Diese Arbeit wurde von den National Institutes of Health (NIH) grant R01 EB008268 unterstützt.

Ein. Chipfertigungstechnologie 12 <ol><li> Machen Sie zwei Plaques aus Zeonex 690R Pellets: verteilen 8-9 Gramm Zeonex Pellets gleichmäßig in der Mitte einer Metallplatte, heizen auf der beheizten Presse bei 198 ° C für 5 min, und wenden Sie dann langsam den Druck bis 2.500 psi für weitere 5 min. Um diesen Schritt abzuschließen, haben wir eine Carver Heißpresse. </li><li> Prägen das mikrofluidischen Kanal in der Plaque mit einem Epoxy-Form. Einzelheiten zur Herstellung der Form an anderer Stelle 12 (Kanal Design in 2b) beschrieben: legte eine Plaque auf den Epoxid-Form; vorzuwärmen sie auf der beheizten Presse bei 157 ° C für 10 min und dann gelten Druck langsam auf 1000 psi für weitere 10 min . Entfernen Sie die Plaque von der Form von Hand oder mit einer Zange, bevor es abkühlt. (Hinweis: Tragen thermische Handschuhe während dieser Schritt.) </li><li> Die Bohrungen in der geprägten Chip am Einlass, Abfall-Port und dem Ausgang des Mikrofluidkanals. Wir verwendeten eine 1,25 mm DurchmesserEter Bohrer, um die drei Löcher machen. </li><li> Bond das geprägte Chip mit anderen Plaque (oben): Waschen Sie die Chips mit IPA, RNAse Auswärts-und VE-Wasser in der Reihenfolge; der Luft trocknen und wärmen sie auf der beheizten Presse bei 131 ° C für 10 min und dann bei 350 psi für eine andere 10 min. </li><li> Bringen Nanoports am Einlass, Abfall und den Auslassöffnungen separat mit JB Weld Epoxy, Heilung für 15 bis 24 Stunden nach den Anweisungen des Herstellers. </li><li> Spülen Sie die SPE-Kanal mit 50 ul RNAse Away, mit 100 ul Nuklease freiem Wasser folgte. </li><li> Laden das SPE Kanal mit 4 ul der Pfropflösung (Methylmethacrylat mit 3% w / v Benzophenon) und dann Vernetzen des Methacrylat durch Inkubation für 10 min in einem UV-Ofen unter 365 nm UV-Wellenlänge und 2000 mJ / cm 2. Entfernen Sie die restliche Pfropfen Lösung mit Vakuum. Wir haben die Wand Vakuum. Für die besten Ergebnisse sollte eine frische Lösung verwendet werden. Es ist üblicherweise jedoch akzeptabel, zum Speichern eineshergestellten Lösung bei 4 ° C für bis zu einer Woche. </li><li> Machen den SPE-Säule Lösung (alle v / v%): 16% Ethylendimethacrylat, 24% Butylmethacrylat, 42% 1-Dodecanol, 18% Cyclohexanol und fügen 1% 2-Dimethylamino-4-(methyl-phenylamino)-phenol Als der Photoinitiator. </li><li> Nehmen Sie das gleiche Volumen von Silica-Mikrokugeln Lösung als SPE-Säule-Lösung (eine 1:1-Mischung zu machen), und vollständig trocknen bei 70 ° C über Nacht in einem Vakuumofen. Break up das Pellet durch Klopfen auf den Schlauch mit dem Deckel verschlossen. Fügen Sie die SPE-Säule Lösung in die getrocknete Kieselsäure Mikrosphären Rohr und Vortex mischen. </li><li> Legen Sie 4 ul der SPE-Lösung in den gleichen Kanal und dann vernetzen sie durch UV-Bestrahlung für 2,5 min. Dann drehen Sie den Chip über und bestrahlen für weitere 2,5 min. Das polymerisierte SPE-Säule sollte eine undurchsichtige weiße Feststoff sein. </li><li> Waschen Sie den Kanal mit 500 ul 100% Methanol zu spülen, das überschüssige Reaktanten. Dieser Waschvorgang entfernt auch intra-Polymer-porogenic Lösungsmittel, wodurch die offene Porenstruktur des SPE-Säule, wenn unter Umgebungsbedingungen getrocknet wird. </li><li> Befestigen zwei Dünnfilmerhitzer zur Unterseite des Chips mit wärmeleitfähigen Band. Die Seiten der Heizungen müssen mit dem Rand der breiten Kanäle für die Denaturierung und Annealing getrennt (siehe Abbildung 1) ausgerichtet werden. </li><li> Platz fünf Thermoelemente in den Chip über den Toten endete offenen Seite Kanäle jeder Chip (siehe Abbildung 2b). Die Thermoelemente Aufenthalt in diesen Kanälen via Presspassung. Band wurde zur weiteren beheben Sie den Speicherort von Thermoelementen. </li></ol> 2. Solid Phase Extraction Method <ol><li> Bereiten Sie 96 ul 70% Ethanol mit DEPC-behandeltem und autoklaviert Wasser (deionisiertes oder Millipore-gefiltert). </li><li> Add 4 ul 25X RNA Sichern auf 96 ul 70% Ethanol in ein Rohr, und duplizieren Sie die gleiche Mischung mit 100% Ethanol in einem separaten Röhrchen. </li><li> Bereiten 300ul von Kanal-Puffer durch Mischen der folgenden: 75 ul 6 M Guanidin Thiocyanat (GuSCN), 150 &amp;mgr; l 2-Propanol, 63 ul von Nuklease freiem Wasser und 12 ul 25X RNA Secure. </li><li> Bereiten 312 ul Lysepuffer durch Mischen: 100 ul von 6 M GuSCN, 200 &amp;mgr; l 2-Propanol und 12 ul 25X RNA Secure. </li><li> Erwärmen der 70% Ethanol, 100% Ethanol, Kanalpuffer und Lyse Puffer bei 60 ° C für 10-20 min, um die RNA Sichere aktivieren. </li><li> Um den Mikrofluidkanal äquilibrieren, führen Kanalpuffer durch den SPE-Kanal bei einer Fließgeschwindigkeit von 0,8 ml / Std. </li><li> Quick-tauen die Probe im VTM bei 37 ° C im Wasserbad, und entfernen Sie die Probe sofort aus dem Wasserbad sobald es aufgetaut ist. </li><li> Zentrifuge Probe bei 13.000 rpm für 10 min und transferieren Sie 100 ul des Überstandes in 300 ul Lysepuffer. Füllen Sie die Mischung durch Zugabe von 6 ul 1 ug / ul Träger RNA. </li><li> Vortex zu mischen, und nach Spin für5 sec, laden Sie das gesamte Lysat in einem Luer-lok 1 cc Spritze. </li><li> Ausführen durch den SPE-Kanal bei einer Fließgeschwindigkeit von 0,8 Lysat ml / Std. </li><li> Waschen Sie die SPE-Kanal mit 100 ul 70% Ethanol und 100 ul OF100% Ethanol bei 1 ml / hr gefolgt. (Es gibt eine 50 ul Totvolumen in der Spitze der Spritze, die nicht aus wird geschoben bekommen, also eigentlich nur 50 ul wird durch den Kanal gehen.) </li><li> Positionieren Sie eine leere Spritze in der 0,5 ml-Markierung und drücken Sie Luft durch den Kanal, um sie bei 1 trocknen ml / hr. </li><li> Führen 67,5 ul Nuklease-freiem Wasser durch den Kanal, um die gebundenen Nukleinsäuren bei 0,5 eluieren ml / h, und sammeln 13,5 ul aus dem nanoport am Abfall-Port. (Der Fluidverlust ist auf die 54 ul Totvolumen in der Spritzenspitze und dem SPE Kanal.) </li></ol> 3. RT-PCR <ol><li> Bereiten 36,5 ul des RT-PCR-Master-Mix in der Luft trocknen Schritt (2,12) mit den Reagenzien in einem Qiagen OneStep RT-PCR-Kits. </li><li> Legen RT-PCR Reagenz in der nanoport am Abfall-Port, und mischen sich mit RNA, die folgenden 50 ul RT-PCR-Reaktion erhalten: 13,5 ul eluierten RNA in Nuklease freiem Wasser, 4 ul RT-PCR Enzym-Mix, 10 ul Q-Lösung, 10 μl1X ein Schritt-Puffer, 2 ul 25 mM MgCl 2, 1 ul 50 uM Vorwärtsprimer 5&#39;-GAC CRA TCC TGT CAC CTC TGA C-3 &#39;, 1 ul 50 uM Rückwärtsprimer 5&#39;-AGG GCA TTY TGG ACA AAK CGT CTA-3 &#39;, 2 ul dNTP, 0,75 ul 1,0% w / v BSA, 0,73 und 5,02 ul PEG8000 ul Nuklease freiem Wasser. </li><li> Legen des RT-PCR-Mischung in die RT-Kanal durch leichtes Vakuum (verwendeten wir die Wand Vakuum) und Abdichtung des Nanoport mit einer geschlossenen Armatur. </li><li> Übernehmen 35 V Heizung 1. Halten der Reagenzien in der RT-Kanal für 30 min nach der Heizer 1 äquilibriert bei 50 ° C. Bewahren Sie die Reagenzien im RT-Kanal für 15 min nach der Heizung 1 äquilibriert bei 95 ° C. </li><li> Übernehmen 27 V Heizung 1 und 50 V am Heizgerät 2, etwa drei Minuten oder bis Heizung 1 äquilibriert warten bei 60 &amp; deg, C und Heizer 2 äquilibriert bei 95 ° C. </li><li> Schieben Sie die Reagenzien in die PCR-Kanal bei 0,5 ul / min. Es dauert etwa 20 min für die Reagenzien durch die Serpentinen Kanal fließen. </li><li> Da die Probe ermöglicht es dem Ende der Serpentinenkanal, sammeln die PCR-Produkte am Austritt unter Verwendung eines entsprechend dimensionierten Pipette und Spitze. </li></ol> 4. PCR Produkte Detektion <ol><li> Wir verwenden ein Agilent High Sensitivity DNA-Test, um die Produkte zu erkennen. So führen Sie diesen Test nehmen Sie die Agilent High Sensitivity DNA Kit aus dem Kühlschrank 30 min vor dem Test. </li><li> Schalten Sie den Bioanalyzer und öffnen Sie die 2100 Expert Software. </li><li> Legen Sie die 1 ul der PCR-Produkte in der Erprobung und folgen Sie den Agilent-Protokoll ( <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> Analysieren und die Daten aufzuzeichnen. </li></ol>

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Die Autoren haben keine konkurrierenden finanziellen Interessen erklärt.

Die diagnostische hier vorgestellte Methode demonstriert die Fähigkeit eines integrierten mikrofluidischen Chip aus Kunststoff mit Influenza A RNA aus Patientenproben mit hoher Spezifität und eine niedrige Nachweisgrenze zu verstärken 13 Wir haben diesen Chip für potenzielle point of care testing:. (A) die Temperatur und Fluidik Steuerung vereinfacht wurden, (b) der Chip ist kostengünstig und eignet sich für einen hohen Durchsatz Herstellung im Spritzgussverfahren, und (c) der Chip ist ein Einwegartikel...

Millionen von Todesfällen während der drei Influenza-Pandemien des 20. Jahrhunderts 1 berichtet. Darüber hinaus wurde die jüngste Influenza-Pandemie durch die Weltgesundheitsorganisation (WHO) zwei im Jahr 2009 erklärt, und zum 1. August 2010, 18.449 Todesfälle wurden von der WHO 3 angegeben. Diese Pandemie gezeigt erneut die hohe Belastung durch Infektionskrankheiten, und die Notwendigkeit für eine schnelle und präzise Erkennung von Influenza schnell Krankheiten Bestätigung entsprechende Reaktion des Gesundheitswesens und wirksame Behandlung 4 zu ermöglichen. Es gibt mehrere Methoden häufig zur Diagnose von Influenza verwendet werden, umfassen diese schnelle Immunoassays, direkte Fluoreszenz-Antigen-Tests (DFA) und Viruskultur Methoden. Schnelle Immuntests drastisch fehlt Empfindlichkeit 5-8, während die beiden anderen Methoden arbeitsintensiv und zeitaufwendig 9 sind. Molekulare Tests bieten mehrere Vorteile, einschließlich einer kurzen Turn-around-Zeit, hohe Sensitduktivität und höhere Spezifität. Mehrere kommerzielle Einrichtungen haben zu schnellen molekularen Tests (auch als Nukleinsäure-Tests oder NATs) arbeitet für Infektionskrankheiten, und einige haben Influenza-Tests in ihren Pipelines. Doch die meisten von ihnen verlangen, Off-Chip-Probenvorbereitung. Keiner der Clinical Laboratory Improvement Amendments (CLIA) verzichtet molekularen Tests beinhalten Probenvorbereitung in die Testkassette oder Modul. Lab-on-a-Chip-Technik spielt eine wichtige Rolle bei der Entwicklung von Point-of-Care-Prüfgeräte. Nach der Einführung des ersten PCR-Chip 1993 10 wurden zahlreiche Anstrengungen in die Entwicklung Nukleinsäure Testchips gesetzt worden. Haben jedoch nur wenige davon integrierten rohe Probenvorbereitung mit nachgeschalteter Verstärkung. Wir haben zuvor die Miniaturisierung einer Festphasenextraktion Säule (SPE) in einem Kunststoff mikrofluidischen Chip 11 demonstriert und die Entment und Optimierung eines kontinuierlichen Stroms PCR-Chip 12. Hier erweitern wir die bisherigen Arbeiten, die SPE mit RT-und PCR-Schritte integriert in einem einzigen Chip für die klinische Diagnostik und demonstrieren ihre Fähigkeit, Nukleinsäuren aus Patienten Nasen-Rachen-(NP) Tupfer und saugt verstärken.

<table border="1"><tbody><tr><td> Name des Reagenz / Material </td><td> Firma </td><td> Katalog-Nummer </td></tr><tr><td> 1-Dodecanol </td><td> Sigma-Aldrich, St. Louis, MO </td><td> 443.816-500G </td></tr><tr><td> 2,2-Dimethoxy-2-phenylacetophenon </td><td> Sigma-Aldrich, St. Louis, MO </td><td> 196.118-50G </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> Benzophenone </td><td> Sigma-Aldrich, St. Louis, MO </td><td> 239.852-50G </td></tr><tr><td> BSA </td><td> Thermo Fisher Scientific, pittsburge, PA </td><td> A7979-50ML </td></tr><tr><td> Butylmethacrylat </td><td> Sigma-Aldrich, St. Louis, MO </td><td> 235865-100 ml </td></tr><tr><td> Carrier RNA </td&gt; <td> Qiagen, Valencia, CA </td><td> 1017647 </td></tr><tr><td> Cyclohexanol </td><td> Sigma-Aldrich, St. Louis, MO </td><td> 105.899-1L </td></tr><tr><td> Ethanol </td><td> Sigma-Aldrich, St. Louis, MO </td><td> E7023 </td></tr><tr><td> Ethylendimethacrylat </td><td> Sigma-Aldrich, St. Louis, MO </td><td> 335861 </td></tr><tr><td> Ethylenglykoldimethacrylat </td><td> Sigma-Aldrich, St. Louis, MO </td><td> 335.681-100ML </td></tr><tr><td> Glasspritze 250 ul </td><td> Hamilton, Reno, NV </td><td> 81127 </td></tr><tr><td> Guanidinthiocyanat </td><td> Sigma-Aldrich, St. Louis, MO </td><td> 50981 </td></tr><tr><td> High Sensitivity DNA Kit </td><td> Agilent Technologies, Santa Clara, CA </td><td> 5067-4626 </td></tr><tr><td> Heißpresse </td><td> Carver, Wabash, IN </td><td> 4386 </td></tr><tr><td> JB Weld Epoxies </td><td> Mcmaster-Carr, Elmhurst, IL </td><td> 7605A11 </td></tr><tr><td> Luer-Lok Spritzen </td><td> BD-Medical, Franklin Lakes, NJ </td><td> 309628 </td></tr><tr><td> Magnesiumchlorid </td><td> Thermo Fisher Scientific, pittsburge, PA </td><td> AB-0359 </td></tr><tr><td> Methanol </td><td> Sigma-Aldrich, St. Louis, MO </td><td> 494437 </td></tr><tr><td> Methylmethacrylat </td><td> Sigma-Aldrich, St. Louis, MO </td><td> M55909 </td></tr><tr><td> Nanoport </td><td> Upchurch Scientific </td><td> N-333-01 </td></tr><tr><td> Nanoport Fitting </td><td> Upchurch Scientific </td><td> F-120x </td></tr><tr><td> Nuclease freies Wasser </td><td> Thermo Fisher Scientific, pittsburge, PA </td><td> PR-p1193 </td></tr><tr><td> OneStep RT-PCR Kit </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> Stromversorgung </td><td> VWR, Radnor, PA </td><td> 300V </td></tr><tr><td> RNase AWAY </td><td> Sigma-Aldrich, St. Louis, MO </td><td> 83.931-250ML </td></tr><tr><td> RNASecure </td><td> Applied Biosystems, Foster City, CA </td><td> AM7005 </td></tr><tr><td> Siliciumoxidmikrokügelchen </td><td> Polysciences, Warrington, PA </td><td> 24324-15 </td></tr><tr><td> Spritzenpumpe </td><td> Harvard Apparatus, Holliston, MA </td><td> HA2000P/10 </td></tr><tr><td> Wärmeleitende Band </td><td> McMaster-Carr, Elmhurst, IL </td><td> 6838A11 </td></tr><tr><td> Thermoelement </td><td> Omega Engineering, Stamford, CT </td><td> 5SRTC-TT-J-40 bis 36 </td></tr><tr><td> Dünnschicht-Heizungen </td><td> Minco, Minneapolis, MN </td><td> HK5166R529L12A </td></tr><tr><td> Ultraviolet Crosslinker </td><td> UPV, Upland, CA </td><td> CL-1000 </td></tr><tr><td> Zeonex </td> &lt;td&gt; Zeon Chemicals, Louisville, KY </td><td> 690R </td></tr></tbody></table>

microfluidic chip fabrication and method to detect influenza

Schnelle und effektive Diagnostik spielen eine wichtige Rolle bei der Steuerung Infektionskrankheit, die durch eine effektive Patienten-Management und Behandlung. Hier präsentieren wir einen integrierten mikrofluidischen thermoplastischen Chip mit der Fähigkeit zur Influenza-A-Virus bei Patienten Nasen-Rachen-(NP) Tupfer und saugt verstärken. Beim Laden der Patientenprobe, die Mikrofluideinheit sequentiell durchführt On-Chip Zelllyse, RNA-Reinigung und Konzentration in der Festphasenextraktion (SPE), die reverse Transkription (RT) und Polymerasekettenreaktion (PCR) in der RT-PCR-Kammern, jeweils. Endpunktbestimmung erfolgt mit einem außerhalb des Chips Bioanalyzer (Agilent Technologies, Santa Clara, CA). Für Peripheriegeräte, verwendeten wir ein einziges Spritzenpumpe, um Reagenz und Proben anzutreiben, während zwei Dünnschichtheizelemente als Wärmequellen für die RT und PCR Kammern wurden verwendet. Der Chip wurde entwickelt, um einzelne Schicht und geeignet für hohen Durchsatz, um die Fertigung zu reduzieren fabricatiZeit und Kosten. Der mikrofluidische Chip bietet eine Plattform für eine Vielzahl von Viren und Bakterien, die nur durch Änderungen im Reagenz-Design benötigt, um neue Krankheitserreger von Interesse erkennen beschränkt zu analysieren.

<html> Ein typisches Ergebnis ist in Abbildung 3 für eine Influenza A infizierten Nasen-Rachen-wash Probe gezeigt. Aufgrund der unterschiedlichen Mengen von Influenza-Virus in jeder Patientenprobe, wird die Endkonzentration des PCR-Produkts variieren. Ein gutes Ergebnis sollte geräuscharm, zwei klare ladder Gipfel (35 und 10380 bp) und ein einzelnes Produkt Peak bei der entworfenen Produkt-Größe (107 bp) für die positive Probe. Während das Produkt Peak theoretisch sollte für negative Kontrollen fe...

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

Eine integrierte Mikrofluidvorrichtung thermoplastischen Chip zur Verwendung als molekulare Diagnostik entwickelt. Der Chip führt Nukleinsäureextraktion, reverse Transkriptase und PCR. Methoden zur Herstellung und den Betrieb der Chip beschrieben.

Mikrofluidischen Chip Herstellung und Verfahren zur Influenza Detect

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

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14.8K Aufrufe.  Boston University. Eine integrierte Mikrofluidvorrichtung thermoplastischen Chip zur Verwendung als molekulare Diagnostik entwickelt. Der Chip führt Nukleinsäureextraktion, reverse Transkriptase und PCR. Methoden zur Herstellung und den Betrieb der Chip beschrieben.

Serie: 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.

Fluoreszenz-Nachweisverfahren für Mikrofluidik-Tropfen-Plattformen

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

Forschung

Biotechnik

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;

Eine mikrofluidische Chip für die vielseitige chemische Analyse von Einzelzellen

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.

Mikrofluidische Herstellung von Polymer- und Biohybridfasern mit vorgefertigter Größe und Form

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.

Eines mikrofluidischen Chips für ICPMS Probenaufgabe

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.

Bildgestützte, laserbasierte Herstellung von Mikrofluidik-Networks-Vascular abgeleitet

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.

Herstellung von dreidimensionalen Papierbasis Microfluidic Devices für Immunoassays

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.

Trockenen Film Photoresist-basierte elektrochemische mikrofluidischen Biosensor Plattform: Gerät Fertigung, On-Chip-Test Vorbereitung und Systembetrieb

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.

Herstellung und Validierung von einer Orgel-on-Chip-System mit integrierten Elektroden, direkt Transendothelial elektrischen Widerstand zu quantifizieren

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.

Skalierbare Fertigung von dehnbar, Dual Channel, mikrofluidischen Orgel Chips

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