Bu araştırma çalışmaları Atomik Seviye Catalyst Tasarım, ABD Enerji Departmanı, Bilim, Ödül sayısı DE-SC0001058 altında Temel Enerji Bilimler Dairesi Başkanlığı tarafından finanse edilen bir Enerji Frontier Araştırma Merkezi Merkezi&#39;nin bir parçası olarak desteklenen ve aynı zamanda Yönetim Kurulu tarafından desteklenmektedir hibe ödül numarası LEQSF (2009-14)-EFRC-MAÇ ve LEDSF-EPS (2012)-OPT-IN-15 altında Vekaleti. MRCAT işlemleri Enerji Bakanlığı ve MRCAT üye kurumlar tarafından desteklenmektedir. ANL de Gelişmiş Foton Kaynak kullanımı, Sözleşme No: DE-AC02-06CH11357 altında ABD Enerji Departmanı, Fen Bilimleri, Temel Enerji Bilimler Dairesi Ofisi tarafından desteklenmektedir. Jtm için mali destek Atom verimli Kimyasal Dönüşümler Enstitüsü (İAİS), ABD Enerji Bakanlığı tarafından finanse edilen bir Enerji Frontier Araştırma Merkezi, Bilim Ofisi, Temel Enerji Bilimler Office&#39;in bir parçası olarak sağlandı.

Millifluidics set-up: 220 mm (L) x 2 mm (W) x 0.15 mm (Y) boyutlarında serpantin kanalları var Microplumbers Microsciences LLC, dan (polyester tereftalat polimerden yapılmış) bir millifluidic çip satın alın. Pompaya çip bağlamak için 0.25 mm ID boyutlarında, OD 1/16, FEP Boru kullanın. İki farklı deneyler için iki farklı pompalar kullanarak. İlk deneyde (bakır nanopartikülleri) ve ikinci deney (altın nanopartiküller) için millifluidic cihaz için kullanın P-Pompa. , Kanalların içinde gaz kabarcıklarının problemi en aza indirmek için, yeni hazırlanmış bir NaBH4 solüsyonu, gaz kabarcıkları çözelti kaçmak böylece yonga içine pompalama önce ~ 10-15 dakika boyunca beklemeye açık bırakıldı. Bu adım bizim deneyler için tüm izledi. 1.. Ultra-küçük Bakır Nano Kümeler (UCNCs) sentezi <ol><li> Gerekli Kimyasallar: bakır elde (II) nitrate hidrat, sodyum borohidrid, sodyum hidroksit peletleri ve O-[2 - (3-mercaptopropionylamino) etil]-O&#39;-metilpolietilen glikol (MW = 5,000) [MPEG] ve daha fazla arıtılmadan tüm kimyasal kullanımı. Deney için nanopure su (M 18.2 cm) kullanın. </li><li> Kullanım P-Pompa deney için azot basıncı altında düzenlenmiştir. Karşılık gelen akış oranı (ml / s) ile ilişkili önce deney için farklı basınçlarda çözücü olarak su ile pompalar edin. Deneyin başlamasından önce deiyonize su ile millifluidic reaktör ve boru durulayın. </li><li> Bakır ve 174 mg (0.95 mmol) çözündürülür (II) nitrat ve O-[2 - (3-mercaptopropionylamino) etil] 610 mg (0.122 mmol) bir in-O&#39;-metilpolietilen nanopure 28 ml su içinde tutmak glikol ve bunların bir giriş kanalı ile bağlanacak şişe </li><li> Farklı bir şişe içinde bir sodyum borohidrit çözeltisi 111 mg (2.93 mmol) ve 102 mg (2.78 mmol) 28 ml sodyum hidroksit (pH ~ 13) tutun ve ile bağlamakDiğer giriş kanalı. </li><li> Farklı akış hızları (aşağıda verilmiştir) de millifluidic reaktör içinde aynı anda her iki çözüm akış ve bir cam şişe içinde çıkışında elde edilen UCNCs toplar. Azot ile temizleyin ve bu çözelti, azot altında depolanır. </li><li> Sentezi için, oda sıcaklığında 50 mbar (6.81 ml / saat), 100 mbar (14.31 ml / saat), 200 mbar (32.7 ml / saat) ve 300 mbar (51.4 ml / saat) farklı sabit basınçlar altında pompaları çalıştırın farklı akış hızlarında UCNCs. </li></ol> Sentez prosedürü P-pompa ile millifluidic düzen kullanılarak gösterilmiştir, aynı zamanda Millifluidica gelen el millifluidic cihazı kullanılarak gerçekleştirilebilir. 2. Zaman Altın Nanopartikül Formation in situ Kinetik Çalışmaları Çözüldü <ol><li> Gerekli Kimyasallar: chloroauric asit (. HAuCl 4 3H 2 O) mezo-2 ,3-dimerkaptosüksinik asit (DMSA) ve sodyum bor hidrür &amp; # Edinme160, ve daha fazla arıtılmadan tüm kimyasal kullanımı. Deney için nanopure su (M 18.2 cm) kullanın. </li><li> Tam otomatik, nabız ücretsiz şırınga pompaları çip içerisindeki sıvıları akmaya, yüksek hassasiyet kullanın. Gerekli akış hızını optimize etmek üzere önceki deney için farklı akış hızları ile, çözücü olarak, su ile pompalar edin. </li><li> (I) 4 HAuCl standart solüsyonları hazırlayın. 3H 2 O (10 mmol, 118.2 mg/30 mi) ve NANOpure içinde bir sodyum hidroksit (pH 12), 50 mg (ii) DMSA (20 mmol, 109.2 mg/30 mi) su. </li><li> Otomatik bir pompa kullanılarak 10 ml / st &#39;lik bir sabit akış hızında millifluidic yonga iki ayrı şırınga yoluyla iki çözüm besleyin. </li><li> Çift XYZ yönde hareket erişimi vardır ve çözeltiler çip pompalandı olarak çip üzerine farklı bölgelere XAS veri toplama, bir metal kademesi kullanılarak senkrotron ışın hattına millifluidic çip. </li></ol> &lt;Süreem&gt; in situ analiz prosedürü P-pompa ile millifluidic düzen kullanılarak gösterilmiştir, aynı zamanda, elde tutulan millifluidic cihazı kullanılarak gerçekleştirilebilir. 3. Sürekli Akış Altın kataliz Bu prosedür, bir el millifluidic cihazı kullanılarak gösterilmiştir. <ol><li> Gerekli Kimya: chloroauric asit (. HAuCl 4 3H 2 O), meso-2 ,3-dimerkaptosüksinik asit (DMSA), sodyum borohidrit, 4-nitrofenol, 4-amino fenol elde edilir ve daha fazla saflaştırma olmadan tüm kimyasallar kullanın. Deney için nanopure su (M 18.2 cm) kullanın. </li><li> Katalizör hazırlanması:. HAuCl 4 standart çözeltiler hazırlayın 3H 2 O NANOpure (10 mmol, 118.2 mg/30 mi), DMSA (20 mmol, 109.2 mg/30 mi) ve NaBH4 (10 mmol, 11.34 mg/30 mi) su. </li><li> Iki ayrı şişelere HAuCl 4 ve DMSA çözeltilerin her biri 10 mi almak ve inci akış45 dakika boyunca 12 ml / st &#39;lik muntazam bir akış hızı ile elde tutulan millifluidic cihazı kullanarak çip içinde em. </li><li> Au Au (I) &#39;e düşürmek için, 15 dakika boyunca 12 ml / saat akış hızı ile çip içinde 10 mmol 4 Akış NaBH (0). </li><li> Son olarak, kataliz deneyler yapmadan önce, aynı akış hızında 30 dakika boyunca nanopure su ile çip yıkayın. </li><li> Kataliz reaksiyonu: aşağıda verilen altın katalizörü (yukarıda hazırlanmıştır) içinde 4-aminofenol (4-AP), 4-nitrofenol kimyasal dönüşüm reaksiyonu (redüksiyon) (4-NP) gerçekleştirme millifluidic kanal kaplama. </li><li> 4-nitrophenolate iyon (4-NPI) oluşturmak için 0.65 mol NaBH4 çözeltisi 3.3 ml 4-NP 9 x 10 -5 mol solüsyonu 15 ml karıştırın. </li><li> Katalitik etkinliğini değerlendirmek için 5 ml / sa &#39;lik bir sabit akış hızında çip içinde biriken altın katalizör üzerinden elde edilen solüsyonu geçirir. 250-50 dalga boyu aralığında toplanan ürünlerin UV-Vis spektrumu Analiz0 nm 4-NP dönüşüm onaylayın. </li><li> 4-NPI kalibrasyon eğrisi elde reaksiyonun katalitik aktivitesini tahmin. Kalibrasyon eğrisi farklı standart konsantrasyonlarda 4-NPI, deneysel olarak gözlemlenen emme yoğunluğunu (I) çizilerek elde edilebilir. UV-Vis absorpsiyon eğrileri için (399 nm) pik yükseklikleri emme yoğunluğunu (I) değerleri temsil ve Bira Lambert&#39;in yasaya göre, tepe yüksekliği değerinde herhangi bir değişiklik konsantrasyonu karşılık gelen değişiklik gösterecektir. Bu nedenle, kalibrasyon eğrisinden reaktifin ilk ve son konsantrasyonlarında fark bularak katalitik aktivitesini tahmin ediyoruz. Pik yüksekliği 1 ünite (Şekil 6) ise, örneğin, bu (kalibrasyon grafiği göre)% 90 katalitik bir dönüşüm karşılık gelir. </li></ol>

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CSSR Kaya hariç tüm yazarlar hiçbir rakip mali çıkarlarını olmadığını beyan ederim. C. SSR Kumar şirketin Millifluidica LLC kurucusudur.

-O&#39;-metilpolietilen glikol (MW = 5,000) [MPEG] - UCNCs polimerik kapatma maddesi O-[(3-Mercaptopropionylamino) etil 2] mevcudiyetinde sodyum borohidrit ile bakır nitrat indirgeme reaksiyonu ile oluşturulmuştur. Reaksiyon, 6.8 ml / saat, 14,3 ml / saat, 32.7 ml / st ve oluşan UCNCs on akış hızlarının etkisini incelemek için 51.4 ml / saat olarak farklı akış-oranlarda millifluidic çip reaktör içinde gerçekleştirilmiştir. Yukarıda akış oranları için, ilgili kalış süreleri 47.49, 24.44, 16.56 ...

Kimyasal sentez için lab-on-a-chip (LOC) cihazları artmış kütle ve ısı transferi, üstün reaksiyon kontrolü, yüksek verim ve daha güvenli bir çalışma ortamı 1. açısından önemli bir avantaj göstermiştir. Bu cihazlar geniş yonga tabanlı akışkansal ve nonchip bazlı akışkan cihazlara ayrılabilir. Çip tabanlı akışkansal arasında, Mikroakiskan iyi incelenmiş ve konu literatürde 2-5 iyi kaplı. Nonchip tabanlı LOC sistemleri tubular reaktörler 6 kullanın. Geleneksel olarak, mikroakışkan sistemleri tam kontrol ve geometrik milimetre altı ölçeğe kısıtlı sıvıların manipülasyonu için kullanılır. Son zamanlarda (genişlik veya derinlik ya da kanalların iki ya da boyutu en az bir milimetre) 7-9 milimetre ölçeğinde kanallarında sıvıların manipülasyonu için kullanılabilecek çipli millifluidics, kavramını var. Ayrıca, millifluidic cips WHI imal etmek nispeten kolaydırle akış oranları ve reaktiflerin manipülasyon üzerinde benzer bir kontrol sunuyor. Bu yongalar, aynı zamanda daha dar bir boyut dağılımına sahip nanopartiküller kontrollü sentezinin ölçek büyütme için olanak sağlar, ve böylece, daha az kalma süreleri oluşturma, daha yüksek akış hızlarında çalıştırılabilir. Örnek olarak, biz son zamanlarda ultra-küçük bakır nanoklastırlaar sentezini gösterdi ve yerinde X-ışını soğurma spektroskopisi kullanarak yanı sıra TEM onları karakterize var. Bakır nanoklastırlar 7 istikrarlı koloitlerin oluşumu için çok etkili iki dişli PEG&#39;lenmiş stabilize edici madde, MPEG, kullanımı ile kombine millifluidic kanal içinde küçük kalma süreleri elde etmek için yeteneği. Kimyasal ve nanomalzemelerin sentezine ek olarak, millifluidics nedeniyle prob alanında yüksek satış hacmi ve konsantrasyonu, daha genel ve verimli bir zaman çözümlü bir kinetik çalışmalar için ve aynı zamanda achie olan sentetik bir platforma, sunabilirakışkan sistemleri 7,10 daha gürültü oranı daha iyi sinyal ves. Biz 5 msn 11 gibi küçük bir zaman çözünürlüğü ile in situ XAS kullanılarak çözeltiden altın Nano büyümesinin zaman çözüldü analizi için bir örnek olarak millifluidic çip kullanımını göstermektedir. Ayrıca, kataliz uygulamaları için bugüne kadar geliştirilen mikro reaktörlerin çoğunluğu silikon 12,13 dayanmaktadır. Oluşturulan küçük birimlere ek olarak kendi pahalı üretim büyük ölçekli üretim için onları uygun hale getirir. Nanocatalysts ile kanal kaplanması için iki genel yöntem - kimyasal ve fiziksel, genellikle silikon kaplama işlemleri olarak adlandırılan, rağbet 14,15 şu anda. Mikro pahalı imalat ek olarak, kanalların tıkanması mikro reaktör kataliz büyük ölçekli üretim için uygun olabilir hale getirir. Microreactors mikro sürekli akış geçiş işlemleri earli heterojen kataliz için kullanılmış olsa da16-18 er, boyut kontrol yeteneği ve sürekli akış kanalları içinde gömülü altın nano yapılı katalizörler morfolojisi önce keşfedilmeyi değildi. Biz son zamanlarda, Au katalizörü ile millifluidic kanalları kaplanması için bir teknoloji geliştirdik nano morfolojisi ve boyutları (Şekil 5) 11 kontrollü olan, endüstriyel önemli kimyasal reaksiyonlar katalizini yürütülmesi için. Bir örnek olarak millifluidic kanalları içinde kaplanmış altın nano yapılı ile katalize 4-aminofenol, 4-nitrofenol dönüşüm göstermiştir. Tek bir millifluidic reaktör çip 50-60 ml / saat, 7 yüksek verimlilik ve kimyasalların kontrollü sentezinin akış oranlarını üretebilir olduğu göz önüne alındığında, ya sürekli akış işletim veya paralel işleme ile mümkündür. Millifluidics yukarıda tarif kaç örnekle, teklif olanakları yararlanmak için, biz de göstermek kullanıcı dostu birtaşınabilir ve var millifluidic cihaz gibi millifluidic cips, manifoldlar, akış kontrol, pompa ve elektrik bağlantıları gibi tüm gerekli bileşenleri entegre. Böyle bir millifluidic cihazı, Şekil 7&#39;de gösterildiği gibi, hemen işletme Millifluidica LLC (edinilebilir <a href="http://www.millifluidica.com" target="_blank">www.millifluidica.com</a> ). Makale ayrıca nanomalzemelerin, tepkime mekanizmaları ve sürekli akış kataliz zaman çözümlü analizi kontrol sentezi için, aşağıda tarif edildiği gibi, elde tutulan cihazı kullanılarak millifluidic protokolleri sağlar.

<table><tbody><tr><td>Copper (II) nitrate hydrate</td><td>Sigma-Aldrich</td><td>13778-31-9</td><td>99.999% pure</td></tr><tr><td>O-[2-(3-mercaptopropionylamino)ethyl]-O&amp;prime;-methylpolyethylene glycol</td><td>Sigma-Aldrich</td><td>401916-61-8</td><td>MW=5,000</td></tr><tr><td>HAuCl&lt;sub&gt;4&lt;/sub&gt;&lt;sup&gt;.&lt;/sup&gt;3H&lt;sub&gt;2&lt;/sub&gt;O (Chloroauric acid)</td><td>Sigma-Aldrich</td><td>27988-77-8</td><td>99.999% pure</td></tr><tr><td>meso-2,3-dimercaptosuccinic acid (DMSA)</td><td>Sigma-Aldrich</td><td>304-55-2</td><td>~98% pure</td></tr><tr><td>4-Nitrophenol</td><td>Sigma-Aldrich</td><td>100-02-7</td><td>spectrophotometric grade</td></tr><tr><td>4-Aminophenol</td><td>Sigma-Aldrich</td><td>123-30-8</td><td>&gt;99% pure (HPLC grade)</td></tr><tr><td>Sodium borohydride</td><td>Sigma-Aldrich</td><td>16940-66-2</td><td>98% pure</td></tr><tr><td>Sodium hydroxide pellets</td><td>Sigma-Aldrich</td><td>1310-73-2</td><td>99.99% pure</td></tr><tr><td colspan="4">&lt;strong&gt;EQUIPMENT&lt;/strong&gt;</td></tr><tr><td>Millifluidic Chips</td><td>Microplumbers Microsciences LLC</td><td>SDC-01</td><td>Made from polyester terephthalate polymer</td></tr><tr><td>Pressure Pump</td><td>Mitos P-Pump, Dolomite</td><td>3200016</td><td></td></tr><tr><td>Automated Syringe Pump</td><td>Cetoni Automation and Microsystems, GmbH</td><td></td><td>Syringe pump neMESYS</td></tr><tr><td>UV-3600 UV-VIS-NIR Spectrophotometer</td><td>Shimadzu</td><td></td><td></td></tr><tr><td>Hand-held Millifluidic Device</td><td>Millifluidica</td><td>SCMD-1008</td><td>&lt;strong&gt;Figure 7&lt;/strong&gt;</td></tr></tbody></table>

millifluidics for chemical synthesis and time-resolved mechanistic studies

Kimyasal sentez ve zaman çözümlü bir mekanik çalışmalar için millifluidic cihazlarını kullanan işlemler üç örnek alınarak açıklanmıştır. İlk olarak, çok küçük bakır nanoklastırlaar sentezi tarif edilmektedir. İkinci örnek yerinde X-ışını soğurma spektroskopi kullanarak altın nanoparçacık oluşumunu analiz kimyasal reaksiyonların zaman çözülmesi kinetik soruşturma için kendi yarar sağlar. Nihai örneğin nano yapılı katalizör ile kaplanmış kanal iç millifluidic reaksiyonlar sürekli akış kataliz gösterir.

Iyi dağıtılmış ve dar bir boyut dağılımı ile muntazam boyutlu bakır nanoklastırlar millifluidic yonga setup (Şekil 1a) kullanılarak elde edildi. Sentezi için kullanılan farklı akış hızları kümelerin boyutu üzerinde önemli bir etkiye sahip değildi. Bununla birlikte, akış hızındaki artış ile, boyut dağılımının daralması gözlenebilir bir gelişme yoktur. En iyi dar bir boyut dağılımına sahip UCNCs 32,7 ml / saatlik bir akış hızı ile elde edildi. 32,7 ml / saat akış hızı ile ...

Watch this Scientific Journal Video about Millifluidics for Chemical Synthesis and Time-resolved Mechanistic Studies at JoVE.com

Millifluidics for Chemical Synthesis and Time-resolved Mechanistic Studies

Millifluidic Nanomalzemelere aygıtları, tepkime mekanizmaları ve sürekli akış kataliz zaman çözümlü bir analiz olarak kontrol edilen sentez için kullanılmaktadır.

Kimyasal Sentez ve Zaman-çözülmesi Mekanistiksel Çalışmaları Millifluidics

Procedures utilizing millifluidic devices for chemical synthesis and time-resolved mechanistic studies are described by taking three examples. In the ...

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11.1K Görüntüleme.  Louisiana State University.  Millifluidic Nanomalzemelere aygıtları, tepkime mekanizmaları ve sürekli akış kataliz zaman çözümlü bir analiz olarak kontrol edilen sentez için kullanılmaktadır. 

Video: Millifluidics for Chemical Synthesis and Time-resolved Mechanistic Studies

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 ...

Biyomühendislik

Real-time Monitoring of Reactions Performed Using Continuous-flow Processing: The Preparation of 3-Acetylcoumarin as an Example

By using inline monitoring, it is possible to optimize reactions performed using continuous-flow processing in a simple and rapid way. It is also possible to ensure consistent product quality over time using this technique. We here show how to interface a commercially available flow unit with a Raman spectrometer. The Raman flow cell is placed after the back-pressure regulator, meaning that it can be operated at atmospheric pressure. In addition, the fact that the product stream passes through a length of tubing before entering the flow cell means that the material is at RT. It is important that the spectra are acquired under isothermal conditions since Raman signal intensity is temperature dependent. Having assembled the apparatus, we then show how to monitor a chemical reaction, the piperidine-catalyzed synthesis of 3-acetylcoumarin from salicylaldehyde and ethyl acetoacetate being used as an example. The reaction can be performed over a range of flow rates and temperatures, the in-situ monitoring tool being used to optimize conditions simply and easily.

Real-time monitoring allows for fast optimization of reactions performed using continuous-flow processing. Here the preparation of 3-acetylcoumarin is used as an example. The apparatus for performing in-situ Raman monitoring is described, as are the steps required to optimize the reaction.

Sürekli akış İşleme kullanma Gerçekleştirilen Reaksiyonları Gerçek zamanlı izleme: Bir Örnek olarak 3-Acetylcoumarin Hazırlanması

By using inline monitoring, it is possible to optimize reactions performed using continuous-flow processing in a simple and rapid way. It is also possible ...

Kimya

Microfluidic Mixers for Studying Protein Folding

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 for this is that folding takes place over a wide range of timescales, from nanoseconds to seconds or longer, depending on the protein1. Conventional stopped-flow mixers have allowed measurement of folding kinetics starting at about 1 ms. We have recently developed a microfluidic mixer that dilutes denaturant ~100-fold in ~8 &mu;s2. Unlike a stopped-flow mixer, this mixer operates in the laminar flow regime in which turbulence does not occur. The absence of turbulence allows precise numeric simulation of all flows within the mixer with excellent agreement to experiment3-4.
Laminar flow is achieved for Reynolds numbers Re &le;100. For aqueous solutions, this requires micron scale geometries. We use a hard substrate, such as silicon or fused silica, to make channels 5-10 &mu;m wide and 10 &mu;m deep (See Figure 1). The smallest dimensions, at the entrance to the mixing region, are on the order of 1 &mu;m in size. The chip is sealed with a thin glass or fused silica coverslip for optical access. Typical total linear flow rates are ~1 m/s, yielding Re~10, but the protein consumption is only ~0.5 nL/s or 1.8 &mu;L/hr. Protein concentration depends on the detection method: For tryptophan fluorescence the typical concentration is 100 &mu;M (for 1 Trp/protein) and for FRET the typical concentration is ~100 nM.
The folding process is initiated by rapid dilution of denaturant from 6 M to 0.06 M guanidine hydrochloride. The protein in high denaturant flows down a central channel and is met on either side at the mixing region by buffer without denaturant moving ~100 times faster (see Figure 2). This geometry causes rapid constriction of the protein flow into a narrow jet ~100 nm wide. Diffusion of the light denaturant molecules is very rapid, while diffusion of the heavy protein molecules is much slower, diffusing less than 1 &mu;m in 1 ms. The difference in diffusion constant of the denaturant and the protein results in rapid dilution of the denaturant from the protein stream, reducing the effective concentration of the denaturant around the protein. The protein jet flows at a constant rate down the observation channel and fluorescence of the protein during folding can be observed using a scanning confocal microscope5.

In this work we explain the fabrication and use of a microfluidic mixer capable of mixing two solutions in ~8 &mu;s. We also demonstrate the use of these mixers with spectroscopic detection using UV fluorescence and fluorescence resonance energy transfer (FRET).

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 Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography

This protocol describes fabricating microfluidic devices with low X-ray background optimized for goniometer based fixed target serial crystallography. The devices are patterned from epoxy glue using soft lithography and are suitable for in situ X-ray diffraction experiments at room temperature. The sample wells are lidded on both sides with polymeric polyimide foil windows that allow diffraction data collection with low X-ray background. This fabrication method is undemanding and inexpensive. After the sourcing of a SU-8 master wafer, all fabrication can be completed outside of a cleanroom in a typical research lab environment. The chip design and fabrication protocol utilize capillary valving to microfluidically split an aqueous reaction into defined nanoliter sized droplets. This loading mechanism avoids the sample loss from channel dead-volume and can easily be performed manually without using pumps or other equipment for fluid actuation. We describe how isolated nanoliter sized drops of protein solution can be monitored in situ by dynamic light scattering to control protein crystal nucleation and growth. After suitable crystals are grown, complete X-ray diffraction datasets can be collected using goniometer based in situ fixed target serial X-ray crystallography at room temperature. The protocol provides custom scripts to process diffraction datasets using a suite of software tools to solve and refine the protein crystal structure. This approach avoids the artefacts possibly induced during cryo-preservation or manual crystal handling in conventional crystallography experiments. We present and compare three protein structures that were solved using small crystals with dimensions of approximately 10-20 &#181;m grown in chip. By crystallizing and diffracting in situ, handling and hence mechanical disturbances of fragile crystals is minimized. The protocol details how to fabricate a custom X-ray transparent microfluidic chip suitable for in situ serial crystallography. As almost every crystal can be used for diffraction data collection, these microfluidic chips are a very efficient crystal delivery method.

Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography

This protocol describes in detail how to fabricate and operate microfluidic devices for X-ray diffraction data collection at room temperature. Additionally, it describes how to monitor protein crystallization by dynamic light scattering and how to process and analyze obtained diffraction data.

Mikrosıvısal çip Situ Crystal x-ışını kırınım ve in Situ dinamik ışık saçılma seri kristalografisi için için

This protocol describes fabricating microfluidic devices with low X-ray background optimized for goniometer based fixed target serial crystallography. The ...

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 ...

A Modular Microfluidic Technology for Systematic Studies of Colloidal Semiconductor Nanocrystals

Colloidal semiconductor nanocrystals, known as quantum dots (QDs), are a rapidly growing class of materials in commercial electronics, such as light emitting diodes (LEDs) and photovoltaics (PVs). Among this material group, inorganic/organic perovskites have demonstrated significant improvement and potential towards high-efficiency, low-cost PV fabrication due to their high charge carrier mobilities and lifetimes. Despite the opportunities for perovskite QDs in large-scale PV and LED applications, the lack of fundamental and comprehensive understanding of their growth pathways has inhibited their adaptation within continuous nanomanufacturing strategies. Traditional flask-based screening approaches are generally expensive, labor-intensive, and imprecise for effectively characterizing the broad parameter space and synthesis variety relevant to colloidal QD reactions. In this work, a fully autonomous microfluidic platform is developed to systematically study the large parameter space associated with the colloidal synthesis of nanocrystals in a continuous flow format. Through the application of a novel translating three-port flow cell and modular reactor extension units, the system may rapidly collect fluorescence and absorption spectra across reactor lengths ranging 3 - 196 cm. The adjustable reactor length not only decouples the residence time from the velocity-dependent mass transfer, it also substantially improves the sampling rates and chemical consumption due to the characterization of 40 unique spectra within a single equilibrated system. Sample rates may reach up to 30,000 unique spectra per day, and the conditions cover 4 orders of magnitude in residence times ranging 100 ms - 17 min. Further applications of this system would substantially improve the rate and precision of the material discovery and screening in future studies. Detailed within this report are the system materials and assembly protocols with a general description of the automated sampling software and offline data processing.

Detailed herein are the operation and assembly protocols of a modular microfluidic screening platform for the systematic characterization of colloidal semiconductor nanocrystal syntheses. Through fully adjustable system arrangements, highly efficient spectra collection may be carried out across 4 orders of magnitude reaction time scales within a mass transfer-controlled sampling space.

Kolloidal yarı iletken Nanocrystals sistematik çalışmalar için modüler bir mikrosıvısal teknolojisi

Colloidal semiconductor nanocrystals, known as quantum dots (QDs), are a rapidly growing class of materials in commercial electronics, such as light ...

Mühendislik

A Femtoliter Droplet Array for Massively Parallel Protein Synthesis from Single DNA Molecules

Advances in spatial resolution and detection sensitivity of scientific instrumentation make it possible to apply small reactors for biological and chemical research. To meet the demand for high-performance microreactors, we developed a femtoliter droplet array (FemDA) device and exemplified its application in massively parallel cell-free protein synthesis (CFPS) reactions. Over one million uniform droplets were readily generated within a finger-sized area using a two-step oil-sealing protocol. Every droplet was anchored in a femtoliter microchamber composed of a hydrophilic bottom and a hydrophobic sidewall. The hybrid hydrophilic-in-hydrophobic structure and the dedicated sealing oils and surfactants are crucial for stably retaining the femtoliter aqueous solution in the femtoliter space without evaporation loss. The femtoliter configuration and the simple structure of the FemDA device allowed minimal reagent consumption. The uniform dimension of the droplet reactors made large-scale quantitative and time-course measurements convincing and reliable. The FemDA technology correlated the protein yield of the CFPS reaction with the number of DNA molecules in each droplet. We streamlined the procedures about the microfabrication of the device, the formation of the femtoliter droplets, and the acquisition and analysis of the microscopic image data. The detailed protocol with the optimized low running cost makes the FemDA technology accessible to everyone who has standard cleanroom facilities and a conventional fluorescence microscope in their own place.

The overall goal of the protocol is to prepare over one million ordered, uniform, stable, and biocompatible femtoliter droplets on a 1 cm2 planar substrate that can be used for cell-free protein synthesis.

Tek DNA Moleküllerinden Kitlesel Paralel Protein Sentezi için Femtolitre Damlacık Dizisi

Advances in spatial resolution and detection sensitivity of scientific instrumentation make it possible to apply small reactors for biological and ...

Use of Dual Optical Tweezers and Microfluidics for Single-Molecule Studies

Visual biochemistry is a powerful technique for observing the stochastic properties of single enzymes or enzyme complexes that are obscured in the averaging that takes place in bulk-phase studies. To achieve visualization, dual optical tweezers, where one trap is fixed and the other is mobile, are focused into one channel of a multi-stream microfluidic chamber positioned on the stage of an inverted fluorescence microscope. The optical tweezers trap single molecules of fluorescently labeled DNA and fluid flow through the chamber and past the trapped beads, stretches the DNA to B-form (under minimal force, i.e., 0 pN) with the nucleic acid being observed as a white string against a black background. DNA molecules are moved from one stream to the next, by translating the stage perpendicular to the flow to enable the initiation of reactions in a controlled manner. To achieve success, microfluidic devices with optically clear channels are mated to glass syringes held in place in a syringe pump. Optimal results use connectors permanently bonded to the flow cell, tubing that is mechanically rigid and chemically resistant, and which is connected to switching valves that eliminate bubbles that prohibit laminar flow.

Visual, single-molecule biochemistry studied through microfluidic chambers is greatly facilitated using glass barrel, gas-tight syringes, stable connections of tubing to flow cells, and elimination of bubbles by placing switching valves between the syringes and tubing. The protocol describes dual optical traps that enable visualization of DNA transactions and intermolecular interactions.

Tek Moleküllü Çalışmalarda Çift Optik Cımbız ve Mikroakışkan Kullanımı

Visual biochemistry is a powerful technique for observing the stochastic properties of single enzymes or enzyme complexes that are obscured in the ...

Biyokimya

A Multilayer Microfluidic Platform for the Conduction of Prolonged Cell-Free Gene Expression

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic regulatory circuits. The analysis of synthetic genetic regulatory networks in vivo is time consuming and suffers from a lack of environmental control, with exogenous synthetic components interacting with host processes resulting in undesired behavior. To overcome these issues, cell-free characterization of novel circuitry is becoming more prevalent. In vitro transcription and translation (IVTT) mixtures allow the regulation of the experimental environment and can be optimized for each unique system. The protocols presented here detail the fabrication of a multilayer microfluidic device that can be utilized to sustain IVTT reactions for prolonged durations. In contrast to batch reactions, where resources are depleted over time and (by-) products accumulate, the use of microfluidic devices allows the replenishment of resources as well as the removal of reaction products. In this manner, the cellular environment is emulated by maintaining an out-of-equilibrium environment in which the dynamic behavior of gene circuits can be investigated over extended periods of time. To fully exploit the multilayer microfluidic device, hardware and software have been integrated to automate the IVTT reactions. By combining IVTT reactions with the microfluidic platform presented here, it becomes possible to comprehensively analyze complex network behaviors, furthering our understanding of the mechanisms that regulate cellular processes.

The fabrication process of a PDMS-based, multilayer, microfluidic device that allows in vitro transcription and translation (IVTT) reactions to be performed over prolonged periods is described. Furthermore, a comprehensive overview of the hardware and software required to automate and maintain these reactions for prolonged durations is provided.

Uzun Süreli Hücresiz Gen Ekspresyonunun İletimi için Çok Katmanlı Mikroakışkan Platform

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

Yüksek Verimli Mikroakışkan Cihaz Kullanarak Dinamik ÇevreSel Koşulların Üretilmesi

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...