondes ou plus, en fonction de la protéine 1. Classiques stopped-flow mélangeurs ont permis de mesurer la cinétique de pliage à partir d&#39;environ 1 ms. Nous avons récemment développé un mélangeur microfluidique qui dilue dénaturant ~ 100 fois dans ~ 8 ms 2. La différence d&#39;un mélangeur de flux arrêté, ce mélangeur fonctionne dans le régime d&#39;écoulement laminaire dans lequel la turbulence ne se produit pas. L&#39;absence de turbulence permet la simulation numérique précise de tous les flux au sein de la table de mixage avec un excellent accord pour expérimenter 3-4. À flux laminaire est atteint pour des nombres de Reynolds Re ≤ 100. Pour les solutions aqueuses, ce qui nécessite des géométries échelle du micron. Nous utilisons un substrat dur, tel que le silicium ou fusionnés siliconeca, pour faire um canaux 5-10 de large et 10 um de profondeur (voir figure 1). Les plus petites dimensions, à l&#39;entrée de la zone de mélange, sont de l&#39;ordre de 1 pm à la taille. La puce est scellé avec une lamelle de verre mince ou en silice fondue pour l&#39;accès optique. Typiques au total des débits linéaires sont ~ 1 m / s, Re cédant ~ 10, mais la consommation de protéines est seulement ~ 0,5 nL / s ou 1,8 pi / h. La concentration en protéines dépend de la méthode de détection: Pour fluorescence du tryptophane la concentration typique est de 100 um (pour 1 Trp / protéine) et pour FRET de la concentration typique est de ~ 100 nM. Le processus de pliage est initiée par dilution rapide de dénaturant de 6 M à 0,06 M de chlorhydrate de guanidine. La protéine en dénaturant haute coule un canal central et est remplie de chaque côté à la zone de mélange par le tampon, sans dénaturant le déplacement ~ 100 fois plus rapide (voir Figure 2). Cette géométrie provoque la constriction rapide de l&#39;écoulement des protéines dans une étroitejet ~ 100 nm de large. Diffusion des molécules légères dénaturant est très rapide, tandis que la diffusion des molécules de protéines lourds est beaucoup plus lente, la diffusion inférieure à 1 um à 1 ms. La différence de constante de diffusion de l&#39;agent de dénaturation des protéines et des résultats de dilution rapide de l&#39;agent de dénaturation des protéines à partir du flux, la réduction de la concentration efficace de l&#39;agent de dénaturation autour de la protéine. Le jet protéine s&#39;écoule à une vitesse constante dans le canal d&#39;observation et de la fluorescence de la protéine pendant le pliage peut être observé à l&#39;aide d&#39;un microscope à balayage confocal 5.

Watch this Scientific Journal Video about Microfluidic Mixers for Studying Protein Folding at JoVE.com

Microfluidic Mixers for Studying Protein Folding

Dans ce travail nous expliquer la fabrication et l&#39;utilisation d&#39;un mélangeur microfluidique capable de mélanger deux solutions dans ~ 8 ms. Nous démontrons également l&#39;utilisation de ces mélangeurs avec détection spectroscopique utilisant la fluorescence UV et de fluorescence de transfert d&#39;énergie par résonance (FRET).

Mélangeurs microfluidiques pour étudier repliement des protéines

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 ...

microfluidic-mixers-for-studying-protein-folding

Research

JoVE Journal

Bioengineering

15.0K vues.  Michigan State University. Dans ce travail nous expliquer la fabrication et l'utilisation d'un mélangeur microfluidique capable de mélanger deux solutions dans ~ 8 ms. Nous démontrons également l'utilisation de ces mélangeurs avec détection spectroscopique utilisant la fluorescence UV et de fluorescence de transfert d'énergie par résonance (FRET).

Vidéo: Microfluidic Mixers for Studying Protein Folding

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 ...

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Bio-ingénierie

Thermodynamics of Membrane Protein Folding Measured by Fluorescence Spectroscopy

Membrane protein folding is an emerging topic with both fundamental and health-related significance. The abundance of membrane proteins in cells underlies the need for comprehensive study of the folding of this ubiquitous family of proteins. Additionally, advances in our ability to characterize diseases associated with misfolded proteins have motivated significant experimental and theoretical efforts in the field of protein folding. Rapid progress in this important field is unfortunately hindered by the inherent challenges associated with membrane proteins and the complexity of the folding mechanism. Here, we outline an experimental procedure for measuring the thermodynamic property of the Gibbs free energy of unfolding in the absence of denaturant, &Delta;G&deg;H2O, for a representative integral membrane protein from E. coli. This protocol focuses on the application of fluorescence spectroscopy to determine equilibrium populations of folded and unfolded states as a function of denaturant concentration. Experimental considerations for the preparation of synthetic lipid vesicles as well as key steps in the data analysis procedure are highlighted. This technique is versatile and may be pursued with different types of denaturant, including temperature and pH, as well as in various folding environments of lipids and micelles. The current protocol is one that can be generalized to any membrane or soluble protein that meets the set of criteria discussed below.

This video article details the experimental procedure for obtaining the Gibbs free energy of membrane protein folding by tryptophan fluorescence.

Thermodynamique du repliement des protéines membranaires mesuré par fluorescence Spectroscopie

Membrane protein folding is an emerging topic with both fundamental and health-related significance.  The abundance of membrane proteins in cells ...

Fluorescence detection methods for microfluidic droplet platforms

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

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

Méthodes de détection par fluorescence pour les plateformes microfluidiques gouttelettes

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

High-throughput Protein Expression Generator Using a Microfluidic Platform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays1. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein 4, protein-RNA5 or protein-DNA6 interactions.
The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins7, DNA8, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

À haut débit Générateur expression des protéines utilisant une plate-forme microfluidique

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

A Microfluidic Device for Studying Multiple Distinct Strains

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a comparative manner. Multiwell plates are routinely used to compare many different strains or cell lines, but allow limited control over the environment dynamics. Microfluidic devices, on the other hand, allow exquisite dynamic control over the surrounding conditions, but it is challenging to image and distinguish more than a few strains in them. Here we describe a method to easily and rapidly manufacture a microfluidic device capable of applying dynamically changing conditions to multiple distinct yeast strains in one channel. The device is designed and manufactured by simple means without the need for soft lithography. It is composed of a Y-shaped flow channel attached to a second layer harboring microwells. The strains are placed in separate microwells, and imaged under the exact same dynamic conditions. We demonstrate the use of the device for measuring protein localization responses to pulses of nutrient changes in different yeast strains.

We present a simple method to produce microfluidic devices capable of applying similar dynamic conditions to multiple distinct strains, without the need for a clean room or soft lithography.

Un dispositif microfluidique pour l&#39;étude de plusieurs souches distinctes

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

A Microfluidic Chip for ICPMS Sample Introduction

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

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

Une puce microfluidique pour ICPMS Présentation échantillon

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

Studying Protein Function and the Role of Altered Protein Expression by Antibody Interference and Three-dimensional Reconstructions

A strict management of protein expression is not only essential to every organism alive, but also an important strategy to investigate protein functions in cellular models. Therefore, recent research invented different tools to target protein expression in mammalian cell lines or even animal models, including RNA and antibody interference. While the first strategy has gathered much attention during the past two decades, peptides &#160;mediating a translocation of antibody cargos across cellular membranes and into cells, obtained much less interest. In this publication, we provide a detailed protocol how to utilize a peptide carrier named Chariot in human embryonic kidney cells as well as in primary hippocampal neurons to perform antibody interference experiments and further illustrate the application of three-dimensional reconstructions in analyzing protein function. Our findings suggest that Chariot is, probably due to its nuclear localization signal, particularly well-suited to target proteins residing in the soma and the nucleus. Remarkably, when applying Chariot to primary hippocampal cultures, the reagent turned out to be surprisingly well accepted by dissociated neurons.

Controlling protein expression is not only essential to every organism alive, but also an important strategy to investigate protein functions in cellular models. The protocol presented shows the application of antibody interference in mammalian cells including primary hippocampal neurons and demonstrates the use of three-dimensional reconstructions in studying protein function.

Etudier la fonction des protéines et le rôle de Altered Expression des protéines par les interférences des anticorps et des reconstructions tridimensionnelles

A strict management of protein expression is not only essential to every organism alive, but also an important strategy to investigate protein functions ...

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.

Entièrement automatisé centrifuge microfluidique Dispositif pour Ultrasensitive Protein Détection de sang total

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

Designing Microfluidic Devices for Studying Cellular Responses Under Single or Coexisting Chemical/Electrical/Shear Stress Stimuli

Microfluidic devices are capable of creating a precise and controllable cellular micro-environment of pH, temperature, salt concentration, and other physical or chemical stimuli. They have been commonly used for in vitro cell studies by providing in vivo like surroundings. Especially, how cells response to chemical gradients, electrical fields, and shear stresses has drawn many interests since these phenomena are important in understanding cellular properties and functions. These microfluidic chips can be made of glass substrates, silicon wafers, polydimethylsiloxane (PDMS) polymers, polymethylmethacrylate (PMMA) substrates, or polyethyleneterephthalate (PET) substrates. Out of these materials, PMMA substrates are cheap and can be easily processed using laser ablation and writing. Although a few microfluidic devices have been designed and fabricated for generating multiple, coexisting chemical and electrical stimuli, none of them was considered efficient enough in reducing experimental repeats, particular for screening purposes. In this report, we describe our design and fabrication of two PMMA-based microfluidic chips for investigating cellular responses, in the production of reactive oxygen species and the migration, under single or coexisting chemical/electrical/shear stress stimuli. The first chip generates five relative concentrations of 0, 1/8, 1/2, 7/8, and 1 in the culture regions, together with a shear stress gradient produced inside each of these areas. The second chip generates the same relative concentrations, but with five different electric field strengths created within each culture area. These devices not only provide cells with a precise, controllable micro-environment but also greatly increase the experimental throughput.

Micro-fabricated devices integrated with fluidic components provide an in vitro platform for cell studies mimicking the in vivo micro-environment. We developed polymethylmethacrylate-based microfluidic chips for studying cellular responses under single or coexisting chemical/electrical/shear stress stimuli.

Conception de dispositifs microfluidiques pour étudier les réponses cellulaires Sous simple ou Coexistence chimique / électrique / Shear Stress Stimuli

Microfluidic devices are capable of creating a precise and controllable cellular micro-environment of pH, temperature, salt concentration, and other ...

Multi-step Variable Height Photolithography for Valved Multilayer Microfluidic Devices

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to entry for non-specialists who would benefit greatly from the ability to develop their own microfluidic devices to address research questions. Particularly lacking has been the open dissemination of protocols related to photolithography, a key step in the development of a replica mold for the manufacture of polydimethylsiloxane (PDMS) devices. While the fabrication of single height silicon masters has been explored extensively in literature, fabrication steps for more complicated photolithography features necessary for many interesting device functionalities (such as feature rounding to make valve structures, multi-height single-mold patterning, or high aspect ratio definition) are often not explicitly outlined. 
Here, we provide a complete protocol for making multilayer microfluidic devices with valves and complex multi-height geometries, tunable for any application. These fabrication procedures are presented in the context of a microfluidic hydrogel bead synthesizer and demonstrate the production of droplets containing polyethylene glycol (PEG diacrylate) and a photoinitiator that can be polymerized into solid beads. This protocol and accompanying discussion provide a foundation of design principles and fabrication methods that enables development of a wide variety of microfluidic devices. The details included here should allow non-specialists to design and fabricate novel devices, thereby bringing a host of recently developed technologies to their most exciting applications in biological laboratories.

Multilayer microfluidic devices often involve the fabrication of master molds with complex geometries for functionality. This article presents a complete protocol for multi-step photolithography with valves and variable height features tunable to any application. As a demonstration, we fabricate a microfluidic droplet generator capable of producing hydrogel beads.

Multi-étape Hauteur variable photolithographie pour dispositifs microfluidiques à valve Multilayer

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to ...