Diese Arbeit wird von der National Science Foundation FIBR (NSF EF-0623664) und IDBR (NSF DBI-0754570) unterstützt. Diese Arbeit wurde teilweise aus Mitteln der National Science Foundation Grants FIBR 0623664 vom Zentrum für Biophotonik, einem NSF Science and Technology Center verwaltet, von der University of California, Davis, unter einem Kooperationsabkommen PHY 0120999 verwaltet unterstützt. Die Forschung von Lisa Lapidus, Ph.D. wird zum Teil durch ein Career Award in der wissenschaftlichen Schnittstelle von der Burroughs Welcome Fonds unterstützt werden.

<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>

Wir haben nichts zu offenbaren.

Eine schnelle Durchmischung war ein Entwicklungsziel für das Gebiet der Proteinfaltung seit vielen Jahren, weil es seit langem anerkannt, dass molekulare Prozesse von Biomolekülen über Zeitskalen von Pikosekunden bis Sekunden auftreten. Konventionelle Stopped-Flow-Mischer haben Totzeiten von 1-5 ms, die vor allem durch Turbulenz ist begrenzt. Turbulente kontinuierlichen Mischern mit Mischzeiten von 30-300 us haben in den vergangenen 15 Jahren von einer kleinen Anzahl von Gruppen entwickelt, sondern erfordern in der Regel hohe Flussraten, um diese zu erreichen Mischzeiten und verwenden daher eine Menge von Probe 6-8. Dieses Protokoll beschreibt die Verwendung von Mikrofluidsystemen Mischen Chips schnelle Verdünnung in der laminaren Strömung zu erreichen. Es gibt mehrere Vorteile, die innerhalb dieses Systems, sondern eine primäre ist die Fähigkeit, die gesamte Mischverfahren mit hoher Genauigkeit, die man, um die gemischte Antwort zu modifizieren simulieren. T-Mischer von anderen Gruppen mit einfachen geom entwickeltetries haben Mischzeiten von 100-200 us, die hauptsächlich von der Größe der Kanäle 9-10 wurden beschränkt gezeigt. Durch Skalieren der Größe der Kanäle bis 10 um Ritter verringert die Mischzeit auf ~ 10 us 11. Yao und Bakajin zeigte, dass kleine Veränderungen in der Geometrie können zu erheblichen Verbesserungen in der Mischzeit 4 führen. Zeigten jedoch, dass Arbeit, die Beschleunigung der Vermischung kann auch zu einer langsameren Phasen als auch führen. Das Design in dieser Arbeit verwendet 12-13 ist ein Kompromiss zwischen den beiden besten Entwürfe in Bezug 4, um die langsameren exponentiellen Abfall in Vergällungsmittel Konzentration zu minimieren und auch um die Funktion besser reproduzierbar in DRIE Ätzen. Es gab einige Kontroversen auf dem Gebiet der ultraschnellen Vermischung über die Definition der Mischzeit. Es ist wichtig, den Unterschied zwischen &quot;Mischzeit&quot; und &quot;tote Zeit&quot; zu erkennen. Die Totzeit wird in einem turbulenten Mischer als die Zeit, während der eine Messung! Definiertt nicht gemacht werden und können in der Tat deutlich länger als die tatsächliche Mischzeit. Es wird üblicherweise erreicht, indem mehrere Messungen eines pseudo-erster Ordnung bimolekularen Reaktion (wie Abschrecken der Tryptophan-Fluoreszenz von N bromosuccinate) bei verschiedenen Konzentrationen bestimmt. Die beobachteten exponentiellen Abfall angebaut, um an einem einzigen Wert angenommen, dass t = 0 s sein konvergieren und die Zeit zwischen diesem Zeitpunkt und dem ersten Messpunkt ist die Totzeit. Für laminare Strömung Mischer, können die Messungen während des Mischprozesses hergestellt sein, so gibt es keine Totzeit nur eine Mischzeit. Die Mischzeit ist einfach die Zeit, um zwei Lösungen kombinieren, bis eine ausreichende Einheitlichkeit erreicht wird. Wir haben zuvor die Mischzeit auf eine 90/10, die Zeit für die Konzentration des Denaturierungsmittels, um von 90% bis 10% des ungemischten Wert verkleinert werden definiert. Jedoch ist die Form des Mischkurve nicht perfekt symmetrisch mit dem Schwanz des Zerfalls mit einer kleinen exponentielle Komponente. Ferner weist ein Proteinfaltungstark nicht-lineare Abhängigkeit von Denaturierungsmittel Konzentration, so dass Falten oft beginnen kann, selbst wenn der Vergällungsstoff nur durch zwei-fache reduziert. Deshalb haben wir in jüngster Zeit die Mischzeit als die Zeit, um die Vergällungsmittel Konzentration um 80% definiert. Sicherlich andere Definitionen können in Abhängigkeit von den Anforderungen des Experiments werden. Die Verwendung dieses Mischpult auf die Proteinfaltung zu studieren hat immer wieder überraschende Ergebnisse zeigten. Die Faltung von Cytochrom c und Apomyoglobin haben lange studiert worden, um mehrere Klapptritt und frühen Ergebnisse mit kontinuierlichen Mischern haben gezeigt, Zusammenbruch auf der ~ 100 us Zeitskala 10,14-15 auftreten. Unsere Messungen mit diesem Mixer zeigte dort auf mindestens 2 Schritte auf dieser Zeitskala zu sein, ein sehr schneller, wahrscheinlich nicht-spezifischen, Kollaps (wie von Trp-Fluoreszenz spektrale Verschiebung gemessen) innerhalb der Mischzeit des Mischers eine längere Phase folgte (wie gemessen durch das Abschrecken von insgesamt Trp Emission) das ist wahrscheinlich das first Bildung von nativen Struktur 5. Wir haben auch ein 50 us-Prozess in der B1-Domäne von Protein L beobachtet, Umsturz der herkömmlichen Interpretation, dass dieses Protein eine 2-Staaten-Ordner 13 ist. Ein Vorteil dieser schnellen Mischer ist, dass es die Faltung von Proteinen zu den am schnellsten Faltung, die normalerweise nur messbar gewesen durch Nanosekunden-T-Sprung Instrumente zu sondieren. T-Sprung erfordert in der Regel Beobachtung der Faltung / Entfaltung Entspannung in der Nähe der Schmelztemperatur des Proteins und somit nie folgt die Faltung der gesamten Bevölkerung. Mit diesem Mischpult haben wir bei der Faltung von γ-Repressor (λ 6-86) sowohl mit Trp Gesamtemission und spektrale Verschiebung angesehen und fand Beweise, dass das Protein kein Hindernis für die Faltung unter Bedingungen, die starke Faltung nicht zugänglich waren zu früheren T hat -Messungen 12 springen. Schließlich haben wir den Falten des Villin Kopfstück HP-35 gemessen, von ter am schnellsten Ordner nicht gemessen und festgestellt, dass die Faltung Rate nach dem Mischen gemessen ~ 5-mal langsamer als mit T-Sprung unter den gleichen Bedingungen 16 gemessen ist. Dies deutet darauf hin, dass Faltungskinetik von den Anfangsbedingungen ab hängt, Kippen eine wichtige Annahme in diesem Bereich.

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td> AZ P4110 Fotolack </td><td> AZ Electronic Materials </td><td></td><td></td></tr><tr><td> AZ 400 K Entwickler </td><td> AZ Electronic Materials </td><td></td><td></td></tr><tr><td> Baker PRS 2000 Photoresist-Stripper </td><td> Avantor Performance Materials </td><td></td><td></td></tr><tr><td> AquaBond </td><td> AquaBond Technologies </td><td> AquaBond 55 </td><td></td></tr><tr><td> 500 pm Abdeckwafers </td><td> SENSOR Prep Dienstleistungen </td><td></td><td> Ø: 100 mm ± 0,5 mm Dicke: 0,5 mm ± 0,025 mm Standard-Toleranzen (Sofern nicht anders angegeben) Oberflächenfinish: SI: 4-8a; SII: 8-12a Wohnungen: Ein primärer SEMI-STD; Ein Scribe Sauber und poliert, beidseitig </td></tr><tr><td> 170 um Abdeckwafers </td><td> SENSOR Prep Dienstleistungen </td><td> 7980 2G Waffeln </td><td> Ø: 100 mm ± 0,5 mm Stärke: 0,17 mm ± 0,025 mm Standard-Toleranzen (Sofern nicht anders angegeben) Oberflächenfinish: SI: 4-8a; SII: 8-12a Wohnungen: Ein primärer SEMI-STD; Ein Scribe Sauber und poliert, beidseitig </td></tr><tr><td> Deep Reactive Ion Radierer für Oxide </td><td> ULVAC </td><td> ULVAC NL-6000 </td><td></td></tr><tr><td> Argon-Ionen-Laser </td><td> Cambridge Laboratories Laser </td><td> Lexel 95-SHG </td><td> λ = 258 nm </td></tr><tr><td> Argon-Ionen-Laser </td><td> Melles Griot </td><td></td><td> λ = 488 nm </td></tr><tr><td> Mikroskop </td><td> Olymp </td><td> IX-51</td><td></td></tr><tr><td> Mikroskopobjektiv </td><td> Thor Labs </td><td> OFR LMU-40X-UVB 0,5 NA </td><td> λ = 258 nm </td></tr><tr><td> Mikroskopobjektiv </td><td> Olymp </td><td> UPLSAPO 60XW 1,2 NA </td><td> λ = 488 nm </td></tr><tr><td> Nanopositionierer </td><td> Mad City Labs </td><td> Nano-LP100 </td><td></td></tr><tr><td> Motorisierte Verschiebetisch </td><td> Semprex Corp </td><td> KL-Serie 12-6436 </td><td></td></tr><tr><td> GaAsP Photonenzähler </td><td> Hamamatsu </td><td> H7421-40 </td><td></td></tr><tr><td> Monochromator </td><td> Horiba Instruments Inc </td><td> MicroHR </td><td></td></tr><tr><td> CCD-Kamera </td><td> Andor Technology </td><td> idus 420A-BU </td><td></td></tr><tr><td> Guanidinhydrochlorid (GuHCl) </td><td> Sigma-Aldrich </td><td&gt; G4505 </td><td></td></tr></tbody></table>

microfluidic mixers for studying protein folding

Der Prozess, durch den sich ein Protein faltet seine native Konformation ist äußerst relevant für Biologie und Gesundheit des Menschen aber immer noch wenig verstanden. Ein Grund dafür ist, dass Faltung erfolgt über einen weiten Bereich von Zeitskalen von Nanosekunden bis Sekunden oder länger, je nach dem Protein 1. Konventionelle Stopped-Flow-Mischer erlaubt haben Messung von Faltungskinetik ab etwa 1 ms. Wir haben kürzlich einen Mikrofluidik-Mixer, der Vergällungsmittel ~ 100-fach in ~ 8 ms 2 verdünnt entwickelt. Im Gegensatz zu einem Stopped-Flow-Mixer arbeitet diese Mischer in der Laminar-Flow-Regime, in dem Turbulenzen nicht auftritt. Das Fehlen von Turbulenz ermöglicht eine präzise numerische Simulation aller Strömungen innerhalb des Mischers mit exzellenter Übereinstimmung zu experimentieren 3-4. Laminar-Flow ist für Reynoldszahlen Re ≤ 100 erreicht. Bei wässrigen Lösungen erfordert diese Geometrien im Mikrometerbereich. Wir verwenden ein hartes Substrat, wie Silizium oder Silizium kondensiertenca, um Kanäle 5-10 &amp;mgr; m breit und 10 um tief (siehe Abbildung 1). Die kleinste Abmessungen, am Eingang zu dem Mischbereich, liegen in der Größenordnung von 1 um in der Größe. Der Chip wird mit einer dünnen Glas-oder Fused-Silica-Deckglas für optische Zugang verschlossen. Typische insgesamt lineare Flussraten sind ca. 1 m / s, was zu Re ~ 10, aber die Protein-Verbrauch beträgt nur ~ 0,5 NL / s oder 1,8 ul / hr. Die Proteinkonzentration wird durch den Nachweis Verfahren: Für Tryptophanfluoreszenz die typische Konzentration 100 pM (1 Trp / Protein) und FRET die typische Konzentration ~ 100 nM. Der Falzvorgang wird durch schnelle Verdünnung des Denaturierungsmittels von 6 M bis 0,06 M Guanidin-Hydrochlorid eingeleitet. Das Protein in hoher Vergällungsmittel fließt einem zentralen Kanal und ist auf beiden Seiten an der Mischbereich durch Puffer ohne Vergällungsmittel bewegt ~ 100-mal schneller (siehe Abbildung 2) erfüllt. Diese Geometrie bewirkt eine schnelle Verengung des Proteins Fluss in ein engesJet ~ 100 nm breit sind. Die Diffusion der Moleküle Licht Vergällungsmittel ist sehr schnell, während die Diffusion der schweren Protein-Moleküle ist viel langsamer, diffundieren weniger als 1 um in 1 ms. Der Unterschied in der Diffusionskonstante des Denaturierungsmittels und den Protein führt zu einer schnellen Verdünnung des Denaturierungsmittels von dem Protein Strom, wodurch die effektive Konzentration des Denaturierungsmittels in der Umgebung des Proteins. Das Protein Strahl fließt mit einer konstanten Geschwindigkeit entlang der Beobachtungskanal und Fluoreszenz des Proteins während der Faltung beobachtet unter Verwendung eines konfokalen 5 beschrieben.

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

Microfluidic Mixers for Studying Protein Folding

In dieser Arbeit erklären wir die Herstellung und Verwendung eines mikrofluidischen Mixer zum Mischen zweier Lösungen in ~ 8 ms. Wir zeigen auch den Einsatz dieser Mischer mit spektroskopischen Nachweis mittels UV-Fluoreszenz-und Fluoreszenz-Resonanz-Energie-Transfer (FRET).

Mikrofluidischen Mischern für ein Studium Proteinfaltung

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 Aufrufe.  Michigan State University. In dieser Arbeit erklären wir die Herstellung und Verwendung eines mikrofluidischen Mixer zum Mischen zweier Lösungen in ~ 8 ms. Wir zeigen auch den Einsatz dieser Mischer mit spektroskopischen Nachweis mittels UV-Fluoreszenz-und Fluoreszenz-Resonanz-Energie-Transfer (FRET).

Serie: 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.

Einem mikrofluidischen-basierte Hydrodynamische Trap für einzelne Teilchen

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

Forschung

Biotechnik

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.

Thermodynamik der Membrane Protein Folding durch Fluoreszenz-Spektroskopie

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.

Fluoreszenz-Nachweisverfahren für Mikrofluidik-Tropfen-Plattformen

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.

High-throughput Protein Expression Generator mit einem mikrofluidischen Plattform

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.

Einer mikrofluidischen Vorrichtung für ein Studium mehrere verschiedene Stämme

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.

Eines mikrofluidischen Chips für ICPMS Probenaufgabe

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.

Das Studium von Proteinfunktionen und die Rolle der Altered Protein Expression von Antikörper-Interferenz und Dreidimensionale Rekonstruktionen

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.

Vollautomatische Schleuder Mikrofluidik-Vorrichtung zur Ultrasensitive Proteindetektion aus Vollblut

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.

Entwerfen Mikrofluidiksysteme für ein Studium zellulären Reaktionen Unter Einzel- oder Coexisting Chemie / Elektro / Schubspannung 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.

Mehrstufige Höhenverstellbarem Lithografie für Valved Mehrschichtige Mikrofluidiksysteme

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