Este trabajo es apoyado por la Fundación Nacional de Ciencias FIBR (NSF EF-0623664) y IDBR (NSF DBI-0754570). Este trabajo fue parcialmente financiado por fondos de la National Science Foundation Grant FIBR 0623664 administrado por el Centro para la Biofotónica, una Ciencia NSF y el Centro de Tecnología, gestionado por la Universidad de California en Davis, conforme al Acuerdo Cooperativo PHY 0120999. La investigación de Lisa Lapidus, Ph.D. está apoyado en parte por un premio a la Trayectoria en la interfaz de la Ciencia con cargo al Fondo 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>

No tenemos nada que revelar.

La mezcla rápida ha sido un objetivo de desarrollo para el campo de plegamiento de las proteínas durante muchos años, ya que hace tiempo se reconoce que los procesos moleculares de las biomoléculas se producen en escalas de tiempo que van desde picosegundos a los segundos. Convencionales de flujo detenido mezcladores tienen tiempos muertos de 5.1 ms, el cual está limitado principalmente por la turbulencia. Turbulentos mezcladores de flujo continuo con tiempos de mezcla de 30-300 mS se han desarrollado durante los últimos 15 años por un pequeño número de grupos, pero generalmente requieren altas velocidades de flujo para lograr estos tiempos de mezcla y por lo tanto, utilizan una gran cantidad de la muestra 6.8. Este protocolo se describe el uso de chips de microfluidos de mezcla para lograr una rápida dilución en el régimen de flujo laminar. Hay múltiples ventajas para trabajar dentro de este régimen, pero un uno primaria es la capacidad para simular el proceso de mezcla con alta precisión que permite una para modificar la respuesta de mezcla. T-mezcladores desarrollados por otros grupos con un simple geometríaetries han demostrado tiempos de mezcla de 100-200 mS que estaban limitados principalmente por el tamaño de los canales 9-10. Por escalar el tamaño de los canales a Knight micras 10 reduce el tiempo de mezcla a 10 ~ 11 mS. Yao y Bakajin mostraron que pequeños cambios en la geometría podría conducir a mejoras significativas en el tiempo de mezclado 4. Sin embargo, ese trabajo muestra que la aceleración de la mezcla también se puede llevar a más lentas fases también. El diseño utilizado en este trabajo 12 a 13 es un compromiso entre los dos mejores diseños en la referencia 4 para minimizar la descomposición más lenta exponencial de la concentración de desnaturalizante y también para hacer que la característica más reproducible en el grabado DRIE. Ha habido una cierta controversia en el campo de la mezcla ultrarrápida sobre la definición del tiempo de mezclado. Es importante reconocer la diferencia entre &quot;tiempo de mezcla&quot; y &quot;tiempo muerto&quot;. El tiempo muerto se define en un mezclador turbulento como el tiempo durante el cual un MEDIDASt no se pueden hacer y de hecho puede ser significativamente más largo que el tiempo de mezcla real. Se determina típicamente por hacer varias mediciones de una reacción de pseudo primer orden bimolecular (tales como enfriamiento de triptófano fluorescencia por N bromosuccinate) a diversas concentraciones. Los decaimientos exponenciales observados están equipados para converger en un solo valor supone que t = 0 s y el tiempo entre dicho punto y el primer punto medido es el tiempo muerto. Para mezcladores de flujo laminar, las mediciones se pueden hacer durante el proceso de mezclado así que no hay tiempo muerto, sólo un tiempo de mezclado. El tiempo de mezclado es simplemente el tiempo para combinar dos soluciones hasta uniformidad suficiente se alcanza. Hemos definido previamente el tiempo de mezcla para ser un tiempo 90/10, el tiempo para la concentración de desnaturalizante para disminuir de 90% a 10% del valor sin mezclar. Sin embargo, la forma de la curva de mezcla no es perfectamente simétrico con la cola de la caries que tiene un componente exponencial pequeño. Además, el plegamiento de proteínas tiene unaltamente no lineal dependencia de la concentración de desnaturalizante de modo que a menudo plegable puede comenzar incluso si el desnaturalizante sólo se reduce por dos veces. Por lo tanto, se han definido más recientemente el tiempo de mezcla como el tiempo para reducir la concentración desnaturalizante en un 80%. Ciertamente otras definiciones se pueden utilizar dependiendo de los requerimientos del experimento. El uso de este mezclador para estudiar el plegamiento de proteínas ha revelado consistentemente resultados sorprendentes. Plegado de citocromo C y apomyoglobin han sido estudiados para tener varios escalones plegables y los primeros resultados con los mezcladores de flujo continuo mostraron colapso que se produzca en el plazo de tiempo ~ 100 mS 10,14-15. Nuestras mediciones con este mezclador mostraba que había un mínimo de 2 pasos en esta escala de tiempo, un muy rápido, probablemente no específica, colapso (medida por fluorescencia Trp cambio espectral) dentro del tiempo de mezcla del mezclador seguido por una fase más lenta (como medido por el enfriamiento del total de emisiones Trp) que es probable que el frimero formación de la estructura nativa 5. También hemos observado un proceso de 50 mS en el dominio B1 de la proteína L, el vuelco a la interpretación convencional de que esta proteína es una carpeta de 2 estados 13. Una ventaja de este mezclador rápido es que se puede investigar el plegamiento de las proteínas más rápidas plegables que han sido por lo general sólo medible por nanosegundos salto de T-instrumentos. T-salto requiere típicamente observación de plegado / desplegado relajación cerca de la temperatura de fusión de la proteína y por lo tanto, nunca sigue el plegamiento de toda la población. Con este mezclador hemos visto en el plegamiento de las γ-represor (λ 6-86) tanto con las emisiones totales Trp y desplazamiento espectral y no han encontrado evidencia de que la proteína no tiene ningún obstáculo para doblar en condiciones de fuerte plegables que no eran accesibles a T anteriores -salta las medidas 12. Finalmente hemos medido el plegado de la pieza de cabeza villina HP-35, una de tél más rápidas carpetas todavía midió y se encontró que la tasa de plegado medido después de la mezcla es de ~ 5 veces más lento que medido por T-salto en las mismas condiciones 16. Esto sugiere que la cinética de plegado depende de las condiciones de partida, el vuelco una suposición importante en el campo.

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> AZ P4110 Fotoresinas </td><td> AZ Electronic Materials </td><td></td><td></td></tr><tr><td> AZ 400 K para desarrolladores </td><td> AZ Electronic Materials </td><td></td><td></td></tr><tr><td> PRS Baker 2000 fotosensible separador </td><td> Avantor Performance Materials </td><td></td><td></td></tr><tr><td> AquaBond </td><td> AquaBond Tecnologías </td><td> AquaBond 55 </td><td></td></tr><tr><td> 500 oblea cubierta de m </td><td> SENSOR Servicios de preparación </td><td></td><td> Ø: 100 mm ± 0,5 mm Espesor: 0,5 mm ± 0,025 mm Tolerancias estándar (a menos que) Acabado de la superficie: SI: 4-8a; SII: 8-12a Pisos: Un Primaria SEMI-STD; un escriba Limpio y pulido, las dos caras </td></tr><tr><td> 170 oblea cubierta de m </td><td> SENSOR Servicios de preparación </td><td> 7980 2G Obleas </td><td> Ø: 100 mm ± 0,5 mm Espesor: 0,17 mm ± 0,025 mm Tolerancias estándar (a menos que) Acabado de la superficie: SI: 4-8a; SII: 8-12a Pisos: Un Primaria SEMI-STD; un escriba Limpio y pulido, las dos caras </td></tr><tr><td> Grabador profunda iones reactivos para los óxidos </td><td> ULVAC </td><td> ULVAC NL-6000 </td><td></td></tr><tr><td> Láser de ion argón </td><td> Cambridge láser Laboratorios </td><td> Lexel 95-SHG </td><td> l = 258 nm </td></tr><tr><td> Láser de ion argón </td><td> Melles Griot </td><td></td><td> l = 488 nm </td></tr><tr><td> Microscopio </td><td> Olimpo </td><td> IX-51</td><td></td></tr><tr><td> Microscopio Objetivo </td><td> Thor Labs </td><td> OFR LMU-40X-UVB 0,5 NA </td><td> l = 258 nm </td></tr><tr><td> Microscopio Objetivo </td><td> Olimpo </td><td> UPLSAPO 60XW 1,2 NA </td><td> l = 488 nm </td></tr><tr><td> Nanopositioner </td><td> Mad Labs Ciudad </td><td> Nano-LP100 </td><td></td></tr><tr><td> Traducción motorizado Etapa </td><td> Semprex Corp </td><td> KL-serie 12-6436 </td><td></td></tr><tr><td> GaAsP Photon Contador </td><td> Hamamatsu </td><td> H7421-40 </td><td></td></tr><tr><td> Monocrómetro </td><td> Horiba Instruments Inc </td><td> MicroHR </td><td></td></tr><tr><td> CCD de la cámara </td><td> Andor Technology </td><td> idus 420A-BU </td><td></td></tr><tr><td> Hidrocloruro de guanidina (GuHCl) </td><td> Sigma-Aldrich </td><td&gt; G4505 </td><td></td></tr></tbody></table>

microfluidic mixers for studying protein folding

El proceso por el cual una proteína se pliega en su conformación nativa es de gran importancia para la biología y la salud humana y aún poco conocidos. Una razón para esto es que plegado tiene lugar en un amplio rango de escalas de tiempo, de nanosegundos a segundos o más, dependiendo de la proteína 1. Convencionales de flujo detenido mezcladores han permitido la medición de la cinética de plegado a partir de alrededor de 1 ms. Recientemente hemos desarrollado un mezclador de microfluidos que diluye desnaturalizante ~ 100 veces en ~ 8 mS 2. A diferencia de un mezclador de flujo detenido, este mezclador opera en el régimen de flujo laminar en el que la turbulencia no se produce. La ausencia de la turbulencia permite la simulación numérica precisa de todos los flujos dentro de la mesa de mezclas con un acuerdo excelente para experimentar 3-4. El flujo laminar se logra para números de Reynolds Re ≤ 100. Para soluciones acuosas, esto requiere geometrías micras escala. Nosotros utilizamos un sustrato duro, tal como el silicio o silicio fundidoCA, para hacer micras canales 5-10 ancho y profundo 10 micras (Ver Figura 1). Las dimensiones más pequeñas, en la entrada a la zona de mezcla, son del orden de 1 micra de tamaño. El chip se sella con un vidrio delgado o cubreobjetos de sílice fundida para el acceso óptico. Las velocidades típicas del flujo total son lineales aproximadamente 1 m / s, dando Re ~ 10, pero el consumo de proteínas es sólo ~ 0,5 nl / s ó 1,8 l / h. La concentración de proteína depende del método de detección: Para triptófano fluorescencia la concentración típica es de 100 micras (1 para Trp / proteína) y para FRET la concentración típica es de ~ 100 nM. El proceso de plegado se inicia por dilución rápida de desnaturalizante de 6 M a 0,06 M de hidrocloruro de guanidina. La proteína en desnaturalizante alta fluye hacia abajo un canal central y se reunieron a ambos lados en la región de mezcla por tampón desnaturalizante sin mover ~ 100 veces más rápido (ver Figura 2). Esta geometría causa constricción rápida del flujo de proteína en un estrechochorro ~ 100 nm de ancho. La difusión de las moléculas desnaturalizantes de luz es muy rápida, mientras que la difusión de las moléculas de proteína pesados ​​es mucho más lenta, se difunden a menos de 1 micra de 1 ms. La diferencia en la constante de difusión del desnaturalizante y los resultados de proteínas en dilución rápida del desnaturalizante de la corriente de la proteína, lo que reduce la concentración efectiva del desnaturalizante alrededor de la proteína. El chorro de proteína fluye a una velocidad constante por el canal de observación y la fluorescencia de la proteína durante el plegado puede observarse usando un microscopio confocal de barrido 5.

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

Microfluidic Mixers for Studying Protein Folding

En este trabajo se explica la fabricación y el uso de un mezclador de microfluidos capaz de mezclar dos soluciones en ~ 8 mS. También demuestran el uso de estos mezcladores con detección espectroscópica utilizando fluorescencia UV y la transferencia de energía de resonancia fluorescente (FRET).

Mezcladores de microfluidos para el estudio plegamiento de proteínas

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 vistas.  Michigan State University. En este trabajo se explica la fabricación y el uso de un mezclador de microfluidos capaz de mezclar dos soluciones en ~ 8 mS. También demuestran el uso de estos mezcladores con detección espectroscópica utilizando fluorescencia UV y la transferencia de energía de resonancia fluorescente (FRET).

Video: 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.

Una de microfluidos basada en hidrodinámica trampa para las partículas individuales

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

Investigación

Bioingeniería

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.

Termodinámica del plegado de proteínas de membrana medido por espectroscopia de fluorescencia

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.

Métodos de detección de fluorescencia para las plataformas de las gotas de microfluidos

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.

Proteínas de alto rendimiento del generador de expresiones utilizando una plataforma de microfluidos

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 dispositivo de microfluidos para el estudio de varias cepas distintas

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.

Un chip de microfluidos para ICPMS Introducción Muestra

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.

El estudio de la función de proteínas y el Papel de la expresión de la proteína alterada por la interferencia de anticuerpos y reconstrucciones tridimensionales

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.

Totalmente automatizada de dispositivos de microfluidos para centrífuga Ultrasensible detección de proteínas a partir de sangre entera

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.

El diseño de dispositivos de microfluidos para el estudio de las respuestas celulares en la sección individual o Coexistencia Química / eléctrica / Shear Stress Los estímulos

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.

Múltiples pasos Fotolitografia variable Altura de múltiples capas con válvula dispositivos de microfluidos

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