Bu çalışma, Ulusal Bilim Vakfı fibr (NSF EF-0.623.664) ve IDBR (NSF DBI-0.754.570) tarafından desteklenmektedir. Bu çalışma kısmen Kooperatif Anlaşması PHY 0120999 altında Kaliforniya Üniversitesi, Davis tarafından yönetilen Biophotonics, bir NSF Bilim ve Teknoloji Merkezi Merkezi tarafından yönetilen Ulusal Bilim Vakfı fibr Hibe 0623664, fon tarafından desteklenmiştir. Lisa Lapidus, doktora ve araştırma Burroughs Hoşgeldiniz Fonundan Bilimsel Arayüz bir Kariyer Ödülü kısmen desteklenmektedir.

<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>

Biz ifşa hiçbir şey yok.

Uzun biyomoleküllerin moleküler süreçlerin, piko saniye kadar zaman ölçeğindeki meydana geldiği kabul edilmiştir, çünkü Hızlı karıştırma yıllarca protein katlanması alan için bir gelişme hedefi olmuştur. Konvansiyonel durdu-akış karıştırıcılar başta türbülans ile sınırlıdır 1-5 ms ölü zamanları vardır. 30-300 ms&#39;den karıştırma süreleri ile Çalkantılı sürekli akış mikserler gruba az sayıda son 15 yılda geliştirilen ancak genellikle bu karıştırma süreleri elde etmek için yüksek debi gerektiren ve bu nedenle örnek 6-8 çok kullanmaya başlamıştır. Bu protokol laminer akış rejimindeki hızlı seyreltme ulaşmak için mikroakışkan karıştırma fiş kullanımı anlatılmaktadır. Orada bu rejim içinde çalışan birden çok avantajı vardır, ancak birincil bir bir karıştırma yanıt değiştirmenize olanak veren yüksek hassasiyet ile tüm karıştırma işlemi taklit yeteneğidir. Basit geom ile diğer gruplar tarafından geliştirilen T-mikserleretries öncelikle kanal 9-10 büyüklüğü ile sınırlı kaldı 100-200 ms&#39;den karıştırma kez gösterilmiştir. 10 mikron Knight kanallarının boyutunu ölçekleme By ~ 10 ms&#39;dan 11 karıştırma süresi azalır. Yao ve Bakajin geometri küçük değişiklikler karıştırma süresi 4 önemli gelişmelere yol açabileceğini gösterdi. Bununla birlikte, bu çalışma karıştırma olduğu ivme zamanda da yavaş fazlar yol açabilir gösterdi. Bu çalışmada 12-13 kullanılan tasarım denaturant konsantrasyon yavaş üstel çürüme en aza indirmek ve aynı zamanda Drie aşındırma bir özelliği daha tekrarlanabilir yapmak için referans 4&#39;te iki iyi tasarımları arasında bir uzlaşmadır. Karıştırma süresi tanımı içinde karıştırılması ultrahızlı bireylerde alanında bazı tartışmalara olmuştur. Bu &quot;karıştırma süresi&quot; ve &quot;ölü zaman&quot; arasındaki farkı fark etmek önemlidir. Ölü zaman zarfında bir ÖLÇME süre olarak çalkantılı bir mikser tanımlanırt yapılamaz ve aslında gerçek karıştırma süreden önemli ölçüde daha uzun olabilir. Bu, tipik olarak, çeşitli konsantrasyonlarda bir sahte-birinci dereceden iki moleküllü reaksiyonu (örneğin, N bromosuccinate tarafından triptofan floresans soğutmadan gibi) çeşitli ölçümler yapmak tarafından belirlenir. Gözlenen üstel çürükleri t = 0 s olarak kabul tek bir değerde yakınsama takılan ve bu noktadan ve ilk ölçülen nokta arasındaki zaman ölü bir zamanı vardır. Herhangi bir ölü zaman, sadece bir karıştırma süresi olacak şekilde laminar akış karıştırıcılar için ölçüm karıştırma işlemi sırasında yapılabilir. Karıştırma süresi gerçekte yeterli homojenlik elde edilene kadar iki çözüm birleştirme zamanı. Biz daha önce 90/10, karıştırılmamış değerinin% 10 ila% 90 azaltmak için denaturant konsantrasyonu için zaman olması karıştırma süresi belirledik. Bununla birlikte, karıştırma eğrisi şekil küçük üstel bileşenine sahip çürüme kuyruk ile mükemmel simetrik değildir. Ayrıca, protein katlanması, bir vardenaturant konsantrasyonu üzerindeki yüksek doğrusal-olmayan bir bağımlılığının katlama genellikle denaturant sadece iki misli azalır bile başlaması böylece. Bu nedenle son zamanlarda% 80 oranında denaturant konsantrasyonunu azaltmak için zaman olarak karıştırma süresi belirledik. Kuşkusuz, diğer tanımları deney gereksinimlerine bağlı olarak kullanılabilir. Protein katlanması incelemek için bu mikser kullanımı sürekli şaşırtıcı sonuçlar ortaya koymuştur. Uzun sürekli akış mikserler ile çoklu katlama adımları ve erken sonuçlar almaya çalışılmıştır sitokrom c ve apomyoglobin katlanması ~ 100 s ölçeğinde 10,14-15 oluşmasına çöküşü gösterdi. Bu mikser ile yaptığımız ölçümler (olarak daha yavaş bir faz takip Mikserin karıştırma süre içinde, çok hızlı, muhtemelen spesifik olmayan, çökmek (as Trp floresans spektral vardiya ile ölçülen) bu ölçeğinde en az 2 adım olacaksa gösterdi ) toplam Trp emisyon soğutma ile ölçülen olasılıkla fdoğal yapısı 5 IRST oluşumu. Ayrıca, bu proteinin, bir 2-klasörün 13 durumu olduğu geleneksel yorumlama iptali için, protein, L B1 alanı içinde bir 50 μs işlemi araştırmacılar tarafından gözlenmiştir. Bu hızlı bir mikser bir avantajı, genellikle sadece nanosaniye T-atlama araçlar ile ölçülebilir olmuştur hızlı katlama proteinlerin katlama yoklamak olmasıdır. T-atlama genellikle katlama / protein erime sıcaklığına yakın rahatlama açılması ve bu nedenle bütün nüfusun katlanması aşağıdaki asla gözlem gerektirir. Bu mikser ile biz Trp toplam emisyon ve spektral vardiya her ikisi ile γ-repressör katlama (λ 6-86) baktım ve protein önceki T tarafından erişilebilir olmadığını güçlü katlama koşullarda katlama için hiçbir engel olmadığını kanıtlar buldular ölçümleri 12-atlamak. Son olarak, bir t villin başlık HP-35 ile katlama ölçülenO hızlı klasörleri henüz ölçülür ve karıştırma sonra ölçülen katlama oranı aynı şartlar altında 16 T-atlama ile ölçülen daha ~ 5 kat daha yavaş olduğunu buldu. Bu katlanma kinetikleri alanda büyük bir varsayımı devrilmesiydi başlangıç ​​şartlarına bağlı olduğunu göstermektedir.

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> AZ P4110 rezist </td><td> AZ Elektronik Malzemeler </td><td></td><td></td></tr><tr><td> AZ 400 K Geliştirici </td><td> AZ Elektronik Malzemeler </td><td></td><td></td></tr><tr><td> Baker PRS 2000 fotorezist striptizci </td><td> Avantor Performans Malzemeleri </td><td></td><td></td></tr><tr><td> AquaBond </td><td> AquaBond Teknolojileri </td><td> AquaBond 55 </td><td></td></tr><tr><td> 500 mikron kapak gofret </td><td> SENSÖR Hazırlık Hizmetler </td><td></td><td> Ø: 100 mm ± 0.5 mm Kalınlık: 0.5 mm ± 0.025 mm Standart Toleranslar (Kaydetti sürece) Yüzey: SI: 4-8A; SII: 8-12a Daire: Bir İlköğretim YARI-STD; Bir Scribe Temiz ve Parlak, Her İki Tarafa </td></tr><tr><td> 170 mikron kapak gofret </td><td> SENSÖR Hazırlık Hizmetler </td><td> 7980 2G Gofret </td><td> Ø: 100 mm ± 0.5 mm Kalınlık: 0.17 mm ± 0.025 mm Standart Toleranslar (Kaydetti sürece) Yüzey: SI: 4-8A; SII: 8-12a Daire: Bir İlköğretim YARI-STD; Bir Scribe Temiz ve Parlak, Her İki Tarafa </td></tr><tr><td> Oksitler için derin reaktif iyon yakıcısı </td><td> ULVAC </td><td> ULVAC NL-6000 </td><td></td></tr><tr><td> Argon Lazer İyon </td><td> Cambridge Lazer Laboratuvarları </td><td> LEXEL 95-SHG </td><td> λ = 258 nm </td></tr><tr><td> Argon Lazer İyon </td><td> Melles Griot </td><td></td><td> λ = 488 nm </td></tr><tr><td> Mikroskop </td><td> Olympus </td><td> IX-51</td><td></td></tr><tr><td> Mikroskop Amaç </td><td> Thor Labs </td><td> OFR LMU-40X-UVB 0.5 NA </td><td> λ = 258 nm </td></tr><tr><td> Mikroskop Amaç </td><td> Olympus </td><td> UPLSAPO 60XW 1,2 NA </td><td> λ = 488 nm </td></tr><tr><td> Nanopositioner </td><td> Mad City Labs </td><td> Nano-LP100 </td><td></td></tr><tr><td> Motorlu Çeviri Sahne </td><td> Semprex Corp </td><td> KL-12-6436 Serisi </td><td></td></tr><tr><td> GaAsP Foton Sayaç </td><td> Hamamatsu </td><td> H7421-40 </td><td></td></tr><tr><td> Monochrometer </td><td> Horiba Instruments Inc </td><td> MicroHR </td><td></td></tr><tr><td> CCD kamera </td><td> Andor Teknoloji </td><td> idus 420A-BU </td><td></td></tr><tr><td> Guanidin hidroklorür (GuHCl) </td><td> Sigma-Aldrich </td><td&gt; G4505 </td><td></td></tr></tbody></table>

microfluidic mixers for studying protein folding

Kendi doğal yapısındaki bir proteini kıvrımlar biyoloji ve insan sağlığı hala henüz tam olarak anlaşılamamıştır oldukça alakalı olan süreçtir. Bunun bir nedeni, bu katlama protein 1 bağlı olarak, nanosaniye ile saniye ya da daha fazla, süresini çok geniş bir aralık içinde yer alan bir. Konvansiyonel durdu-akış mikserler katlama kinetik ölçümü yaklaşık 1 ms başlayan sağladı. Biz son zamanlarda ~ 8 μs 2 denaturant ~ 100 kat sulandırır bir mikroakışkan karıştırıcı geliştirdik. Durmuş bir akış mikser aksine, bu mikser türbülans oluşmaz hangi laminer akış rejimi çalışır. Türbülans olmaması 3-4 deneme için mükemmel bir anlaşma ile mikser içindeki tüm akımları hassas sayısal simülasyonu sağlar. Laminar akış ≤ 100 Re Reynolds sayıları için elde edilir. Sulu çözelti, bu mikron ölçeğinde geometri gerektirir. Bu tür silikon veya erimiş silikonlu yağla olarak, sert bir yüzey kullanımıca, (Bakınız Şekil 1) kanalları 5-10 mikron genişliğinde ve 10 mikron derin yapmak. Küçük boyutları, karıştırma bölgeye girişinde, boyutu 1 mikron mertebesinde vardır. Çip optik erişim için ince bir cam veya silika coverslip ile örtülmüştür. Tipik toplam doğrusal akım hızları ~ 1 m / s, akma Re ~ 10, fakat protein tüketimi sadece 0.5 ~ nL / s veya 1.8 uL / saat. Protein konsantrasyonu tespit yöntemi bağlıdır: triptofan floresans için tipik derişim 100 uM (1 Trp / protein) ve tipik konsantrasyonu ~ 100 nM&#39;dirki FRET için. Katlama işlemi 6 M 0.06 M guanidin hidroklorür denaturant hızlı seyreltme tarafından başlatılır. Yüksek denaturant bulunan protein merkezi bir kanal aşağı akar ve denaturant (bkz. Şekil 2) ~ 100 kat daha hızlı hareket ettirmeden tampon ile karıştırma bölgede iki tarafında karşılanmaktadır. Bu dar bir geometri içine proteini akışının hızlı daralma nedenjet ~ 100 nm çapında. Ağır protein moleküllerinin difüzyon çok yavaş 1 ms difüzyon az 1 mikron iken ışık denaturant moleküllerin difüzyon, çok hızlıdır. Proteini yaklaşık denaturant etkili konsantrasyonu azaltarak denaturant ve protein akışından denaturant hızlı dilüsyonu proteini sonuçların sabit bir difüzyon farkı,. Protein jet bir tarama mikroskobu konfokal 5 kullanılarak tespit edilebilir katlama sırasında protein gözlem kanalı ve floresans aşağı doğru sabit bir hızda hareket eder.

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

Microfluidic Mixers for Studying Protein Folding

Bu çalışmada biz ~ 8 μs iki çözüm karıştırma yeteneğine sahip mikroakışkan mikser imalat ve kullanımının açıklanması. Ayrıca UV floresan ve floresan rezonans enerji transferi (FRET) kullanarak spektroskopik tespiti ile bu karıştırıcı kullanımını göstermektedir.

Protein Katlama Öğrenmenin mikroakışkan Mikserler

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 Görüntüleme.  Michigan State University. Bu çalışmada biz ~ 8 μs iki çözüm karıştırma yeteneğine sahip mikroakışkan mikser imalat ve kullanımının açıklanması. Ayrıca UV floresan ve floresan rezonans enerji transferi (FRET) kullanarak spektroskopik tespiti ile bu karıştırıcı kullanımını göstermektedir.

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.

Tek Parçacıklar için mikroakışkan tabanlı Hidrodinamik Tuzak

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

Biyomühendislik

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.

Membran Protein katlanması, Termodinamik Floresans Spektroskopisi ile ölçülür.

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.

Mikroakışkan damlacık platformları için Floresan algılama yöntemleri

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.

Bir mikroakışkan Platform kullanarak Yüksek throughput Protein Ekspresyonu Jeneratör

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.

Birden Farklı Suşlarının incelenmesi için bir mikroakışkan Cihazı

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.

ICPMS Numune Giriş için mikroakışkan Chip

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.

Incelenmesi Protein Fonksiyonu ve Antikor Girişim ve üç boyutlu Rekonstrüksiyonunda tarafından değiştirilmiş Protein Ekspresyonu Rolü

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.

Tüm Blood adlı ultrasensitive Protein Algılama için Tam Otomatik Santrifüj mikroakışkan Cihazı

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.

Tek ya da Eşlik Kimya / Elektrik / Kesme Stres Uyaranların altında Hücresel Yanıtları incelenmesi için mikroakışkan Cihazlar Tasarımı

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.

Ventilli Katmanlı mikroakışkan Cihazlar için çok adımlı Değişken Yükseklik Fotolitografi

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