Bu araştırma, Ulusal Sağlık Enstitüleri (NIH) hibe R01 EB008268 tarafından desteklenmiştir.

1. Chip Fabrikasyon 12 <ol><li> Zeonex 690R pelet iki plaklar edin: 5 dakika süreyle 198 ° C&#39;de ısıtılmış pres üzerine bir metal plaka, ön ısıtma merkezinde eşit olarak 8-9 gram Zeonex granül, dağıtılması ve daha sonra başka bir 5 dakika boyunca yavaş yavaş 2,500 psi basınç uygulanır. Bu adımı tamamlamak için, biz bir Carver sıcak pres kullanılmıştır. </li><li> Epoksi kalıp ile plak mikroakışkan kanal Emboss. Kalıp imalatı ile ilgili ayrıntılar başka 12 (Şekil 2b kanal dizaynı) özetlenmiştir: epoksi kalıp üzerine bir plak koydu, sonra 157 de ısıtılmış basın üzerinde ön ısıtma onları ° 10 dk için C ve başka bir 10 dakika süreyle yavaş 1,000 psi basınç uygulayın . Elle kalıptan plak Kaldır veya soğuyuncaya önce bir maşa kullanarak. (Not: Bu adım sırasında termal eldiven giyin.) </li><li> Giriş, atık bağlantı noktası ve mikroakışkan kanal çıkışındaki kabartmalı çip içinde delik delin. Biz 1.25 mm çap kullanılırüç delik yapmak için matkap ucu etre. </li><li> Bond başka bir plak (üstte) ile kabartmalı çip:; diğeri için 350 psi basın 10 dakika süreyle 131 ° C&#39;de ısıtılmış basına Hava kuru ve ön ısıtma onları ve IPA, RNAse Deplasman ve sırayla deiyonize su ile cips yıkayın 10 dk. </li><li> Girişi, atık ve ayrı JB Weld Epoxy kullanarak çıkış limanlarında Nanoports takın; üreticinin talimatlarına göre 15 ila 24 saat boyunca tedavi. </li><li> 100 ul nükleaz ücretsiz su ile izlenen 50 ul RNAse takım ile SPE kanal, durulayın. </li><li> Aşılama çözeltisi (% 3 w / v benzofenon ile metil metakrilat) 4 ul SPE kanal yerleştirin ve daha sonra 365 nm dalga boyu UV ve 2.000 mJ / cm 2 UV altında bir fırın içinde 10 dakika süreyle kuluçkaya alarak metakrilat çapraz bağlar. Vakum ile rezidüel greftleme çözüm çıkarın. Bu duvar vakum kullanılır. En iyi sonuçlar için, taze bir çözüm kullanılmalıdır. Genellikle kabul edilebilir, ancak, bir depolamak içinbir haftaya kadar 4 ° C &#39;de hazırlanan çözelti. </li><li> % 16 etilen dimetakrilat,% 24 oranında bütil metakrilat,% 42 1-dodecanol,% 18 sikloheksanol,% 1 2-dimetilamino-4-(metil-fenilamino)-fenol ekleyin: SPE kolon çözeltisi (her v / v%) yapmak foto-başlatıcı olarak. </li><li> SPE kolon solüsyonu (1:1 &#39;lik bir karışım yapmak için) olarak silis mikrosferler çözelti aynı hacimde çıkar, ve tamamen ° C&#39;da gece boyunca bir vakumlu fırın içinde 70 kurutun. Kapak kapalıyken tüp dokunarak pelet kadar Break. Kuru silika mikroküre tüp ve karıştırmak için girdap içine SPE kolonundan çözüm ekleyin. </li><li> Aynı kanal içine SPE çözümü 4 ul yükleyin ve sonra 2,5 dakika UV ışını tarafından çapraz. Sonra, fazla fiş çevirmek ve başka 2,5 dakika için ışın. Polimerize SPE kolonundan bir opak beyaz katı olmalıdır. </li><li> Fazla reaktifler uzak durulama için% 100 metanol 500 ul ile kanal yıkayın. Bu yıkama da intra-polimer po kaldırırçevre koşulları ve kurutulduğunda rogenic çözücüler, böylece SPE sütun gözenekli bir yapı oluşturmaktır. </li><li> Termal olarak iletken bant ile çip altına iki ince-film ısıtıcılar takın. Isıtıcıların taraf denatürasyon ve (bkz. Şekil 1) ayrı tavlama için geniş kanal kenarı ile uyumlu hale getirilmesi gerekmektedir. </li><li> Ölü aracılığıyla çip içine yerleştirin beş termokupl her çipin açık yan kanallar (Şekil 2b bakın) sona erdi. Termokupl basın uyum yoluyla bu kanallar içinde kalmak. Bant daha termokupl yerini düzeltmek için kullanıldı. </li></ol> 2. Katı Faz Ekstraksiyonu Metodu <ol><li> DEPC işlenmiş ve otoklava su (deiyonize veya Millipore-filtre) ile% 70 etanol 96 ul hazırlayın. </li><li> 25X RNA ekleme 4 ul bir tüp içinde% 70 etanol ile ve 96 ul, güvenli ve bir ayrı tüp içinde% 100 etanol ile aynı karışım çoğaltmak için kullanılır. </li><li> 300 hazırlayınAşağıdaki karıştırma ile kanal tampon ul: 6 M guanidin tiyosiyanat (GuSCN), 2-propanol ve 150 ul, nükleaz serbest su 63 ul, ve güvenli 25X RNA&#39;nın 12 ul of 75 ul. </li><li> 6 M GuSCN, 2-propanol ve 200 ul, güvenli ve 12 ul 25X RNA, 100 ul: karıştırma ile lizis tamponu 312 ul hazırlayın. </li><li> RNA Güvenli aktive etmek için 10-20 dk için 60 ° C sıcaklıkta% 70 etanol ile ısıtılır,% 100 etanol, kanal tampon ve lizis tamponu. </li><li> Mikroakışkan kanal dengelemek için, 0.8 ml / saat &#39;lik bir akış hızında SPE kanalı ile kanalın tampon çalıştırın. </li><li> Su banyosunda 37 ° C&#39;de VTM içinde test örneği Hızlı çözülme, ve kısa sürede çözülmüş gibi su banyosundan hemen örneği kaldırmak. </li><li> 10 dakika için 13,000 rpm&#39;de santrifüje örnek, ve lizis tamponu içine 300 ul süpernatant 100 ul aktarın. 1 ug / ml &#39;den taşıyıcı RNA 6 ul ilave edilerek karışım doldurun. </li><li> Karıştırmak için Vortex, ve spin sonrası5 saniye, bir Luer-lok 1 cc şırınga içine tüm lizat yükleyin. </li><li> 0.8 &#39;lik bir akış hızında SPE kanalı ile lizat çalıştırma ml / sa. </li><li> 1 ml / saatte 100 ul of100% etanol, ardından% 70 etanol ile 100 ul ile SPE kanal, yıkayın. (Dışarı itti almazsınız şırınga ucu bir 50 ul ölü hacim var, bu yüzden aslında sadece 50 ul kanalı geçiyor.) </li><li> 0.5 ml işareti boş bir şırınga yerleştirin ve 1 kurumaya kanalından hava itmek ml / saat. </li><li> 0.5 Bağımlı nükleik asitlerin Zehir ml / saat ve atık limanda nanoport 13.5 ul toplamak kanalıyla nükleaz ücretsiz su 67.5 ul çalıştırın. (Sıvı kaybı enjektör ucu ve SPE kanal içinde 54 ul ölü hacim nedeniyle). </li></ol> 3. RT-PCR <ol><li> Bir Qiagen OneStep RT-PCR kitleri reaktifleri kullanılarak hava kuru adım (2.12) sırasında RT-PCR master miks 36.5 ul hazırlayın. </li><li> RT-PC yükleyinR atık limanda nanoport olarak reaktif ve RNA ile karışım takip eden 50 ul RT-PCR tepkimesinde elde etmek için: nükleaz serbest su, 4 ul RT-PCR enzim karışımı, 10 ul Q çözelti, 10 μl1X bir 13.5 ul yıkandı, RNA Adım tampon, 2 ul 25 mM MgCl2, 1 ul 50 uM ileri primeri 5&#39;-GAC CRA TGT TCC CAC TGA C CTC-3 &#39;, 1 ul 50 uM ters primerler 5&#39;-GCA AGG TTY TGG ACA AAK CGT-3 CTA &#39;, 2 ul dNTP, 0.75 ul% 1.0 w / v BSA, 0.73 ul PEG8000 ve 5.02 ul nükleaz serbest su. </li><li> Hafif vakum ile RT kanal (biz duvar vakum kullanılır) içine RT-PCR karışımı yükleyin ve kapalı bir montaj ile Nanoport mühür. </li><li> Isıtıcı 1 35 V uygulayın. Isıtıcı 1 50 ° C&#39;de equilibrates sonra 30 dakika için RT kanal içinde reaktiflerin tutun 95 ısıtma tertibatından 1 çıkan equilibrates 15 dakika sonra ° C&#39;de için RT kanal içinde reaktiflerin tutun </li><li> Isıtıcı 1 ve ısıtıcı 2 ila 50 V 27 V uygulayın, 60 ve de az üç dakika ya da ısıtıcı 1 equilibrates kadar bekleying; C ve ısıtıcı 2 95 equilibrates ° C </li><li> 0.5 ul / dk &#39;PCR kanal içine reaktifleri bastırın. Reaktifler serpantin kanalı akmaya için yaklaşık 20 dakika sürer. </li><li> Örnek serpantin kanalın sonuna kadar ettiği gibi, uygun boyutta bir pipet ucu kullanılarak ve çıkışındaki PCR ürünleri toplamak. </li></ol> 4. PCR Ürünleri Algılama <ol><li> Biz ürünleri tespit etmek için Agilent Yüksek Hassasiyet DNA testi kullanın. Bu test buzdolabından test etmeden önce 30 dakika Agilent Yüksek Hassasiyet DNA Kiti almak çalıştırmak için. </li><li> Bioanalyzer açın ve 2100 Uzman yazılımını açın. </li><li> Test içine PCR ürünlerinin 1 ul yükleyin ve Agilent protokolü (takip <a href="http://gcf.pbrc.edu/docs/Agilent/Agilent%20Manual.pdf" target="_blank">http://gcf.pbrc.edu/docs/Agilent/Agilent% 20Manual.pdf</a> ). </li><li> Analiz ve veri kaydedebilirsiniz. </li></ol>

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Yazarlar hiçbir rakip mali çıkarlarının ilan ettiler.

Tanı yöntemi yüksek özgüllük ve düşük algılama sınırı ile hasta örneklerinde, influenza A RNA yükseltmek için entegre bir mikroakışkan plastik çipin yeteneği göstermiştir Burada sunulan 13 Biz bakım test potansiyeli noktası için bu çip tasarlanmıştır:. (A) sıcaklık ve akışkan kontrol basitleştirilmiştir, (b) yonga düşük maliyet ve enjeksiyon kalıplama kullanılarak yüksek akış üretim için uygun olan ve (c) yonga ve böylece numune çapraz kirlenmenin azaltılması e...

Ölümlerin Milyonlarca 20. yüzyılın 1. üç influenza pandemisi sırasında bildirilmiştir. Ayrıca, en son grip salgını 2009 yılında Dünya Sağlık Örgütü (WHO) 2 tarafından ilan edildi, ve 1 Ağustos 2010 arasında, 18.449 ölüm DSÖ 3 tarafından rapor edilmiştir. Bu salgın yine bulaşıcı hastalıkların yüksek yük ve hızlı hastalığın teyidi, uygun halk sağlığı yanıtı ve etkili tedavi 4 etkinleştirmek için grip hızlı ve doğru algılanması için ihtiyaç göstermiştir. Yaygın influenza teşhisi için kullanılan birkaç yöntem vardır, bu hızlı immunoassay, direkt floresan antijen testi (DFA) ve viral kültür yöntemleri içerir. Diğer iki yöntem emek-yoğun ve 9 zaman alıcı iken Hızlı immunoassay dramatik, hassasiyet 5-8 yoksundur. Moleküler testler kısa bir geri dönüş süresine, yüksek sensit dahil birçok avantajlar sunarivity ve spesifitesi yüksek. Çeşitli ticari kuruluşlar bulaşıcı hastalıklar için hızlı moleküler testler (ayrıca nükleik asit testleri veya NAT olarak adlandırılır) doğru çalışıyor olması ve birkaç kendi hatlarında grip deneyleri var. Ancak çoğu çip dışı numune hazırlama gerektirir. Moleküler testler test kartuşu veya modülün içine numune hazırlama dahil feragat Klinik Laboratuar Geliştirme Değişiklikleri (CLIA) Yok. Lab-on-a-çip teknolojisi noktası bakım test cihazları gelişiminde önemli bir rol oynar. 1993 10 yılında ilk PCR çip girişten sonra, çeşitli girişimlere nükleik asit testi cips gelişmekte girmiştir. Ancak, bunlardan sadece birkaç aşağı amplifikasyon ile entegre ham numune hazırlama var. Biz daha önce bir plastik mikroakışkan çip 11 içine bir katı faz ekstraksiyonu sütun (SPE) ve minyatür gösterdi ve geliştirmek varsürekli bir akış PCR çip 12 Ment ve optimizasyonu. Burada, klinik teşhis için tek bir yonga içine RT ve PCR adımlarla SPE entegre ve hasta nazofaringeal (NP) bezlerden ve aspirat nükleik asitlerin yükseltmek için kapasitesini göstermek için önceki çalışmaları genişletmek.

<table border="1"><tbody><tr><td> Reaktif / Malzeme Adı </td><td> Şirket </td><td> Katalog Numarası </td></tr><tr><td> 1-dodecanol </td><td> Sigma-Aldrich, St Louis, MO </td><td> 443816-500G </td></tr><tr><td> 2,2-Dimetoksi-2-fenilasetofenon </td><td> Sigma-Aldrich, St Louis, MO </td><td> 196118-50G </td></tr><tr><td> 2100 Bioanalyzer </td><td> Agilent Technologies, Santa Clara, CA </td><td> G2943CA </td></tr><tr><td> 2-Propanol </td><td> Sigma-Aldrich, St Louis, MO </td><td> 19516 </td></tr><tr><td> Benzofenon </td><td> Sigma-Aldrich, St Louis, MO </td><td> 239852-50G </td></tr><tr><td> BSA </td><td> Thermo Fisher Scientific, pittsburge, Pensilvanya </td><td> A7979-50ML </td></tr><tr><td> Bütil metakrilat </td><td> Sigma-Aldrich, St Louis, MO </td><td> 235865-100 mL </td></tr><tr><td> Taşıyıcı RNA </td&gt; <td> Qiagen, Valencia, CA </td><td> 1017647 </td></tr><tr><td> Sikloheksanol </td><td> Sigma-Aldrich, St Louis, MO </td><td> 105899-1L </td></tr><tr><td> Etanol </td><td> Sigma-Aldrich, St Louis, MO </td><td> E7023 </td></tr><tr><td> Etilen dimetakrilat </td><td> Sigma-Aldrich, St Louis, MO </td><td> 335861 </td></tr><tr><td> Etilen glikol dimetakrilat </td><td> Sigma-Aldrich, St Louis, MO </td><td> 335681-100ML </td></tr><tr><td> Cam şırınga 250 ul </td><td> Hamilton, Reno, NV </td><td> 81127 </td></tr><tr><td> Guanidin tiyosiyanat </td><td> Sigma-Aldrich, St Louis, MO </td><td> 50981 </td></tr><tr><td> Yüksek Hassasiyet DNA Kiti </td><td> Agilent Technologies, Santa Clara, CA </td><td> 5067-4626 </td></tr><tr><td> Sıcak pres </td><td> Carver, Wabash, IN </td><td> 4386 </td></tr><tr><td> JB Weld Epoksiler </td><td> McMaster-Carr, Elmhurst, IL </td><td> 7605A11 </td></tr><tr><td> Luer-Lok şırıngalar </td><td> BD-Tıbbi, Franklin Lakes, NJ </td><td> 309628 </td></tr><tr><td> Magnezyum Klorür </td><td> Thermo Fisher Scientific, pittsburge, Pensilvanya </td><td> AB-0359 </td></tr><tr><td> Metanol </td><td> Sigma-Aldrich, St Louis, MO </td><td> 494437 </td></tr><tr><td> Metil metakrilat </td><td> Sigma-Aldrich, St Louis, MO </td><td> M55909 </td></tr><tr><td> Nanoport </td><td> Upchurch Bilimsel </td><td> N-333-01 </td></tr><tr><td> Nanoport Montaj </td><td> Upchurch Bilimsel </td><td> F-120x </td></tr><tr><td> Nükleaz ücretsiz su </td><td> Thermo Fisher Scientific, pittsburge, Pensilvanya </td><td> PR-P1193 </td></tr><tr><td> OneStep RT-PCR kiti </td><td> Qiagen, Valencia, CA </td><td> 210210 </td></tr><tr><td> PEG8000 </td><td> Sigma-Aldrich, St Louis, MO </td><td> 41009 </td></tr&gt; <tr><td> Güç kaynağı </td><td> VWR, Radnor, PA </td><td> 300V </td></tr><tr><td> Deplasman RNAse </td><td> Sigma-Aldrich, St Louis, MO </td><td> 83931-250ML </td></tr><tr><td> RNASecure </td><td> Applied Biosystems, Foster City, CA </td><td> AM7005 </td></tr><tr><td> Silika mikroküreler </td><td> Polysciences, Warrington, PA </td><td> 24324-15 </td></tr><tr><td> Şırınga pompası </td><td> Harvard Apparatus, Holliston, MA </td><td> HA2000P/10 </td></tr><tr><td> Termal İletken Bant </td><td> McMaster-Carr, Elmhurst, IL </td><td> 6838A11 </td></tr><tr><td> Isıl çift </td><td> Omega Mühendislik, Stamford, CT </td><td> 5SRTC-TT-J-40-36 </td></tr><tr><td> İnce film Isıtıcılar </td><td> Minco, Minneapolis, MN </td><td> HK5166R529L12A </td></tr><tr><td> Ultraviyole Çapraz Bağlayıcı </td><td> UPV, Upland, CA </td><td> CL-1000 </td></tr><tr><td> Zeonex </td> &lt;td&gt; Zeon Kimyasallar, Louisville, KY </td><td> 690R </td></tr></tbody></table>

microfluidic chip fabrication and method to detect influenza

Hızlı ve etkili bir teşhis etkili hasta yönetimi ve tedavi sağlayarak bulaşıcı hastalık kontrolünde önemli bir rol oynar. Burada, hastanın nazofarengeal (NP) bezlerden ve aspirat olarak influenza A virusu yükseltmek için yeteneği ile entegre bir mikroakışkan termoplastik çip sunuyoruz. Hasta örneği yükleme üzerine, mikroakışkan cihaz sırayla, on-chip hücre parçalama, RNA saflaştırılması ve katı faz ekstraksiyonu (SPE), ters transkripsiyon (RT) ve RT-PCR odalarına polimeraz zincir reaksiyonu (PCR) içindeki konsantrasyonu adımları yürütmektedir sırasıyla. Bitiş noktası algılama bir off-çip Bioanalyzer (Agilent Technologies, Santa Clara, CA) kullanılarak yapılır. İki ince film ısıtıcılar RT ve PCR odaları için ısı kaynağı olarak kullanılabilir iken çevre için, biz, reaktif ve numune sürücü tek bir şırınga pompası kullanılır. Yonga fabricati azaltmak için yüksek kapasiteli imalatı için tek tabaka ile uygun olacak şekilde tasarlanmıştırzaman ve maliyet. Mikroakışkan çip ilgi yeni patojenlerin saptanması için gerekli reaktif tasarım değişiklikleri ile sınırlıdır virüs ve bakterilerin geniş bir yelpazede, analiz için bir platform sağlar.

<html> Tipik bir sonuç, bir grip A enfekte nazofarenks yıkama örnek için Şekil 3&#39;te de gösterilmiştir. Her bir hasta örneği olarak grip virüsünün farklı miktarlarda nedeniyle, PCR ürününün nihai konsantrasyon olarak değişecektir. İyi bir sonuç düşük gürültü, iki açık merdiveni tepe (35 ve 10380 bp) ve pozitif numune için tasarlanan ürün boyutu (107 bp) tek bir ürün tepe değeri olmalıdır. Ürün pik teorik negatif kontroller için devamsızlık olması gerekirken, ...

Watch this Scientific Journal Video about Microfluidic Chip Fabrication and Method to Detect Influenza at JoVE.com

Microfluidic Chip Fabrication and Method to Detect Influenza

Entegre mikroakışkan termoplastik çip bir moleküler tanı olarak kullanılmak üzere geliştirilmiştir. Çip nükleik asit ekstraksiyon, ters transkriptaz ve PCR gerçekleştirir. Imalatı ve yonga çalıştırmak için yöntemler tarif edilmiştir.

Mikroakışkan Chip İmalat ve İnfluenza Algılama Yöntemi

Fast  and effective diagnostics play an important role in controlling infectious  disease by enabling effective patient management and treatment. Here, we ...

microfluidic-chip-fabrication-and-method-to-detect-influenza

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

14.9K Görüntüleme.  Boston University. Entegre mikroakışkan termoplastik çip bir moleküler tanı olarak kullanılmak üzere geliştirilmiştir. Çip nükleik asit ekstraksiyon, ters transkriptaz ve PCR gerçekleştirir. Imalatı ve yonga çalıştırmak için yöntemler tarif edilmiştir.

Video: Microfluidic Chip Fabrication and Method to Detect Influenza

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

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described in detail. The PLBR can be utilized for the analysis of single bacteria and microcolonies to investigate biotechnological and microbiological related questions concerning, e.g.&#160;cell growth, morphology, stress response, and metabolite or protein production on single-cell level. The device features continuous media flow enabling constant environmental conditions for perturbation studies, but in addition allows fast medium changes as well as oscillating conditions to mimic any desired environmental situation. To fabricate the single use devices, a silicon wafer containing sub micrometer sized SU-8 structures served as the replication mold for rapid polydimethylsiloxane casting. Chips were cut, assembled, connected, and set up onto a high resolution and fully automated microscope suited for time-lapse imaging, a powerful tool for spatio-temporal cell analysis. Here, the biotechnological platform organism Corynebacterium glutamicum&#160;was seeded into the PLBR and cell growth and intracellular fluorescence were followed over several hours unraveling time dependent population heterogeneity on single-cell level, not possible with conventional analysis methods such as flow cytometry. Besides insights into device fabrication, furthermore, the preparation of the preculture, loading, trapping of bacteria, and the PLBR cultivation of single cells and colonies is demonstrated. These devices will add a new dimension in microbiological research to analyze time dependent phenomena of single bacteria under tight environmental control. Due to the simple and relatively short fabrication process the technology can be easily adapted at any microfluidics lab and simply tailored towards specific needs.

In this protocol the fabrication, setup and basic operation of a microfluidic picoliter bioreactor (PLBR) for single-cell analysis of prokaryotic microorganisms is introduced. Industrially relevant microorganisms were analyzed as proof of principle allowing insights into growth rate, morphology, and phenotypic heterogeneity over certain time periods, hardly possible with conventional methods.

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described ...

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this purpose, the cells are passively trapped in the middle of a microchamber. Upon activation of the control layer, the cell is isolated from the surrounding volume in a small chamber. The surrounding volume can then be exchanged without affecting the isolated cell. However, upon short opening and closing of the chamber, the solution in the chamber can be replaced within a few hundred milliseconds. Due to the reversibility of the chambers, the cells can be exposed to different solutions sequentially in a highly controllable fashion, e.g. for incubation, washing, and finally, cell lysis. The tightly sealed microchambers enable the retention of the lysate, minimize and control the dilution after cell lysis. Since lysis and analysis occur at the same location, high sensitivity is retained because no further dilution or loss of the analytes occurs during transport. The microchamber design therefore enables the reliable and reproducible analysis of very small copy numbers of intracellular molecules (attomoles, zeptomoles) released from individual cells. Furthermore, many microchambers can be arranged in an array format, allowing the analysis of many cells at once, given that suitable optical instruments are used for monitoring. We have already used the platform for proof-of-concept studies to analyze intracellular proteins, enzymes, cofactors and second messengers in either relative or absolute quantifiable manner.

In this article we present a microfluidic chip for single cell analysis. It allows the quantification of intracellular proteins, enzymes, cofactors, and second messengers by means of fluorescent assays or immunoassays.&#160;

Tek Hücrelerinin yönlü Kimyasal Analiz için mikroakışkan Chip

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this ...

Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned Size and Shape

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and used to dictate the shape as well as the diameter of a core stream.&#160;Grooves in the top and bottom of a microfluidic channel were designed to direct the sheath fluid and shape the core fluid.&#160;By matching the viscosity and hydrophilicity of the sheath and core fluids, the interfacial effects are minimized and complex fluid shapes can be formed.&#160;Controlling the relative flow rates of the sheath and core fluids determines the cross-sectional area of the core fluid.&#160;Fibers have been produced with sizes ranging from 300 nm to ~1 mm, and fiber cross-sections can be round, flat, square, or complex as in the case with double anchor fibers. Polymerization of the core fluid downstream from the shaping region solidifies the fibers.&#160;Photoinitiated click chemistries are well suited for rapid polymerization of the core fluid by irradiation with ultraviolet light.&#160;Fibers with a wide variety of shapes have been produced from a list of polymers including liquid crystals, poly(methylmethacrylate), thiol-ene and thiol-yne resins, polyethylene glycol, and hydrogel derivatives. Minimal shear during the shaping process and mild polymerization conditions also makes the fabrication process well suited for encapsulation of cells and other biological components.

Two adjacent fluids passing through a grooved microfluidic channel can be directed to form a sheath around a prepolymer core; thereby&#160;determining both shape and cross-section. Photoinitiated polymerization, such as thiol click chemistry, is well suited for rapidly solidifying the core fluid into a microfiber with predetermined size and shape.

Önceden Tasarlanmış Boyut ve Şekle Sahip Polimerik ve Biyohidred Elyafların Mikroakışkan İmalatı

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and ...

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 ...

Image-guided, Laser-based Fabrication of Vascular-derived Microfluidic Networks

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic networks embedded in PEGDA hydrogels. Here, we describe the creation of virtual masks that allow for image-guided laser control; the photopolymerization of a micromolded PEGDA hydrogel, suitable for microfluidic network fabrication and pressure head-driven flow; the setup and use of a commercially available laser scanning confocal microscope paired with a femtosecond pulsed Ti:S laser to induce hydrogel degradation; and the imaging of fabricated microfluidic networks using fluorescent species and confocal microscopy. Much of the protocol is focused on the proper setup and implementation of the microscope software and microscope macro, as these are crucial steps in using a commercial microscope for microfluidic fabrication purposes that contain a number of intricacies. The image-guided component of this technique allows for the implementation of 3D image stacks or user-generated 3D models, thereby allowing for creative microfluidic design and for the fabrication of complex microfluidic systems of virtually any configuration. With an expected impact in tissue engineering, the methods outlined in this protocol could aid in the fabrication of advanced biomimetic microtissue constructs for organ- and human-on-a-chip devices. By mimicking the complex architecture, tortuosity, size, and density of in vivo vasculature, essential biological transport processes can be replicated in these constructs, leading to more accurate in vitro modeling of drug pharmacokinetics and disease.

This protocol outlines the implementation of image-guided, laser-based hydrogel degradation to fabricate vascular-derived, biomimetic microfluidic networks embedded in poly(ethylene glycol) diacrylate (PEGDA) hydrogels. These biomimetic microfluidic systems may be useful for tissue engineering applications, generation of in vitro disease models, and fabrication of advanced "on-a-chip" devices.

Vasküler kaynaklı mikroakışkan Ağların görüntü kılavuzluğunda, Lazer tabanlı Fabrikasyon

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic ...

Fabrication of Three-dimensional Paper-based Microfluidic Devices for Immunoassays

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and directed within a layer of paper. Moreover, stacking multiple layers of patterned paper creates sophisticated three-dimensional microfluidic networks that can support the development of analytical and bioanalytical assays. Paper-based microfluidic devices are inexpensive, portable, easy to use, and require no external equipment to operate. As a result, they hold great promise as a platform for point-of-care diagnostics. In order to properly evaluate the utility and analytical performance of paper-based devices, suitable methods must be developed to ensure their manufacture is reproducible and at a scale that is appropriate for laboratory settings. In this manuscript, a method to fabricate a general device architecture that can be used for paper-based immunoassays is described. We use a form of additive manufacturing (multi-layer lamination) to prepare devices that comprise multiple layers of patterned paper and patterned adhesive. In addition to demonstrating the proper use of these three-dimensional paper-based microfluidic devices with an immunoassay for human chorionic gonadotropin (hCG), errors in the manufacturing process that may result in device failures are discussed. We expect this approach to manufacturing paper-based devices will find broad utility in the development of analytical applications designed specifically for limited-resource settings.

We detail a method to fabricate three-dimensional paper-based microfluidic devices for use in the development of immunoassays. Our approach to device assembly is a type of multilayer, additive manufacturing. We demonstrate a sandwich immunoassay to provide representative results for these types of paper-based devices.

İmmüno için üç boyutlu Paper-based mikroakışkan Cihazlar İmalat

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and ...

Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An easy-to-use and low-cost sensor platform is highly desired to measure various types of analytes (e.g., biomarkers, hormones, and drugs) quantitatively and specifically. For this reason, dry film photoresist technology - enabling cheap, facile, and high-throughput fabrication - was used to manufacture the microfluidic biosensor presented here. Depending on the bioassay used afterwards, the versatile platform is capable of detecting various types of biomolecules. For the fabrication of the device, platinum electrodes are structured on a flexible polyimide (PI) foil in the only clean-room process step. The PI foil serves as a substrate for the electrodes, which are insulated with an epoxy-based photoresist. The microfluidic channel is subsequently generated by the development and lamination of dry film photoresist (DFR) foils onto the PI wafer. By using a hydrophobic stopping barrier in the channel, the channel is separated into two specific areas: an immobilization section for the enzyme-linked assay and an electrochemical measurement cell for the amperometric signal readout.
The on-chip bioassay immobilization is performed by the adsorption of the biomolecules to the channel surface. The glucose oxidase enzyme is used as a transducer for electrochemical signal generation. In the presence of the substrate, glucose, hydrogen peroxide is produced, which is detected at the platinum working electrode. The stop-flow technique is applied to obtain signal amplification along with rapid detection. Different biomolecules can quantitatively be measured by means of the introduced microfluidic system, giving an indication of different types of diseases, or, in regard to therapeutic drug monitoring, facilitating a personalized therapy.

A microfluidic biosensor platform was designed and fabricated using low-cost dry film photoresist technology for the rapid and sensitive quantification of various analytes. This single-use system allows for the electrochemical readout of on-chip-immobilized enzyme-linked assays by means of the stop-flow technique.

Kuru zar elektrokimyasal mikrosıvısal Biyoalgılayıcı fotorezist tabanlı Platform: Cihaz imalat, On-chip tahlil hazırlama ve sistemin çalışması

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An ...

Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful research tools for studying human health and disease. To characterize the barrier function of cell layers cultured inside organ-on-chip devices, often transendothelial or transepithelial electrical resistance (TEER) is measured. To this end, electrodes are usually integrated into the chip by micromachining methods to provide more stable measurements than is achieved with manual insertion of electrodes into the inlets of the chip. However, these electrodes frequently hamper visual inspection of the studied cell layer or require expensive cleanroom processes for fabrication. To overcome these limitations, the device described here contains four easily integrated electrodes that are placed and fixed outside of the culture area, making visual inspection possible. Using these four electrodes the resistance of six measurement paths can be quantified, from which the TEER can be directly isolated, independent of the resistance of culture medium-filled microchannels. The blood-brain barrier was replicated in this device and its TEER was monitored to show the device applicability. This chip, the integrated electrodes and the TEER determination method are generally applicable in organs-on-chips, both to mimic other organs or to be incorporated into existing organ-on-chip systems.

This publication describes the fabrication of an organ-on-chip device with integrated electrodes for direct quantification of transendothelial electrical resistance (TEER). For validation, the blood-brain barrier was mimicked inside this microfluidic device and its barrier function was monitored. The presented methods for electrode integration and direct TEER quantification are generally applicable.

İmalat ve Organ-on-chip sistemin doğrudan Transendothelial elektrik direncini ölçmek için entegre elektrotlar ile doğrulama

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful ...

Scalable Fabrication of Stretchable, Dual Channel, Microfluidic Organ Chips

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human patients. Human Organ-on-a-Chip (Organ Chip) microfluidic cell culture devices, which provide an experimental in vitro platform to assess efficacy, toxicity, and pharmacokinetic (PK) profiles in humans, may be better predictors of therapeutic efficacy and safety in the clinic compared to animal studies. These devices may be used to model the function of virtually any organ type and can be fluidically linked through common endothelium-lined microchannels to perform in vitro studies on human organ-level and whole body-level physiology without having to conduct experiments on people. These Organ Chips consist of two perfused microfluidic channels separated by a permeable elastomeric membrane with organ-specific parenchymal cells on one side and microvascular endothelium on the other, which can be cyclically stretched to provide organ-specific mechanical cues (e.g., breathing motions in lung). This protocol details the fabrication of flexible, dual channel, Organ Chips through casting of parts using 3D printed molds, enabling combination of multiple casting and post-processing steps. Porous poly (dimethyl siloxane) (PDMS) membranes are cast with micrometer sized through-holes using silicon pillar arrays under compression. Fabrication and assembly of Organ Chips involves equipment and steps that can be implemented outside of a traditional cleanroom. This protocol provides researchers with access to Organ Chip technology for in vitro organ- and body-level studies in drug discovery, safety and efficacy testing, as well as mechanistic studies of fundamental biological processes.

Here, we present a protocol that describes the fabrication of stretchable, dual channel, organ chip microfluidic cell culture devices for recapitulating organ-level functionality in vitro.

Ölçeklenebilir gerilebilir, Çift kanal, mikrosıvısal Organ cips imalatı

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human ...