Name: taking advantage of reduced droplet-surface interaction to optimize transport of bioanalytes in digital microfluidics
Uploaded: 2014-11-10T15:00:00.000+00:00
Duration: 7 min 57 s
Description: Watch this Scientific Journal Video about Taking Advantage of Reduced Droplet-surface Interaction to Optimize Transport of Bioanalytes in Digital Microfluidics at JoVE.com

Biz C ile yardım için mali destek için Lindback Vakfı, Dr. Alexander Sidorenko ve verimli tartışmalar ve teknik yardım için Elza Chu, ve Profesör Robert Smith&#39;e teşekkür C. elegans deneyleri.

NOT: Aşağıda açıklanan prosedürlerde, laboratuvar güvenlik kurallarına mutlaka uyulmalıdır. Özel öneme sahip yüksek gerilim (&gt; 500 V) ve taşıma kimyasallar ile ilgili güvenliğidir. Mum İs ile İletken Yüzey 1. Kaplama <ol><li> Dikdörtgenler halinde kesilmiş bakır metal (0,5 mm kalınlığında 75 x 43 mm). , Yaklaşık 30 saniye boyunca bakır dağlayıcıda batırılarak her bakır yüzeyin temizliği yaklaşık 20 saniye musluk suyu ile yıkayın ve kağıt ile kurulayın. NOT: Yöntem 1 altında kullanılıyorsa, makinenin içine sığacak şekilde 75 x 25 mm boyutları değiştirin. </li><li> (Yaklaşık 40 mikron kalınlığında) yaklaşık olarak eşit kurum kaplama elde etmek için, 30-45 saniye boyunca bakır tabaka altında yanan parafin mum süpürün. Alev içine ~ 1 cm tabakayı tutun. Kırılgan kurum yüzeyine dokunmayın. </li></ol> 2. Kaplama ile Kurum Tabakasının Korunmasına NOT: is tabakası çok kırılgan olduğunuVe korunması için kaplanabilir. İki basit alternatifleri (yöntemleri 1 ve 2) Burada önerilen, ancak daha sağlam protokoller şu anda geliştirilme aşamasındadır. <ol><li> Yöntem 1 <ol><li> Metal buharlaştırıcı veya püskürtme sistemine örnek yükleyin. Sistemin çalışma prosedürleri izlenerek, bölmeyi tahliye ve kurum katman (150-200 nm) üzerinde altın depozisyonu başlar. Cihazın oda sıcaklığına kadar soğumasını bekleyin. </li><li> Dip kaplama, kimyasal bir bölme içine 10 dakika süre ile (% 95 etanol, ACS / USP olarak% 1 h / h) 1-dodekantiol çözelti içinde metalize tabaka. Daha sonra, 60 ° ye yakın bir açı ile tutucularına, hafifçe ancak etanolün büyük damlacıklar ile yüzeyini yıkamak. Gecede, cihazlar kurumaya bırakın. </li></ol></li><li> Yöntem 2 <ol><li> Taban madde mum alevden sıcakken kimyasal bir kaput, derhal kurum ile kaplanmasından sonra, bir tarafında fluorlu bir miktar sıvı damlalarını bırakmak vesubstrat ve 90 ° yakın bir açıya tabakayı eğin. Daha damlalarını bırakmak ve onları tüm kurum yüzey üzerinde çevirelim. NOT: damlacık bir nokta üzerine düştüğünde, kurum uzakta o bölgeden yıkanmış olur. Mümkün olan fluorlu yayılmış sıvının damlacıklar kadar bildirin. </li><li> Kimyasal bir kaput içerisinde sıcak bir plaka (15 dakika boyunca 160 ° C) üzerindeki alt-tabaka pişirin. </li><li> Alt-tabaka kullanımdan önce gece boyunca oda sıcaklığında bekletin. Süresiz saklayın. </li></ol></li></ol> 3. Üst Elektrotlar Fabrikasyon (Abdelgawad ark uyarlanmıştır. 12) <ol><li> Grafik tasarım yazılımları kullanarak elektrotları çizin. Her bir elektrot 0.3 mm genişliğinde, 2 mm uzunluğunda ve elektrot arasındaki fark 0.3 mm&#39;dir. Rehber (aşağıya bakınız, konnektörüne oturana) arasındaki boşluk 2.3 mm (Şekil 1). </li><li> Monarch formatında (3.87 x 7.5 inç) bir esnek bakır laminat (kalın 35 mikron) Trim. Diğer boyutları kullanın iyazıcıyla uyumlu f. Renkli yazıcının elle besleme tepsisine laminat yükleyin. </li><li> Bakır levha üzerine baskı yaparken, dağlama sırasında baskılı desen koruyan, bakır tabaka üzerine siyah mürekkep yoğun bir tabaka sağlamak için &quot;zengin siyah&quot;, ya da &quot;kayıt siyah&quot; kullanmak için emin olun (detaylar için Abdelgawad ve ark. 12 bakınız) olun . Gecede, tamamen basılı tabaka kurumasını bekleyin. </li><li> Kimyasal bir kaput içinde, (40 ° C) bakır dağlayıcıda 50 ml ile bir beher ısınmak. Beher içinde baskılı laminat Dip ve bu, hafifçe yaklaşık 10 dakika boyunca çözelti içinde sallayın. Dağlama zaman bakır dağlayıcıları çözümüne bağlıdır. Her birkaç dakikada, korozyon kontrol ve desen sağlam olup olmadığını görmek. </li><li> Dikkatle su ile laminat yıkayın ve kimyasal kaput aseton ve etanol ile kaplama çıkarın. Bir kez daha yıkayın ve yavaşça kağıt havlu ile laminat kurulayın. </li><li> Dikkatli bir GLA için elektrotlar ile laminat takmakss slayt (75 x 25 mm kalınlığı ~ 1 mm), çift taraflı bant kullanarak. Hava cepleri kaçının. </li><li> Bant kullanarak elektrotlara perfloroalkoksi PFA bir film takın. Bu damlacık ile elektrotların yanlışlıkla temas önlemek için hizmet eden dolayı kısa devre zararlar üst elektrotlar. </li></ol> 4. (Şekil 2&#39;de devre) elektronik arabirim <ol><li> Evrensel bir devre kartına röleleri ve kapasitörler C lehim. </li><li> Elektronik devreler için bir kaynaksız breadboard üzerinde 10 röle sürücüleri kalanını birleştirin. </li><li> Tel kumanda panosunda bir kanala her röle sürücü girişi. </li><li> Dikkatlice bir bağlantı yerine (Şekil 3) üst elektrotlar oturtun. Tel Şekilde gösterildiği gibi bir üst elektrot her röle sürücü çıkışı. Elektriksel gürültüyü en aza indirmek için, röleleri teller bir çift arasında bir topraklı konnektör temas olduğunu unutmayın. NOT: konnektör t kontrol ayarlanabilir bir platform üzerinde otururüst ve alt (kurum-kaplamalı) alt tabaka arasındaki o mesafe (0.1-0.5 mm). </li><li> Daha sonra 2345, 3456, vb; 0.8 sn, actuate 1234, yani hareket (yönünde 1 elektrot kayması, aynı anda 4 elektrotlara yüksek voltaj (HV) uygulama (yaklaşık 0.8 sn) için zamanlama kontrol etmek için bir programı kullanın ., 0.8 her bir grup için sn ve daha sonra geriye doğru ters yönde çok damlacık hareket de). </li></ol> 5. Damlacık Görselleştirme ve Taşıma <ol><li> CCD kamera ile kombine 96x büyütme montaj - damlacık hareketini kaydetmek için, bir 24X oluşan görselleştirme sistemi kullanırlar. S-video kullanarak kameraya video kaydedici bağlayın. </li><li> C ihtiva eden, 4 ul damlacık Pipet kurum kaplanmış alt-tabakanın altındaki ortam elegans&#39;da. </li><li> ~ Damlacık üzerinde 0.3 mm için üst elektrotlar getirin. Damlacık daha kolay işlem için, beşinci elektrotun altında ortadan yakın olmalıdır. </li><li> Elektronik arayüzü ve yüksek gerilim (500 V RMS) açın ve hareket başlayana kadar damlacık üst elektrot mesafeyi ayarlayın. Üst elektrotlar damlacık dokunmasına izin vermeyin. </li><li> Elektrik darbeleri yanıt cihaz üzerinde başarılı damlacık transferlerin sayısını kaydederek verileri toplayın. Başarılı bir deney, en az 700 damlacık transferi ile karakterize, yani her elektrik nabız sonra bir transferi. </li><li> Damlacık 5-10 darbeleri yanıt artık hareket etmiyor kadar, sürekli veri toplamak. NOT: Yüzey aşağılamak başladığında, hareket yakın damlacık üst elektrotlar getirerek restore edilebilir. </li></ol>

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The authors declare that they have no competing financial interests.

Protokolün en önemli nokta doğrudan damlacıkları hareketli başarı ile ilişkili kurum tabakanın korumasıdır. Kurum katmanı (yukarıdaki yöntem 1) metallenmesi fabrikasyon başarı% 100&#39;e yakın verir. Bununla birlikte, maksimum işletim süresi yaklaşık 10 dakikadır; muhtemelen, damlacık fraksiyonlar metal katmandaki delikler içinden kurum ıslatma edilir. Florlu sıvı ile kurum tabakayla kaplanması kolay ve hızlı bir alternatif olduğunu ve en az kaynak gerektirir, ancak fabrikasyon yüzeylerde...

Sıvı ile çalışan cihaz boyut küçültme &quot;lab-on-a-chip&quot; platformlar gelişimi için büyük önem taşımaktadır. Bu doğrultuda, son iki yıl çeşitli uygulamalar ile, Mikroakiskan alanında önemli ilerlemeler kaydedilmiştir. 1-5 kapalı kanallarda (kanal Mikroakiskan) sıvı taşınması ile zıt, DMF elektrotlar diziler üzerinde damlacıklar yönetir. Bu tekniğin en cazip yararları biri sıvıları taşımak üzere hareket edebilen parçaların olmaması ve hareket anında elektrik sinyallerini kapatarak durdurulur. Ancak, damlacık hareket evrensel bir &quot;lab-on-a-chip&quot; platformu için kesinlikle istenmeyen bir özellik, damlacık içeriğine bağlıdır. Proteinler ve diğer analitlerinin içeren damlacıklar kımıldamaz hale, cihaz yüzeylere sopa. Muhtemelen, bu DMF uygulamaların kapsamının genişletilmesi için önemli bir sınırlama olmuştur; 6-8İstenmeyen yüzey tıkanmayı en aza indirmek için alternatifler, potansiyel olarak damlacık içeriğini etkileyebilecek damlacık ya da çevresi ile ilave kimyasal türlerin eklenmesini içerir. Daha önce, bir grup ilave katkı maddeleri (alan-DW cihazlar olmadan), DMF içinde hücreleri ve proteinlerin taşınmasını sağlamak için bir cihaz geliştirdiler. 9 bu damlacık haddeleme çıkaran bir elemanın geometrisine sahip mum is, 10 dayalı bir yüzey birleştirilmesi ile elde edilmiştir ve daha fazla damlacık yüzey etkileşimi azaltmak, damla üstünde yukarı doğru kuvvete yol açar. Bu yaklaşımda, damlacık hareket yüzey ıslaklığı ile ilişkili değildir. 11 Aşağıda tarif edilen ayrıntılı yöntemin amacı, ilave katkı maddesi olmadan proteinleri, hücreleri ve bütün organizmaları içeren damlacıkların taşıma yeteneğine sahip bir DMF cihazı üretmektir. Saha-DW cihazlar damlacık kimyager büyük ölçüde bağımsız çalışan tam kontrollü platformları için önünügelmelidir. Burada, aynı zamanda bu simülasyonlar cihazın çalışması için gerekli yüksek gerilim rağmen gösteren, damlacık boyunca gerilim düşüşü damlacık içinde bioanalytes üzerinde ihmal edilebilir etkilere işaret, uygulanan voltajın bir küçük bir bölümünü oluşturur. Aslında, Caenorhabditis elegans (C. elegans), biyolojide çalışmaların çeşitli için kullanılan bir nematod ile ön testler, gerilimler uygulandığı gibi solucanlar rahatsız yüzmek olduğunu göstermektedir.

<table><tbody><tr><td>Paraffin candle</td><td></td><td></td><td>Any paraffin candle</td></tr><tr><td>Sputtering system</td><td>Denton Vacuum, Moorestown, NJ</td><td></td><td>Sputter coater Desk V HP equipped with an Au target.&amp;nbsp;</td></tr><tr><td>1-dodecanethiol</td><td>Sigma-Aldrich</td><td>471364</td><td></td></tr><tr><td>Teflon</td><td>Dupont</td><td>AF-1600</td><td></td></tr><tr><td>Fluorinert FC-40</td><td>Sigma-Aldrich</td><td>F9755</td><td>Fluorinated liquid: Prepare Teflon-AF resin in Fluorinert FC-40, 1:100 (w/w), to create the hydrophobic coating.</td></tr><tr><td>Graphic design software -Adobe Illustrator</td><td>Adobe Systems</td><td></td><td>Other softwares might be used as well.</td></tr><tr><td>Copper laminate</td><td>Dupont</td><td>LF9110</td><td></td></tr><tr><td>Laser Printer</td><td>Xerox</td><td>Phaser 6360 or similar</td><td>Check for the compatibility with &quot;rich black&quot; or &quot;registration black&quot; (see text).</td></tr><tr><td>Copper Etchant</td><td>Transene</td><td>CE-100</td><td></td></tr><tr><td>Perfluoroalkoxy (PFA) film</td><td>McMaster-Carr</td><td>84955K22</td><td></td></tr><tr><td>Breadboard</td><td>Allied Electronics</td><td>70012450 or similar</td><td>Large enough to allow the assemble of 10 drivers.</td></tr><tr><td>Universal circuit board</td><td>Allied Electronics</td><td>70219535 or similar</td><td></td></tr><tr><td>Connector</td><td>Allied Electronics</td><td>5145154-8 or similar</td><td></td></tr><tr><td>Control board and control program (LabView software)</td><td>National Instruments</td><td>NI-6229 or similar</td><td></td></tr><tr><td>High-voltage amplifier</td><td>Trek</td><td>PZD700</td><td></td></tr><tr><td>Capacitors C and C1, 100 nF, 60 V</td><td>Allied&amp;nbsp;</td><td>8817183</td><td></td></tr><tr><td>Transistor T, NPN</td><td>Allied&amp;nbsp;</td><td>9350289</td><td></td></tr><tr><td>Diode D, 1N4007</td><td>Allied&amp;nbsp;</td><td>2660007</td><td></td></tr><tr><td>Relay&amp;nbsp;</td><td>Allied&amp;nbsp;</td><td>8862527</td><td></td></tr><tr><td>Visualization system</td><td>Edmund Optics</td><td>VZM 200i or similar</td><td>System magnification 24X &amp;#8211; 96X. It is combined with a Hitachi KP-D20B 1/2 in CCD Color Camera.</td></tr><tr><td>Recorder</td><td>Sony</td><td>GV-D1000 NTSC or similar</td><td>It is connected to the camera by an S-video cable.</td></tr><tr><td>Simulations</td><td>COMSOL Multiphysics</td><td>V. 4.4</td><td></td></tr></tbody></table>

taking advantage of reduced droplet-surface interaction to optimize transport of bioanalytes in digital microfluidics

Digital microfluidics (DMF), a technique for manipulation of droplets, is a promising alternative for the development of &#8220;lab-on-a-chip&#8221; platforms. Often, droplet motion relies on the wetting of a surface, directly associated with the application of an electric field; surface interactions, however, make motion dependent on droplet contents, limiting the breadth of applications of the technique.
Some alternatives have been presented to minimize this dependence. However, they rely on the addition of extra chemical species to the droplet or its surroundings, which could potentially interact with droplet moieties. Addressing this challenge, our group recently developed Field-DW devices to allow the transport of cells and proteins in DMF, without extra additives.
Here, the protocol for device fabrication and operation is provided, including the electronic interface for motion control. We also continue the studies with the devices, showing that multicellular, relatively large, model organisms can also be transported, arguably unaffected by the electric fields required for device operation.

Daha önce, DMF içinde proteinlerin hareketini sağlamak için alan-DW cihazları kullanılmıştır. Özellikle, sığır serumu albümini (BSA) ile damlacıklar daha önce başka yazarlar tarafından (katkı maddesi olmadan) rapor 2.000 kat daha yüksek bir konsantrasyonda taşınmış olabilir. Bu damlacık ve yüzey arasındaki azaltılmış etkileşim nedeniyle oldu; Şekil 4 (. Bakınız Freire ve ark 9 deneyler hakkında daha fazla bilgi için) floresan etiketli BSA ...

Watch this Scientific Journal Video about Taking Advantage of Reduced Droplet-surface Interaction to Optimize Transport of Bioanalytes in Digital Microfluidics at JoVE.com

Taking Advantage of Reduced Droplet-surface Interaction to Optimize Transport of Bioanalytes in Digital Microfluidics

The protocol for fabrication and operation of field dewetting devices (Field-DW) is described, as well as the preliminary studies of the effects of electric fields on droplet contents.

Azaltılmış Damlacık-yüzey etkileşiminden yararlanarak Dijital Mikroakiskan içinde Bioanalytes ulaşımı optimize etmek

Digital microfluidics (DMF), a technique for manipulation of droplets, is a promising alternative for the development of &#8220;lab-on-a-chip&#8221; ...

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7.8K Görüntüleme.  University of the Sciences. The protocol for fabrication and operation of field dewetting devices (Field-DW) is described, as well as the preliminary studies of the effects of electric fields on droplet contents.

Video: Taking Advantage of Reduced Droplet-surface Interaction to Optimize Transport of Bioanalytes in Digital Microfluidics

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

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 Pipette-Tip Based Method for Seeding Cells to Droplet Microfluidic Platforms

Amongst various microfluidic platform designs frequently used for cellular analysis, droplet-microfluidics provides a robust tool for isolating and analyzing cells at the single-cell level by eliminating the influence of external factors on the cellular microenvironment. Encapsulation of cells in droplets is dictated by the Poisson distribution as a function of the number of cells present in each droplet and the average number of cells per volume of droplet. Primary cells, especially immune cells, or clinical specimens can be scarce and loss-less encapsulation of cells remains challenging. In this paper, we present a new methodology that uses pipette-tips to load cells to droplet-based microfluidic devices without the significant loss of cells. With various cell types , we demonstrate efficient cell encapsulation in droplets that closely corresponds to the encapsulation efficiency predicted by the Poisson distribution. Our method ensures loss-less loading of cells to microfluidic platforms and can be easily adapted for downstream single cell analysis, e.g., to decode cellular interactions between different cell types.

This article presents a protocol for seeding scarce population of cells using pipette-tips to droplet microfluidic devices in order to provide higher encapsulation efficiency of cells in droplets.

Pipet Ucu yöntemine damlacık mikrosıvısal platformlar tohum hücrenin temel

Amongst various microfluidic platform designs frequently used for cellular analysis, droplet-microfluidics provides a robust tool for isolating and ...

Mühendislik

Digital Microfluidics for Automated Proteomic Processing

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and prognosis of disease. While clinical proteomic methods vary widely, a common characteristic is the need for (i) extraction of proteins from extremely heterogeneous fluids (i.e. serum, whole blood, etc.) and (ii) extensive biochemical processing prior to analysis. Here, we report a new digital microfluidics (DMF) based method integrating several processing steps used in clinical proteomics. This includes protein extraction, resolubilization, reduction, alkylation and enzymatic digestion. Digital microfluidics is a microscale fluid-handling technique in which nanoliter-microliter sized droplets are manipulated on an open surface. Droplets are positioned on top of an array of electrodes that are coated by a dielectric layer - when an electrical potential is applied to the droplet, charges accumulate on either side of the dielectric. The charges serve as electrostatic handles that can be used to control droplet position, and by biasing a sequence of electrodes in series, droplets can be made to dispense, move, merge, mix, and split on the surface. Therefore, DMF is a natural fit for carrying rapid, sequential, multistep, miniaturized automated biochemical assays. This represents a significant advance over conventional methods (relying on manual pipetting or robots), and has the potential to be a useful new tool in clinical proteomics.
Mais J. Jebrail, Vivienne N. Luk, and Steve C. C. Shih contributed equally to this work.
Sergio L. S. Freire's current address is at the University of the Sciences in Philadelphia located at 600 South 43rd Street, Philadelphia, PA 19104.

Digital Microfluidics is a technique characterized by the manipulation of discrete droplets (~nL - mL) on an array of electrodes by the application of electrical fields. It is well-suited for carrying out rapid, sequential, miniaturized automated biochemical assays. Here, we report a platform capable of automating several proteomic processing steps.

Otomatik Proteomik İşleme Dijital Mikroakiskan

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and ...

Biyoloji

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 ...

Kimya

Microfluidic Buffer Exchange for Interference-free Micro/Nanoparticle Cell Engineering

Engineering cells with active-ingredient-loaded micro/nanoparticles (NPs) is becoming an increasingly popular method to enhance native therapeutic properties, enable bio imaging and control cell phenotype. A critical yet inadequately addressed issue is the significant number of particles that remain unbound after cell labeling which cannot be readily removed by conventional centrifugation. This leads to an increase in bio imaging background noise and can impart transformative effects onto neighboring non-target cells. In this protocol, we present an inertial microfluidics-based buffer exchange strategy termed as Dean Flow Fractionation (DFF) to efficiently separate labeled cells from free NPs in a high throughput manner. The developed spiral microdevice facilitates continuous collection (&#62;90% cell recovery) of purified cells (THP-1 and MSCs) suspended in new buffer solution, while achieving &#62;95% depletion of unbound fluorescent dye or dye-loaded NPs (silica or PLGA). This single-step, size-based cell purification strategy enables high cell processing throughput (106 cells/min) and is highly useful for large-volume cell purification of micro/nanoparticle engineered cells to achieve interference-free clinical application.

This protocol describes the use of an inertial microfluidics-based buffer exchange strategy to purify micro/nanoparticle engineered cells with efficient depletion of unbound particles.

Parazit Mikro / Nanoparçacık Hücre Mühendisliği mikroakışkan Tampon Değişim

Engineering cells with active-ingredient-loaded micro/nanoparticles (NPs) is becoming an increasingly popular method to enhance native therapeutic ...

Electrowetting-based Digital Microfluidics Platform for Automated Enzyme-linked Immunosorbent Assay

Electrowetting is the effect by which the contact angle of a droplet exposed to a surface charge is modified. Electrowetting-on-dielectric (EWOD) exploits the dielectric properties of thin insulator films to enhance the charge density and hence boost the electrowetting effect. The presence of charges results in an electrically induced spreading of the droplet which permits purposeful manipulation across a hydrophobic surface. Here, we demonstrate EWOD-based protocol for sample processing and detection of four categories of antigens, using an automated surface actuation platform, via two variations of an Enzyme-Linked Immunosorbent Assay (ELISA) methods. The ELISA is performed on magnetic beads with immobilized primary antibodies which can be selected to target a specific antigen. An antibody conjugated to HRP binds to the antigen and is mixed with H2O2/Luminol for quantification of the captured pathogens. Assay completion times of between 6 and 10 min were achieved, whilst minuscule volumes of reagents were utilized.

Electrowetting-based digital microfluidic is a technique that utilizes a voltage-driven change in the apparent contact angle of a microliter-volume droplet to facilitate its manipulation. Combining this with functionalized magnetic beads enables the integration of multiple laboratory unit operations for sample preparation and identification of pathogens using Enzyme-linked Immunosorbent Assay (ELISA).

Otomatik Enzimbağlantılı İmmünosorbent Assay için Elektrowetting tabanlı Dijital Mikroakışkanplatformu

Electrowetting is the effect by which the contact angle of a droplet exposed to a surface charge is modified. Electrowetting-on-dielectric (EWOD) exploits ...

Kombinatoryal Plug Üretimi için Mikroakışkan Bir Cihazın Kurulumu

Bilayer Microfluidic Device for Combinatorial Plug Production

Droplet microfluidics is a versatile tool that allows the execution of a large number of reactions in chemically distinct nanoliter compartments. Such systems have been used to encapsulate a variety of biochemical reactions - from incubation of single cells to implementation of PCR reactions, from genomics to chemical synthesis. Coupling the microfluidic channels with regulatory valves allows control over their opening and closing, thereby enabling the rapid production of large-scale combinatorial libraries consisting of a population of droplets with unique compositions. In this paper, protocols for the fabrication and operation of a pressure-driven, PDMS-based bilayer microfluidic device that can be utilized to generate combinatorial libraries of water-in-oil emulsions called plugs are presented. By incorporating software programs and microfluidic hardware, the flow of desired fluids in the device can be controlled and manipulated to generate combinatorial plug libraries and to control the composition and quantity of constituent plug populations. These protocols will expedite the process of generating combinatorial screens, particularly to study drug response in cells from cancer patient biopsies.

Yazar Spotlight: Hesaplamalı ve Deneysel Yaklaşımları Hassas Onkolojiye Entegre Etme

The fabrication of a polydimethylsiloxane (PDMS)-based bilayer device for the production of combinatorial libraries in water-in-oil emulsions (plugs) is presented here. The necessary hardware and software required to automate plug production are detailed in the protocol, and the production of a quantitative library of fluorescent plugs is also demonstrated.

Kombinatoryal Fiş Üretimi için İki Katmanlı Mikroakışkan Cihaz

Droplet microfluidics is a versatile tool that allows the execution of a large number of reactions in chemically distinct nanoliter compartments. Such ...

Azaltılmış Damlacık-yüzey etkileşiminden yararlanarak Dijital Mikroakiskan içinde Bioanalytes ulaşımı optimize etmek

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Mikroakışkan damlacık platformları için Floresan algılama yöntemleri

ICPMS Numune Giriş için mikroakışkan Chip

Pipet Ucu yöntemine damlacık mikrosıvısal platformlar tohum hücrenin temel

Otomatik Proteomik İşleme Dijital Mikroakiskan

Tek DNA Moleküllerinden Kitlesel Paralel Protein Sentezi için Femtolitre Damlacık Dizisi

Parazit Mikro / Nanoparçacık Hücre Mühendisliği mikroakışkan Tampon Değişim

Otomatik Enzimbağlantılı İmmünosorbent Assay için Elektrowetting tabanlı Dijital Mikroakışkanplatformu

Kombinatoryal Fiş Üretimi için İki Katmanlı Mikroakışkan Cihaz

Kombinatoryal Fiş Üretimi için İki Katmanlı Mikroakışkan Cihaz

Kombinatoryal Fiş Üretimi için İki Katmanlı Mikroakışkan Cihaz