This work was supported by the National Institute of Biomedical Imaging and Bioengineering grant #EB003307 and by the National Science Foundation Career Award to A.F.

Microfluidic device design using CorelDraw or AutoCAD software
Principle of PDMS microvalves-based devices: The devices consist of three layers: a fluidic layer containing microchambers of various sizes, a "control layer" containing the microchannels necessary to actuate the fluidic path with microvalves, and a middle thin PDMS membrane that is bound to the control layer. At rest, due to the compliance and hydrophobicity of PDMS, the membrane seals (reversibly) against its seat, therefore the chambers remain isolated from each other without energy input. Valves can be opened by applying negative pressure (e.g., house vacuum), so the PDMS membrane deflects down and separates from the surface that supports the wall between two fluidic chambers, thus connecting the fluidic path. Valve closure can be achieved by switching the pressure setting from vacuum to atmospheric pressure.
Fluidic layer and control layer patterns were designed using CorelDraw or AutoCAD software. Masks containing these designs were printed at high resolution (8,000 to 20,000 dpi) on transparency films through commercial services (CAD/Art services, Bandon, OR) (masks not shown).
Fabrication of silicon masters using standard SU-8 photolithography
<ol>
<li>Standard SU-8 photolithography methods were used to create SU-8 "masters" (SU-8 2050, MicroChem, Newton, MA) for the microfluidic layer and the valve control layer in a cleanroom (not shown in this video). </li>
<li>To facilitate release, prior to PDMS replication the SU-8, masters were silanized by exposure to a vapor of a fluorosilane ((tridecafluoro-1,1,2,2,-tetrahydrooctyl)-1-trichlorosilane (TFOCS)), in a desiccator jar (without drying pellets) attached to a vacuum source. The desiccator chamber must be located inside a chemical fume hood owing to the corrosive nature of TFOCS vapors. </li>
<li>Place a small portion of absorbent paper towel inside the desiccator chamber. Add a drop of TFOCS to the paper towel and evacuate the air from the chamber. Apply vacuum for 1 min and turn off. Close the vacuum and allow 30 min for deposition. Keep the masters in closed containers for future use.</li>
</ol>
Replica molding of PDMS from the masters
<ol>
<li>The fluidic layer and the control layer are made by replica molding of PDMS from SU-8 masters. </li>
<li>Thoroughly mixing PDMS pre-polymer and cross-linker (10:1 wt. Ratio), de-bubble in a desiccator for 10-15 min until bubbles clear. </li>
<li>Cut silicone tubing into 1-2 cm long pieces. Choose the appropriate size of tubing according to application. We use 1.14 mm I.D. tubings here for easy connection to 1/16 inch O.D tubing later. </li>
<li>Use Duco&reg; Cement to glue tubing onto the inlet regions of the SU-8 master of the control layer. Be careful not to use too much glue, as the silicone tubing is made of the same components as PDMS, and the tubing will be embedded into the PDMS microfluidic device, creating air and fluidic tight inlets/outlets. </li>
<li>In our device, inlet regions are designed on the masks of both the fluidic and the control layers, but silicone tubing inlets are molded only into one layer (for example, control layer) of the device. To create inlets to the fluidic layer, we manually remove or puncture the few sections of the membrane that cover the inlet regions. Therefore, after alignment and assembly, all microchannels (those that carry flow as well as those that control the valves) are accessible from the top of the device so that the bottom surface is planar, enabling imaging of the device on a conventional microscope stage. </li>
<li>Carefully pour de-bubbled PDMS onto both masters, around the tubing in the control-layer master. De-bubble again in a desiccator. After de-bubbling is complete, put into 65&deg;C oven for &gt; 1 hour for curing. </li>
<li>Remove cured PDMS-covered masters from the oven. </li>
<li>Cut individual devices from the masters (each master contains three identical devices) and peel off. </li>
<li>Remove glue from the inlet regions using a needle or a pair of forceps. </li>
<li>Take the control layer PDMS into the cleanroom.</li>
</ol>
Thin PDMS Membrane Manufacturing
<ol>
<li>As shown in the device principle, the middle layer consists of a ~12 &mu;m-thick PDMS membrane. </li>
<li>Mix 10:1 wt. ratio of PDMS prepolymer/curing agent mixture with hexane (3:1 wt. ratio) by vortexing. </li>
<li>Move into a clean room. (A dust-free environment is critical for ensuring that the PDMS membranes are free of defects; dust particles may result in membranes containing holes and/or defectively bound to the replica mold.) </li>
<li>Put a silanized 3 inch -diameter wafer onto the vacuum chuck of a Solitec spinner. The wafer has to be silanized (derivatized with fluorosilane) prior to PDMS spinning to facilitate the release of PDMS from silicon surfaces. The Teflon bowl outside of the chuck was wrapped with plastic film for easy cleaning. </li>
<li>Dispense 2-3 ml of PDMS/Hexane mixture onto the wafer using an 18-Gauge syringe needle (for minimizing bubbles). </li>
<li>Set spin parameters. Spin at 7000 rpm for 30 sec, resulting in a PDMS film of ~ 12 &mu;m thickness. </li>
<li>Heat the wafer at 85&deg;C for 4 min on a hot plate to cure the PDMS film.</li>
</ol>
Multilayer PDMS device bonding and assembly
<ol>
<li>Put the control layer and the PDMS membrane into an oxygen plasma chamber. Turn on plasma for 30 sec (oxygen pressure 30 psi, flow rate 3-5 SCFH, 550W). Bring the control layer into contact with the PDMS membrane immediately (within 5 minutes) after oxygen activation. System parameters, such as oxygen pressure, flow rate, and plasma power and treatment time, are empirically configured according to different applications. </li>
<li>Wait for 5 min, and remove the control layer from the wafer along with the membrane. </li>
<li>Remove membranes on the inlets areas so that both control and fluidic layers are accessible from the top via tubing. </li>
<li>Align the control layer (with tubings as inlets) with the fluidic layer (planar) under a stereoscope. Because PDMS seals onto PDMS, no permanent bonding is required.</li>
</ol>
Computer-controlled opening and closing of PDMS microvalves by vacuum or pressure
<ol>
<li>After device alignment and assembly, insert 1/16 inch O.D. (1/32 inch I.D.) Tygon tubing into the 1.14 mm I.D. silicone inlets and connect the inlets to the pressure sources or the fluidic reservoirs. </li>
<li>For opening and closing valves, pressures are controlled by a vacuum line and an air pressure line connected through two pressure regulators to an array of miniature three-way solenoid valves. </li>
<li>The solenoid valves are connected to National Instruments data acquisition hardware controlled via Labview software. </li>
<li>Device operation and membrane deflection are visualized with a color CCD camera (SPOT RT, Diagnostic Instruments, Sterling Heights, MI).</li>
</ol>
Parallel mixing of two different color dyes in different defined nanoliter volumes
We demonstrate the operation of a parallel mixer that allows for storing and mixing precise sub-nanoliter volumes of aqueous solutions at various mixing ratios:
<ol>
<li>The fluidic layer contains two arrays of microchambers: Along array A, the size of the microchambers decreases, starting from the left, from 200 &micro;m x 400 &micro;m to 200 &micro;m x 40 &micro;m; A10 is a 500 &micro;m x 40 &micro;m chamber and is used just for fluidic connection in Array A; to the right of chamber A10 is a set of chambers symmetrically increasing in sizes. The chambers in array B are designed such that the added volume of any two adjacent chambers in different rows always equal to. A0, A0r and B10, B10r are designed as respective controls for solutions A and B without mixing. </li>
<li>The control layer has two independently-controlled sets of valves. A set of valves {V1} is used to connect two chamber arrays with their respective inlets, whereas a second set of valves {V2} is used to connect each pair of chambers in the two arrays. </li>
<li>Fill the microchambers by opening valve set {V1} to allow flow of two dye solutions to arrays A and B, respectively. Flow of solutions can be achieved either by hand or by vacuum pulling controlled with solenoid valves. If air bubbles form in the microchambers, more solution can be pushed to remove the bubbles, or the device can be left for a few minutes and bubbles will disappear due to the air permeability of PDMS. </li>
<li>Close the valve set {V1} to isolate each chamber in both arrays. </li>
<li>Open valve set {V2} to allow fluid mixing between adjacent chambers in different arrays. Mixing only takes ~1-2 min to complete for these volumes. </li>
<li>Close {V2} to push the fluid back to each fluidic chamber and chambers deform back to their original shape. Since the two fluidic arrays are designed with chambers of 11 different sizes, 11 different mixing ratios are produced in a single mixing step.</li>
</ol>
An integrated microfluidic system for computer-controlled perfusion of microfluidic cell cultures
We demonstrate a microfluidic system that is capable of the automated perfusion of multiple solutions to a single cell culture chamber. The inlets are controlled by microvalves, which can be activated in any sequence of single inlets, various combinations, or all at once. The device is capable of producing gradients or mixtures of the various solutions.
This device also consists of three layers: a fluidic layer, a control layer, and a middle thin PDMS membrane.
Alternative fabrication steps for this device:
<ol>
<li>The inlet ports for the fluidic channels and control channels are "punched" using a 1.2 mm diameter Harris Micro-Punch (Ted Pella, Inc.). Tubing is connected to the inlets by using blunted 18 gauge needles which are inserted in to the PDMS through the control layer. This allows for a denser packing of inlets than the silicone tubing. The compliance of the PDMS provides a tight seal around the needles to effectively deliver fluid or pneumatic pressure. </li>
<li>As previously described, the bonding of the thin PDMS membrane to the control layer is accomplished using exposure to oxygen plasma. </li>
<li>The fluidic layer is prepared by replica molding with PDMS pre-polymer and cross-linker at a ratio of 5:1 and partially curing for 25 minutes at 60&deg;C in a convection oven. At this point, the partially cured fluidic layer is still tacky, yet it can be removed from the master. </li>
<li>The fluidic layer is manually aligned to the pre-assembled control and membrane layers using a stereoscope. The assembled device is then placed on a hotplate for 5 minutes at 80&ordm;C. Next, the valve control lines are connected to the automated controller and the valves are actuated until the membrane detaches from fluidic layer at all the valve seats by applying vacuum. After "detaching" the valves, the computer controller is set to cycle the valves on and off while the device is further cured on the hotplate at 80&ordm;C for at least 1 hour. </li>
</ol>
The features of our integrated microfluidic system: The device is capable of automated perfusion of 16 different solutions to a cell culture chamber using a multiplexed valving scheme. The channel design ensures that the resistance of all the inlets is balanced. Our microvalve design isolates solutions and controls rinsing through integrated channels for the rapid removal of fluid, which limits cross-contamination. An integrated herringbone mixer can be activated to produce mixtures of different inlets. Additionally, there are four varying resistance channels that can be activated to alter the flow rate.

<ol>
	<li>Li, N., Hsu, C. H., Folch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parallel+mixing+of+photolithographically-defined+nanoliter+volumes+using+elastomeric+microvalve+arrays.">Parallel mixing of photolithographically-defined nanoliter volumes using elastomeric microvalve arrays.</a> Electrophoresis. 26 (19), 3858-3864 (2005).</li><li>Thorsen, T., Maerkl, S. J., Quake, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+large-scale+integration.">Microfluidic large-scale integration.</a> Science. 298 (5593), 580-584 (2002).</li></ol>

Main advantages of our microvalve design:
<ol>
<li>No extra energy source is required to close the fluidic path, hence the loaded device is highly portable; and</li>
<li>The device can be built by PDMS replicas from photolithographically-patterned SU-8 molds, allowing for microfabricating deep (up to 1 mm) channels with vertical sidewalls (i.e. the height of the features can be specified independently of their width) and resulting in very precise features.</li>
</ol>
Advantages of the par...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Clean silicon wafers</td>
<td>Supplies</td>
<td>Silicon Sense Inc.</td>
<td>3P0110TEST</td>
<td>3-inch diameter, P/Boron</td>
</tr>
<tr>
<td>"Master" wafers containing SU-8 patterns</td>
<td>Supplies</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Fabricated in house using standard photolithography procedures </td>
</tr>
<tr>
<td>Desiccators (2)</td>
<td>Equipment</td>
<td>VWR</td>
<td>24987-048</td>
<td>One for silanization, one for PDMS de-bubbling.</td>
</tr>
<tr>
<td>Balance</td>
<td>Equipment</td>
<td>OHAUS Corp.</td>
<td>SC6010</td>
<td>&nbsp;</td>
<tr>
<td>Oven</td>
<td>Equipment</td>
<td>Sheldon Mfg. Inc.</td>
<td>1330GM</td>
<td>&nbsp;</td>
<tr>
<td>MiniVortexer</td>
<td>Equipment</td>
<td>VWR</td>
<td>58816-121</td>
<td>&nbsp;</td>
<tr>
<td>Spinner</td>
<td>Equipment</td>
<td>Headway Research Inc.</td>
<td>PWM32</td>
<td>&nbsp;</td>
<tr>
<td>Plasma etcher</td>
<td>Equipment</td>
<td>Plasmatic Systems Inc. </td>
<td>Plasma Preen II-973</td>
<td>&nbsp;</td>
<tr>
<td>Hot Plate</td>
<td>Equipment</td>
<td>Torre Pines Scientific</td>
<td>HP30A</td>
<td>&nbsp;</td>
<tr>
<td>Stereoscope</td>
<td>Microscope</td>
<td>Nikon</td>
<td>TMZ1500</td>
<td>&nbsp;</td>
<tr>
<td>CCD camera</td>
<td>Equipment</td>
<td>Diagnostic Instruments</td>
<td>SPOT RT</td>
<td>&nbsp;</td>
<tr>
<td>Solenoid valves</td>
<td>Equipment</td>
<td>Lee Company</td>
<td>LHDA0511111H</td>
<td>&nbsp;</td>
<tr>
<td>Data acquisition board</td>
<td>Hardware</td>
<td>National Instruments</td>
<td>PCI 6025E, CB-50LP</td>
<td>&nbsp;</td>
<tr>
<td>LabView</td>
<td>Software</td>
<td>National Instruments</td>
<td>Version 8.0</td>
<td>&nbsp;</td>
<tr>
<td>Tridecafluoro-1,1,2,2,-tetrahydrooctyl)-1-trichlorosilane</td>
<td>Reagent</td>
<td>United Chemical Technologies</td>
<td>T2492</td>
<td>Silanization must be done in a chemical fume hood.</td>
</tr>
<tr>
<td>PDMS prepolymer and crosslinker</td>
<td>Reagent</td>
<td>Dow-Corning</td>
<td>Sylgard 184</td>
<td>&nbsp;</td>
<tr>
<td>Hexane</td>
<td>Reagent</td>
<td>EMD	</td>
<td>HX0295-6	</td>
<td>

</td>
</tr>
<tr>
<td>Color Dyes</td>
<td>Reagent</td>
<td>Spectrum Chemical Mfg. Corp.</td>
<td>FD&C 110, 135, 150</td>
<td>Blue #1, Yellow #5, Red #3. 
</td>
</tr>
<tr>
<td>3 ml disposable transfer pipets</td>
<td>Supplies</td>
<td>Fisher Scientific</td>
<td>13-711-20</td>
<td>&nbsp;</td>
<tr>
<td>Kimwipes</td>
<td>Supplies</td>
<td>Kimberly-Clark</td>
<td>34155</td>
<td>



</td>
</tr>
<tr>
<td>Weighing boats</td>
<td>Supplies</td>
<td>VWR </td>
<td>12577-027</td>
<td>




</td>
</tr>
<tr>
<td>Tongue depressor</td>
<td>Supplies	</td>
<td>Fisher Scientific	</td>
<td>11-700-555	</td>
<td>&nbsp;</td>
<tr>
<td>P100 dishes</td>
<td>Supplies</td>
<td>Fisher Scientific</td>
<td>08-772E</td>
<td>		</td>
</tr>
<tr>
<td>Silicone tubing (1.14 mm inner diameter (I.D.))</td>
<td>Supplies	</td>
<td>Cole-Palmer Instrument Co.</td>
<td>07625-30</td>
<td>&nbsp;</td>
<tr>
<td>Tygon tubing (O.D. 1/16 in; I.D. 1/32 in)</td>
<td>Supplies	</td>
<td>Cole-Palmer Instrument Co.	</td>
<td>06418-02</td>
<td>&nbsp;</td>
<tr>
<td>Duco Cement</td>
<td>Supplies</td>
<td>Devcon</td>
<td>6245</td>
<td>



</td>
</tr>
<tr>
<td>Razor blade</td>
<td>Tools</td>
<td>VWR</td>
<td>55411-050</td>
<td>



</td>
</tr>
<tr>
<td>Needles</td>
<td>Tools</td>
<td>Fisher Scientific</td>
<td>0053482 (25 Gauge)</td>
<td>




</td>
</tr>
<tr>
<td>#5 Forceps</td>
<td>Tools</td>
<td>Fine Science Tools</td>
<td>11251-20</td>
<td>



</td>
</tr>
<tr>
<td>50 ml centrifuge tube</td>
<td>Supplies</td>
<td>Fisher Scientific</td>
<td>05-526B</td>
<td>




</td>
</tr>
<tr>
<td>Seal wrap film</td>
<td>Supplies	</td>
<td>AEP Industries Inc.	</td>
<td>0153877</td>
<td>&nbsp;</td>
<tr>
<td>1.5 ml microcentrifuge tubes</td>
<td>Supplies</td>
<td>Fisher Scientific</td>
<td>05-406-16</td>
<td>




</td>
</tr>
<tr>
<td>15 ml centrifuge tubes	</td>
<td>Supplies	</td>
<td>BD Falcon	</td>
<td>352097</td>
<td>&nbsp;</td>
<tr>
<td>Purple nitrile power-free gloves</td>
<td>Supplies 	</td>
<td>VWR 	</td>
<td>40101-348</td>
<td>&nbsp;</td>
<tr>
<td>1.2 mm Harris biopsy punch	</td>
<td>Tools</td>
<td>Ted Pella, Inc.</td>
<td>15074	</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

microfluidic chips controlled with elastomeric microvalve arrays

Miniaturized microfluidic systems provide simple and effective solutions for low-cost point-of-care diagnostics and high-throughput biomedical assays. Robust flow control and precise fluidic volumes are two critical requirements for these applications. We have developed microfluidic chips featuring elastomeric polydimethylsiloxane (PDMS) microvalve arrays that: 1) need no extra energy source to close the fluidic path, hence the loaded device is highly portable; and 2) allow for microfabricating deep (up to 1 mm) channels with vertical sidewalls and resulting in very precise features.

The PDMS microvalves-based devices consist of three layers: a fluidic layer containing fluidic paths and microchambers of various sizes, a control layer containing the microchannels necessary to actuate the fluidic path with microvalves, and a middle thin PDMS membrane that is bound to the control layer. Fluidic layer and control layers are made by replica molding of PDMS from SU-8 photoresist masters, and the thin PDMS membrane is made by spinning PDMS at specified heights. The control layer is bonded to the thin PDMS membrane after oxygen activation of both, and then assembled with the fluidic layer. The microvalves are closed at rest and can be opened by applying negative pressure (e.g., house vacuum). Microvalve closure and opening are automated via solenoid valves controlled by computer software.

Here, we demonstrate two microvalve-based microfluidic chips for two different applications. The first chip allows for storing and mixing precise sub-nanoliter volumes of aqueous solutions at various mixing ratios. The second chip allows for computer-controlled perfusion of microfluidic cell cultures.

The devices are easy to fabricate and simple to control. Due to the biocompatibility of PDMS, these microchips could have broad applications in miniaturized diagnostic assays as well as basic cell biology studies.

Watch this Scientific Journal Video about Microfluidic Chips Controlled with Elastomeric Microvalve Arrays at JoVE.com

Microfluidic Chips Controlled with Elastomeric Microvalve Arrays

We demonstrate protocols for manufacturing and automating elastomeric polydimethylsiloxane (PDMS)-based microvalve arrays that need no extra energy to close and feature photolithographically defined precise volumes. A parallel subnanoliter-volume mixer and an integrated microfluidic perfusion system are presented.

Miniaturized microfluidic systems provide simple and effective solutions for low-cost point-of-care diagnostics and high-throughput biomedical assays.  ...

microfluidic-chips-controlled-with-elastomeric-microvalve-arrays

Research

JoVE Journal

Biology

20.9K vues.  University of Washington. Nous démontrons protocoles pour la fabrication et l'automatisation des élastomères polydiméthylsiloxane (PDMS) des tableaux basés sur microvanne qui n'ont pas besoin d'énergie supplémentaire de fermer et de fonctionnalité de photolithographie définie volumes précis. Un parallèle subnanoliter-volume du mélangeur et d'un système de perfusion intégré microfluidique sont présentés.

Vidéo: Microfluidic Chips Controlled with Elastomeric Microvalve Arrays

A Microfluidic Device with Groove Patterns for Studying Cellular Behavior

We describe a microfluidic device with microgrooved patterns for studying cellular behavior. This microfluidic platform consists of a top fluidic channel and a bottom microgrooved substrate. To fabricate the microgrooved channels, a top poly(dimethylsiloxane) (PDMS) mold containing the impression of the microfluidic channels was aligned and bonded to a microgrooved substrate. Using this device, mouse fibroblast cells were immobilized and patterned within microgrooved substrates (25, 50, 75, and 100 &mu;m wide). To study apoptosis in a microfluidic device, media containing hydrogen peroxide, Annexin V, and propidium iodide was perfused into the fluidic channel for 2 hours. We found that cells exposed to the oxidative stress became apoptotic. These apoptotic cells were confirmed by Annexin V that bound to phosphatidylserine at the outer leaflet of the plasma membrane during the apoptosis process. Using this microfluidic device with microgrooved patterns, the apoptosis process was observed in real-time and analyzed by using an inverted microscope containing an incubation chamber (37&deg;C, 5% CO2). Therefore, this microfluidic device incorporated with microgrooved substrates could be useful for studying the cellular behavior and performing high-throughput drug screening.

We describe a protocol for the fabrication of microfluidic devices that can enable cell capture and culture. In this approach patterned microstructures such as grooves within microfluidic channels are used to create low shear stress regions within which cell can dock.

We describe a microfluidic device with microgrooved patterns for studying cellular behavior.  This microfluidic platform consists of a top fluidic channel ...

A Gradient-generating Microfluidic Device for Cell Biology

 The fabrication and operation of a gradient-generating microfluidic device for studying cellular behavior is described.  A microfluidic platform is an enabling experimental tool, because it can precisely manipulate fluid flows, enable high-throughput experiments, and generate stable soluble concentration gradients.  Compared to conventional gradient generators, poly(dimethylsiloxane) (PDMS)-based  microfluidic devices can generate stable concentration gradients of growth factors with well-defined profiles.  Here, we developed simple gradient-generating microfluidic devices with three separate inlets.  Three microchannels combined into one microchannel to generate concentration gradients.  The stability and shape of growth factor gradients were confirmed by fluorescein isothyiocyanate (FITC)-dextran with a molecular weight similar to epidermal growth factor (EGF).  Using this microfluidic device, we demonstrated that fibroblasts exposed to concentration gradients of EGF migrated toward higher concentrations.  The directional orientation of cell migration and motility of migrating cells were quantitatively assessed by cell tracking analysis.  Thus, this gradient-generating microfluidic device might be useful for studying and analyzing the behavior of migrating cells.

We describe a protocol for the microfabrication of the gradient-generating microfluidic device that can generate spatial and temporal gradients in well-defined microenvironment. In this approach, the gradient-generating microfluidic device can be used to study directed cell migration, embryogenesis, wound healing, and cancer metastasis.

 The fabrication and operation of a gradient-generating microfluidic device for studying cellular behavior is described.  A microfluidic platform is an ...

Cell Capture Using a Microfluidic Device

Fabrication of the Thermoplastic Microfluidic Channels

In our lab, we have successfully isolated nucleic acids directly from microliter and submicroliter volumes of human blood, urine and stool using polymer/nanoparticle composite microscale lysis and solid phase extraction columns. The recovered samples are concentrated, small volume samples that are PCRable, without any additional cleanup. Here, we demonstrate how to fabricate thermoplastic microfluidic chips using hot embossing and heat sealing. Then, we demonstrate how to use in situ light directed surface grafting and polymerization through the sealed chip to form the composite solid phase columns. We demonstrate grafting and polymerization of a carbon nanotube/polymer composite column for bacterial cell lysis. We then show the lysis process followed by solid phase extraction of nucleic acids from the sample on chip using a silica/polymer composite column. The attached protocols contain detailed instructions on how to make both lysis and solid phase extraction columns.

Here we demonstrate how to fabricate thermoplastic microfluidic chips using hot embossing and heat sealing. Then we demonstrate how to use in situ light directed surface grafting and polymerization through the sealed chip to form the composite solid phase columns.

In our lab, we have successfully isolated nucleic acids directly from microliter and submicroliter volumes of human blood, urine and stool using ...

Microfluidic Applications for Disposable Diagnostics

In this interview, Dr. Klapperich discusses the fabrication of thermoplastic microfluidic devices and their application for development of new diagnostics.

In this interview, Dr. Klapperich discusses the fabrication of thermoplastic microfluidic devices and their application for development of new diagnostics.

In this interview, Dr. Klapperich discusses the fabrication of thermoplastic microfluidic devices and their application for development of new ...

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Phenotypes are determined by a complex series of physical (e.g. protein-protein) and functional (e.g. gene-gene or genetic) interactions (GI)1. While physical interactions can indicate which bacterial proteins are associated as complexes, they do not necessarily reveal pathway-level functional relationships1. GI screens, in which the growth of double mutants bearing two deleted or inactivated genes is measured and compared to the corresponding single mutants, can illuminate epistatic dependencies between loci and hence provide a means to query and discover novel functional relationships2. Large-scale GI maps have been reported for eukaryotic organisms like yeast3-7, but GI information remains sparse for prokaryotes8, which hinders the functional annotation of bacterial genomes. To this end, we and others have developed high-throughput quantitative bacterial GI screening methods9, 10.
Here, we present the key steps required to perform quantitative E. coli Synthetic Genetic Array (eSGA) screening procedure on a genome-scale9, using natural bacterial conjugation and homologous recombination to systemically generate and measure the fitness of large numbers of double mutants in a colony array format. Briefly, a robot is used to transfer, through conjugation, chloramphenicol (Cm) - marked mutant alleles from engineered Hfr (High frequency of recombination) 'donor strains' into an ordered array of kanamycin (Kan) - marked F- recipient strains. Typically, we use loss-of-function single mutants bearing non-essential gene deletions (e.g. the 'Keio' collection11) and essential gene hypomorphic mutations (i.e. alleles conferring reduced protein expression, stability, or activity9, 12, 13) to query the functional associations of non-essential and essential genes, respectively. After conjugation and ensuing genetic exchange mediated by homologous recombination, the resulting double mutants are selected on solid medium containing both antibiotics. After outgrowth, the plates are digitally imaged and colony sizes are quantitatively scored using an in-house automated image processing system14. GIs are revealed when the growth rate of a double mutant is either significantly better or worse than expected9. Aggravating (or negative) GIs often result between loss-of-function mutations in pairs of genes from compensatory pathways that impinge on the same essential process2. Here, the loss of a single gene is buffered, such that either single mutant is viable. However, the loss of both pathways is deleterious and results in synthetic lethality or sickness (i.e. slow growth). Conversely, alleviating (or positive) interactions can occur between genes in the same pathway or protein complex2 as the deletion of either gene alone is often sufficient to perturb the normal function of the pathway or complex such that additional perturbations do not reduce activity, and hence growth, further. Overall, systematically identifying and analyzing GI networks can provide unbiased, global maps of the functional relationships between large numbers of genes, from which pathway-level information missed by other approaches can be inferred9.

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Systematic, large-scale synthetic genetic (gene-gene or epistasis) interaction screens can be used to explore genetic redundancy and pathway cross-talk. Here, we describe a high-throughput quantitative synthetic genetic array screening technology, termed eSGA that we developed for elucidating epistatic relationships and exploring genetic interaction networks in Escherichia coli.

Phenotypes  are determined by a complex series of physical (e.g. protein-protein) and  functional (e.g. gene-gene or genetic) interactions (GI)1. While ...

Design and Use of Multiplexed Chemostat Arrays

Chemostats are continuous culture systems in which cells are grown in a tightly controlled, chemically constant environment where culture density is constrained by limiting specific nutrients.1,2 Data from chemostats are highly reproducible for the measurement of quantitative phenotypes as they provide a constant growth rate and environment at steady state. For these reasons, chemostats have become useful tools for fine-scale characterization of physiology through analysis of gene expression3-6 and other characteristics of cultures at steady-state equilibrium.7 Long-term experiments in chemostats can highlight specific trajectories that microbial populations adopt during adaptive evolution in a controlled environment. In fact, chemostats have been used for experimental evolution since their invention.8 A common result in evolution experiments is for each biological replicate to acquire a unique repertoire of mutations.9-13 This diversity suggests that there is much left to be discovered by performing evolution experiments with far greater throughput. 
We present here the design and operation of a relatively simple, low cost array of miniature chemostats&mdash;or ministats&mdash;and validate their use in determination of physiology and in evolution experiments with yeast. This approach entails growth of tens of chemostats run off a single multiplexed peristaltic pump. The cultures are maintained at a 20 ml working volume, which is practical for a variety of applications. It is our hope that increasing throughput, decreasing expense, and providing detailed building and operation instructions may also motivate research and industrial application of this design as a general platform for functionally characterizing large numbers of strains, species, and growth parameters, as well as genetic or drug libraries.

We developed and validated a small-footprint array of miniature chemostats built from readily available parts for low cost. Physiological and experimental evolution results were similar to larger volume chemostats. The ministat array provides a compact, inexpensive, and accessible platform for traditional chemostat experiments, functional genomics, and chemical screening applications.

Chemostats are continuous culture systems in which cells are grown in a tightly controlled, chemically constant environment where culture density is ...

Assembly of Nucleosomal Arrays from Recombinant Core Histones and Nucleosome Positioning DNA

Core histone octamers that are repetitively spaced along a DNA molecule are called nucleosomal arrays. Nucleosomal arrays are obtained in one of two ways: purification from in vivo sources, or reconstitution in vitro from recombinant core histones and tandemly repeated nucleosome positioning DNA. The latter method has the benefit of allowing for the assembly of a more compositionally uniform and precisely positioned nucleosomal array. Sedimentation velocity experiments in the analytical ultracentrifuge yield information about the size and shape of macromolecules by analyzing the rate at which they migrate through solution under centrifugal force. This technique, along with atomic force microscopy, can be used for quality control, ensuring that the majority of DNA templates are saturated with nucleosomes after reconstitution. Here we describe the protocols necessary to reconstitute milligram quantities of length and compositionally defined nucleosomal arrays suitable for biochemical and biophysical studies of chromatin structure and function.

A method is presented for the reconstitution of model nucleosomal arrays from recombinant core histones and tandemly repeated nucleosome positioning DNA. We also describe how sedimentation velocity experiments in the analytical ultracentrifuge, and atomic force microscopy (AFM) are used to monitor the extent of nucleosomal array saturation after reconstitution.

Core histone octamers that are repetitively spaced along a DNA molecule  are called nucleosomal arrays. Nucleosomal arrays are obtained in one of two  ...

A Rapid Protocol for Integrating Extrachromosomal Arrays With High Transmission Rate into the C. elegans Genome

Microinjecting DNA into the cytoplasm of the syncytial gonad of Caenorhabditis elegans is the main technique used to establish transgenic lines that exhibit partial and variable transmission rates of extrachromosomal arrays to the next generation. In addition, transgenic animals are mosaic and express the transgene in a variable number of cells. Extrachromosomal arrays can be integrated into the C. elegans genome using UV irradiation to establish nonmosaic transgenic strains with 100% transmission rate of the transgene. To that extent, F1 progenies of UV irradiated transgenic animals are screened for animals carrying a heterozygous integration of the transgene, which leads to a 75% Mendelian transmission rate to the F2 progeny. One of the challenges of this method is to distinguish between the percentage of transgene transmission in a population before (X% transgenic animals) and after integration (&#8805;75% transgenic F2 animals). Thus, this method requires choosing a nonintegrated transgenic line with a percentage of transgenic animals that is significantly lower than the Mendelian segregation of 75%. Consequently, nonintegrated transgenic lines with an extrachromosomal array transmission rate to the next generation &#8804;60% are usually preferred for integration, and transgene integration in highly transmitting strains is difficult. Here we show that the efficiency of extrachromosomal arrays integration into the genome is increased when using highly transmitting transgenic lines (&#8805;80%). The described protocol allows for easy selection of several independent lines with homozygous transgene integration into the genome after UV irradiation of transgenic worms exhibiting a high rate of extrachromosomal array transmission. Furthermore, this method is quite fast and low material consuming. The possibility of rapidly generating different lines that express a particular integrated transgene is of great interest for studies focusing on gene expression pattern and regulation, protein localization, and overexpression, as well as for the development of subcellular markers.

A Rapid Protocol for Integrating Extrachromosomal Arrays With High Transmission Rate into the C. elegans Genome

This protocol describes a rapid and low material consuming procedure for the integration of transgenic extrachromosomal arrays into the Caenorhabditis elegans genome using ultra violet (UV) irradiation. Furthermore, this protocol is particularly well suited for transgenic lines that transmit extrachromosomal arrays at a high rate.

Microinjecting DNA into the cytoplasm of the syncytial gonad of Caenorhabditis elegans is the main technique used to establish transgenic lines that ...

Identifying Protein-protein Interaction Sites Using Peptide Arrays

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may result in disease, making protein interactions important drug targets. It is thus highly important to understand these interactions at the molecular level. Protein interactions are studied using a variety of techniques ranging from cellular and biochemical assays to quantitative biophysical assays, and these may be performed either with full-length proteins, with protein domains or with peptides. Peptides serve as excellent tools to study protein interactions since peptides can be easily synthesized and allow the focusing on specific interaction sites. Peptide arrays enable the identification of the interaction sites between two proteins as well as screening for peptides that bind the target protein for therapeutic purposes. They also allow high throughput SAR studies. For identification of binding sites, a typical peptide array usually contains partly overlapping 10-20 residues peptides derived from the full sequences of one or more partner proteins of the desired target protein. Screening the array for binding the target protein reveals the binding peptides, corresponding to the binding sites in the partner proteins, in an easy and fast method using only small amount of protein.
In this article we describe a protocol for screening peptide arrays for mapping the interaction sites between a target protein and its partners. The peptide array is designed based on the sequences of the partner proteins taking into account their secondary structures. The arrays used in this protocol were Celluspots arrays prepared by INTAVIS Bioanalytical Instruments. The array is blocked to prevent unspecific binding and then incubated with the studied protein. Detection using an antibody reveals the binding peptides corresponding to the specific interaction sites between the proteins.

Peptide array screening is a high throughput assay for identifying protein-protein interaction sites. This allows mapping multiple interactions of a target protein and can serve as a method for identifying sites for inhibitors that target a protein. Here we describe a protocol for screening and analyzing peptide arrays.

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may ...