Name: a microfluidic chip for the versatile chemical analysis of single cells
Uploaded: 2013-10-15T17:00:00
Description: Watch this Scientific Journal Video about A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells at JoVE.com

Forfatterne takker Tom Robinson for korrekturlæsning af manuskriptet, C. Bärtschi og H. Benz til opførelse af den specialbyggede pres kontrolsystem. Vi vil også gerne anerkende brugen af ​​rentrumsfacilitet første og Light Microscopy Center (LMC), både på ETH Zürich. Arbejdet blev finansieret af Merck Serono og Det Europæiske Forskningsråd (ERC) under det 7. rammeprogram (ERC Starting Grant, projekt nr. 203.428, nμLIPIDs).

1.. SU-8 Master Fabrication Forbered begge master forme til kanalerne (fluidiske og kontrol, for diagrammer og dimensioner jf. figur 2) med følgende protokol, men med forskellige maske mønstre. Processen er vist i figur 3a. <ol><li> Start ved opvarmning af en 4 tommer siliciumwafer i 10 minutter ved 180 ° C. Indlæse dehydreret wafer på en spin-coater og bruge følgende protokol for spin-coating SU-8 2015: <ol><li> Spin wafer ved 100 rpm i 20 sek (åben l...

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MicroFluidics teknologi har åbnet nye og spændende muligheder for enkelt-celle analyse. I særdeleshed, har mulighed for at fælde og immobilisere celler individuelt ved microfluidic værktøjer tilladt systematiske kort-og langsigtede undersøgelser af encellede egenskaber og respons. Derudover indkapsling af celler i højfrekvente mikrodråber, genereres på en mikrochip, har gjort det muligt encellede sekretion undersøgelser, som ikke kan udføres med konventionelle cytometri enheder. Den mikrosmådråbe tilgang h...

Mange undersøgelser i fortiden har vist celle-til-celle forskelle inden en stor celle population 1-3, især signaleringsprocesser 4, eller mængden af intracellulære biomolekyler såsom proteiner 5,6, metabolitter og cofaktorer 7,8. Disse heterogeneities anses for at være fundamentalt vigtigt for celle tilpasning og evolution 9, men også spille en central rolle i fremkomsten og behandling af sygdomme såsom kræft 10-13. Derfor undersøgelser af enkelt-celle niveau er af stor interesse i biologisk og farmakologisk forskning, især hvis disse undersøgelser afslører de forskellige celle respons efter behandling med bioaktive kemiske stoffer. I de seneste år er der blevet udviklet mange analytiske platforme, der letter analyse af enkelte levende celler eller den kemiske sammensætning af celleindholdet. Mens fluorescerende cellesortering (FACS) er guld standard for meget high-throughput analyse af enkelte levende celler, kan metoden ikke være ansat til kvantificering af intracellulære eller sekreterede forbindelser. Fremkomsten af ​​mikrofluide platforme har lovet nye analytiske strategier for positionering, behandling og observation af enkeltceller. En milepæl i microfluidics blev nået med integrationen af fleksible PDMS ventiler realiseret af Quake og kollegaer 14,15. Disse ventiler er nyttige, da de kan isolere regioner på chip, fx adskille to kulturer 16. Desuden er de især anvendes til enkelt-celle analyse og dermed bidrage til at reducere analyt udvanding problemer. Effekten af denne tilgang til enkelt-celle analyse er for nylig blevet demonstreret af Hansen og kolleger, der analyserede genekspression fra hundredvis af enlige celler i parallel 17. Når målretning proteiner og metabolitter, er meget vanskeligt at analysen på grund af manglen på egnede amplification metoder, det store antal af forskellige forbindelser til stede, og deres variationer i kemisk natur. Endvidere er de fleste intracellulære biomolekyler forventes at være til stede i lavt antal kopi i størrelsesordenen nogle få titusinder 18, hvorfor den anvendte analysemetode skal have en høj følsomhed. Kraftigere assays, såsom immunoassays og enzymkoblede immunoassays (ELISA) er vanskelige at integrere i mikrofluidenheder da de kræver flere vask og inkubationstrinnene samt overfladebehandling immobilisering. På grund af disse udfordringer, er det ikke overraskende, at der er rapporteret om kun et par eksempler, hvor proteiner eller metabolitter blev kvantificeret på enkelt-celle niveau. For eksempel har undersøgelser af udskillelsen af fluorescerende forbindelser er rapporteret 19,20. For nylig blev implementering med ELISA præsenteret for analysen af udskilles (fluorescerende) proteiner fra en cellekultur (THP-1 celler) 21 og single (immune) celler 10. Målretning intracellulære proteiner, Shi et al. Udviklet en mikrofluid anordning, lettes identifikation af intracellulære proteiner til analyse af signalveje i tumorceller ved hjælp af et immunoassay 11. Imidlertid blev kun relative mængder af proteiner bestemmes og ingen enzymatisk opformering blev anvendt til at øge signal til lav tæthed proteiner. For nylig var vi i stand til at kombinere en enkelt celle fældefangst microdevice med fluorescensanalyser 8 og immunoassays 22 (figur 1). Celler passivt fanget i microsized hurdle strukturer, som tillader forsyning og (hurtig) udveksling af mellemstore og andre kemiske midler uden nogen bevægelse af cellerne. En ringformet ventil omkring hver fælde muliggør isolering af cellen i en meget lille mængde (&quot;den mikrokammer&quot;). Denne ventil aktiveres umiddelbart efter indførelsen af ​​en celle-lysering (hypoosmolar) puffer, hannce forhindrer intracellulære molekyler eller udskilles molekyler diffundere væk. Vigtigst er det, på grund af den lille størrelse af volumen (625 pl) store fortynding af molekylerne undgås. Da lysis og analyse udføres i på den samme position på chippen, er der ingen tab af analysander grund til transport. Chippen design her beskrevet består af 8 alternerende rækker af enten 7 eller 8 microchambers, i alt 60 microchambers. Kamrene aktiveres i rækker, således at krydskontaminering langs en linje er udelukket. Platformen kan anvendes i kombination med fluorescensanalyser samt immunologiske assays (figur 1d). For sidstnævnte har vi etableret protokoller til immobilisering af antistoffer, der er forenelige med chip produktion og montage proces. Derfor platformen åbner vejen for følsomme, pålidelige og kvantificerbare analyser på enkelt celle niveau. Indtil nu har vi brugt apparatet til analyse af intracellular og udskilles enzymer (relativ kvantificering af enzymatiske assays), intracellulære cofaktorer, proteiner og små molekyler (absolut kvantificering af endpoint assays eller ELISA). I det følgende beskriver vi processen chip fremstilling ved hjælp af flerlags blød litografi og protokoller for mønstret af antistofferne ved hjælp af microcontact trykning og overfladekemi. Derudover er nogle eksempler på chip brug og drift givet.

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			<td height="21" style="height:21px;width:344px;">REAGENTS:</td>
			<td style="width:284px;"></td>
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			<td height="43" style="height:43px;width:344px;">Name of the Reagent</td>
			<td style="width:284px;">Company</td>
			<td style="width:103px;">Catalogue Number</td>
			<td style="width:153px;">Comments (optional)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">SU-8 2015</td>
			<td style="width:284px;">MicroChem Corp. (Netwon, MA)</td>
			<td style="width:103px;">n.a.</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">1H,1H,2H,2H-Perfluorodecyl-dimethylchloro-silane</td>
			<td style="width:284px;">ABCR (Karlsruhe, Germany)</td>
			<td style="width:103px;">AB103608</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">4-Methylumbelliferyl &#946;-D-N,N&#8242;-diacetylchitobioside hydrate</td>
			<td style="width:284px;">Sigma Aldrich</td>
			<td style="width:103px;">M9763</td>
			<td style="width:153px;"></td>
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			<td height="22" style="height:22px;width:344px;">Acetone</td>
			<td style="width:284px;">Merck VWR (Darmstadt, Germany)</td>
			<td style="width:103px;">100014</td>
			<td style="width:153px;"></td>
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		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Avidin</td>
			<td style="width:284px;">AppliChem (Axon Lab AG)</td>
			<td style="width:103px;">A-2568</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">AZ 1518</td>
			<td style="width:284px;">AZ Electronic Materials (Wiesbaden, Germany)</td>
			<td style="width:103px;">n.a.</td>
			<td style="width:153px;"></td>
		</tr>
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			<td height="22" style="height:22px;width:344px;">AZ 726 developer</td>
			<td style="width:284px;">AZ Electronic Materials (Wiesbaden, Germany)</td>
			<td style="width:103px;">n.a.</td>
			<td style="width:153px;"></td>
		</tr>
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			<td height="22" style="height:22px;width:344px;">Bovine serum albumin</td>
			<td style="width:284px;">Sigma Aldrich</td>
			<td style="width:103px;">A-4503</td>
			<td style="width:153px;"></td>
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			<td height="22" style="height:22px;width:344px;">Bovine serum albumin, biotin</td>
			<td style="width:284px;">Sigma Aldrich</td>
			<td style="width:103px;">A-8549</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Cell dissociation buffer</td>
			<td style="width:284px;">Invitrogen</td>
			<td style="width:103px;">13151-014</td>
			<td style="width:153px;"></td>
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			<td height="22" style="height:22px;width:344px;">Hexamethyldisilazane (HDMS)</td>
			<td style="width:284px;">Sigma Aldrich</td>
			<td style="width:103px;">40215</td>
			<td style="width:153px;"></td>
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			<td height="22" style="height:22px;width:344px;">Hydrochloric acid</td>
			<td style="width:284px;">Fluka</td>
			<td style="width:103px;">84422</td>
			<td style="width:153px;"></td>
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			<td height="22" style="height:22px;width:344px;">Isopropanol</td>
			<td style="width:284px;">Merck VWR (Darmstadt, Germany)</td>
			<td style="width:103px;">109634</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Magnesium chloride hexahydrate</td>
			<td style="width:284px;">Fluka</td>
			<td style="width:103px;">63068</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">MR developer 600</td>
			<td style="width:284px;">Microresist technology GmbH (Berlin, Germany)</td>
			<td style="width:103px;">n.a.</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">PBS</td>
			<td style="width:284px;">Invitrogen</td>
			<td style="width:103px;">10010-031</td>
			<td style="width:153px;"></td>
		</tr>
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			<td height="22" style="height:22px;width:344px;">PLL-g-PEG grafted</td>
			<td style="width:284px;">SuSoS, (D&#252;bendorf, Switzerland)</td>
			<td style="width:103px;">n.a.</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">PLL-g-PEG grafted biotin</td>
			<td style="width:284px;">SuSoS, (D&#252;bendorf, Switzerland)</td>
			<td style="width:103px;">n.a.</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Potassium chloride</td>
			<td style="width:284px;">Fluka</td>
			<td style="width:103px;">60132</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Protein G, biotin</td>
			<td style="width:284px;">Sigma Fine Chemicals</td>
			<td style="width:103px;">41624</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Silicon wafer</td>
			<td style="width:284px;">Si-Mat (Kaufering, Germany)</td>
			<td style="width:103px;">n.a.</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Sylgard 184 Silicone Elastomer Kit (PDMS)</td>
			<td style="width:284px;">Dow Corning</td>
			<td>39100000</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Tris(hydroxymethyl)-aminomethan</td>
			<td style="width:284px;">Biorad</td>
			<td style="width:103px;">1610716</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Tween 20</td>
			<td style="width:284px;">Biorad</td>
			<td style="width:103px;">1706531</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EQUIPMENT:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:344px;">Material Name</td>
			<td style="width:284px;">Company</td>
			<td style="width:103px;">Catalogue Number</td>
			<td style="width:153px;">Comments (optional)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">0.22 &#181;m PES syringe filter</td>
			<td style="width:284px;">TRP</td>
			<td style="width:103px;">99722</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">1/1.5 mm biopsy puncher</td>
			<td style="width:284px;">Miltex, York PA</td>
			<td style="width:103px;">33-31AA/33-31A</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Cell Trics filter 20 &#181;m</td>
			<td style="width:284px;">Partec</td>
			<td style="width:103px;">04-004-2325</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Centrifuge Sigma 3-18K</td>
			<td style="width:284px;">Kuehner</td>
			<td style="width:103px;">n.a.</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Hotplate HP 160 III BM</td>
			<td style="width:284px;">Sawatec, Sax, Switzerland</td>
			<td style="width:103px;">n.a.</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">MA-6 mask aligner</td>
			<td style="width:284px;">Karl Suess</td>
			<td style="width:103px;">n.a.</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Multizoom AZ100M microscope</td>
			<td style="width:284px;">Nikon Corporation</td>
			<td style="width:103px;">n.a.</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Photomask</td>
			<td style="width:284px;">Microlitho, Essex, U.K.</td>
			<td style="width:103px;">n.a</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Plasma Cleaner PDC-32G</td>
			<td style="width:284px;">Harrick</td>
			<td style="width:103px;">n.a.</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Spin coater Modell WS-400 BZ-6NPP/LITE</td>
			<td style="width:284px;">Laurell</td>
			<td style="width:103px;">n.a.</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Spin Modules SM 180 BM</td>
			<td style="width:284px;">Sawatec, Sax, Switzerland</td>
			<td style="width:103px;">n.a.</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Step profiler Dektak XT Advanced</td>
			<td style="width:284px;">Bruker</td>
			<td style="width:103px;">n.a.</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:344px;">Syringe pump neMESYS</td>
			<td style="width:284px;">Cetoni</td>
			<td style="width:103px;">n.a.</td>
			<td style="width:153px;"></td>
		</tr>
	</tbody>
</table>

a microfluidic chip for the versatile chemical analysis of single cells

Vi præsenterer en mikrofluid anordning, der muliggør kvantitativ bestemmelse af intracellulære biomolekyler flere enlige celler i parallel. Til dette formål er cellerne passivt fanget i midten af ​​en mikrokammer. Ved aktivering af kontrol lag cellen isoleret fra den omgivende mængde i et lille kammer. Det omgivende volumen kan derefter udveksles uden at påvirke den isolerede celle. På kort åbning og lukning af kammeret kan udskiftes dog, at løsningen i kammeret inden for et par hundrede millisekunder. På grund af reversibiliteten af kamrene, kan cellerne udsættes for forskellige løsninger sekventielt i en i høj grad kontrollerbar måde, f.eks til inkubation, vask og endelig cellelysis. De tætsluttende microchambers muliggøre fastholdelse af lysatet, minimere og styre fortynding efter cellelysis. Da lysis og analyse forekomme på samme sted, er høj følsomhed bevares, fordi ingen yderligere dilution eller tab af analytterne opstår under transport. Den mikrokammer design gør derfor pålidelig og reproducerbar analyse af meget små kopi antal intracellulære molekyler (attomol, zeptomoles) frigives fra de enkelte celler. Desuden kan mange microchambers arrangeres i et array format, som tillader analyse af mange celler på en gang, eftersom passende optiske instrumenter anvendes til overvågning. Vi har allerede brugt platformen for proof-of-concept studier til at analysere intracellulære proteiner, enzymer, cofaktorer og sekundære budbringere i enten relativ eller absolut kvantificerbar måde.

Vores platform er i stand til at analysere en række intracellulære samt secernerede molekyler til stede i eller dannes af enkeltceller. Her vil vi gerne præsentere forskellige eksempler undersøgelser for at understrege de forskellige mulige analyser. Vi vil give et eksempel for et secerneret enzym (figur 5a) samt et intracellulært enzym (figur 5b) og protein (figur 5c og d). For flere eksempler, såsom cofaktorer eller små molekyler, henvises til ...

Watch this Scientific Journal Video about A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells at JoVE.com

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

I denne artikel præsenterer vi en mikrofluid chip til enkelt celle analyse. Det giver mulighed for kvantificering af intracellulære proteiner, enzymer, cofaktorer, og sekundære budbringere ved hjælp af fluorescerende eller immunassays.

En Mikrofluid Chip til Alsidig kemisk analyse af enkelte celler

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

Research

JoVE Journal

Bioengineering

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in ...

A Microfluidic-based Hydrodynamic Trap for Single Particles

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

Fluorescence detection methods for microfluidic droplet platforms

Fluorescenspåvisning metoder for mikrofluide dråbe platforme

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

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 ...

A Microfluidic Chip for ICPMS Sample Introduction

En Mikrofluid Chip for ICPMS Sample Introduktion

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma ...

The Multi-organ Chip - A Microfluidic Platform for Long-term Multi-tissue Coculture

Multi-orgel Chip - En Mikrofluid platform for langsigtet Multi-væv cokultur

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need ...

Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

Electrotaxis Studier af lungecancerceller ved hjælp af en Multichannel Dual-elektriske-felt Mikrofluid Chip

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of ...

Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device

Co-kultur af Living microbiome med Microengineered Menneskelig Intestinal Villi i en Gut-on-a-Chip mikrofluidanordning

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human ...

Designing Microfluidic Devices for Studying Cellular Responses Under Single or Coexisting Chemical/Electrical/Shear Stress Stimuli

Design mikrofluidenheder for at studere cellulære reaktioner under ét eller samtidig eksisterende Chemical / El / Shear Stress Stimuli

Microfluidic devices are capable of creating a precise and controllable cellular micro-environment of pH, temperature, salt concentration, and other ...

A Versatile Method of Patterning Proteins and Cells

Et alsidigt Metode til mønstring Proteiner og celler

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning ...