This work was supported by Marie Curie international reintegration grant.

1. Device Fabrication 
<ol>
 <li>Purchased DTPA-D SU-8 control mold and SPR220-7 flow mold from the Stanford Microfluidics Foundry (<a href="http://www.stanford.edu/group/foundry" target="_blank">www.stanford.edu/group/foundry</a>).</li>
 <li>Expose the silicone molds to chlorotrimethylsilane (TMCS) vapor for 10 min to promote elastomer release after the baking steps9. </li>
 <li>Prepare a mixture of silicone based elastomer and curing agent (mix well) in two different ratios 5:1 and 20:1 for the control and flow molds, respectively. The different ratios are necessary for proper bonding of multiple layers. </li>
 <li>Pour the 5:1 PDMS on the control layer (approximately 5 mm height) . Degas the control layer and bake it for 30 min at 80 &deg;C. Spin coat (Laurell, USA) the 20:1 PDMS mixture on the flow layer at 2,600 rpm for 60 sec and then bake it at 80 &deg;C for 30 min. The spin coater velocity is optimized for valve actuation at a pressure of 15 psi. Faster spinning will result in a thinner layer with lower activation pressure and vice versa. The devices we use have a limit of 25 psi in the control and 10 psi in the flow, above which detachment from the substrate may occur. </li>
 <li>Separate the control layer from the mold. Do it slowly and be careful not to peel the SU8 pattern. Then cut the device around its perimeter and punch holes to access the control channels.</li>
 <li>Align the flow and control layers manually under a stereoscope. Start by aligning the upper left corner; the button valve (control layer) must be in the middle of the reaction chamber (flow layer). Next, align the first row and gently release the control layer row by row. Make sure that all the button valves are in the middle of the reaction chambers and that the address and input valves cross the flow channels at the correct position. The process can be repeated locally until all rows are aligned. At the end, release any tension in the PDMS by lifting it carefully from the sides and corners. Do not lift too much or misalignment may occur. </li>
 <li>Bake it for 2 hr at 80 &deg;C.</li>
 <li>Cut around the perimeter of the device and peel the two layer device (chip) from the flow mold. Punch holes to access the flow channels (Figure 1). </li>
</ol>
2. DNA Arraying and Device Alignment
<ol>
 <li>Produce synthetic genes by assembly PCR. The synthetic genes are compose of T7 promoter, ribosome binding site (RBS), ORF with two epitope tags (one at each end), and T7 terminator 4. The genes can vary in length from 100 bp up to, at least, 5,000 bp.</li>
 <li>Prepare the synthetic genes for arraying: prepare a mixture of poly ethylene glycole (1.25%) and D-trehalose dihydrate (125 mg/ml) and dispense 2 &mu;l per reaction into 384 wells plate. This solution will reduce irreversible binding of the DNA to the glass as well as for visualization during alignment 10. </li>
 <li>Transfer the synthetic genes to the 384 wells plate. Usually DNA concentrations can range from 10 ng/&mu;l to 100 ng/&mu;l final concentration. Add dH2O to a final volume of 20 &mu;l (depends on microarrayer and pin type). </li>
 <li>Spot a series of synthetic genes onto epoxy coated glass substrates using a microarrayer. We use MicroGrid 610 (Bio Robotics) with SMT-S75 silicone pins (Parallel Synthesis, USA). Contact printing of the DNA, with these specific pins, results in spots with diameter of about 100 &mu;m on the glass surface. Each pin load is enough for approximately 100 spots. Make sure the column and row pitches correspond to the specific device used. The device we use contains 16 columns and 40 rows with a pitch of 680 &mu;m by 320 &mu;m, respectively. </li>
 <li>Manually align the microfluidic device to the gene array under a stereoscope. The DNA spot should be in the middle of the DNA chambers. Start the alignment by locating the first row of spots below the first row of DNA chambers. Then, bring into line the rest of the rows finishing with fine tuning of the whole device. Once the array is aligned care should be taken to alleviate any stress in the PDMS, by lifting it locally. If there is tension left in the PDMS it may not bond well to the microarray. </li>
 <li>Finally, bond the device to the glass slide by overnight incubation on a hot plate at 80 &deg;C. </li>
</ol>
3. Priming and Activating the Device
<ol>
 <li>The valves in the control layer are actuated from the computer using LabVIEW. The LabVIEW script controls a series of solenoid micro valves via an electronic control box (purchased from Stanford Microfluidics Foundry). The solenoid valve manifold is connected to compressed air, which control the air flow and pressure into the control valves on the device. </li>
 <li>Connect the device to the solenoid valves manifold using a flexible plastic tubing with internal diameter of 0.02" (Tygon) and stainless steel pin (New England Small Tubes Corporation). </li>
 <li>Fill the tubes with DDW and insert the pin into the access holes of the control layer. Make sure that each tube is connected to its corresponding control channel. </li>
 <li>Run the LabVIEW application on the computer to activate the control channels. By activating the valve from the LabVIEW script, we apply air pressure, which in turn will push the water into the PDMS control channels. Set the air pressure to 5 psi. It is recommended to fill the 'sandwich' and the address valves first, in order to identify cross-talks between control channels of defective devices. In case of cross-talks, actuation of the 'sandwich' control line will lead to the actuation of either the neck or button control lines. This is similar to a short in an electrical circuit. A device with cross-talk is defective and cannot be used. </li>
 <li>After all the control channels and valves are full of DDW, the valves are primed and ready to block the flow channels underneath them. Increase the air pressure to 15 psi. Valve activation is controlled from a LabVIEW program through a set of on/off switches connected to individual solenoid valves. Each switch controls a specific control line through its corresponding solenoid valve. Turning on the switch button (in the script) will apply air pressure in the corresponding tube. The air pressure will push the DDW in the control line and will result in expansion of the microfluidic valves, blocking the channel underneath. Turning off a control valve, will release the air pressure and subsequently release the blocking of the respective flow channel. </li>
 <li>To ensure that all the valves are open, connect a tube to one of the flow inputs and flow air into the device at 4-5 psi. This will release any sticky valves from the glass slide. </li>
 <li>Finally, the device is primed and ready. Close all the input and 'neck' valves, to start the surface chemistry. </li>
</ol>
4. Surface Chemistry
<ol>
 <li>In order to facilitate the self-assembly of a protein array on the surface and prevent non-specific adsorption within the microfluidic device, the surface is chemically modified: </li>
 <li>To flow a component through the device, connect a new tube with the required solution to one of the flow channels in the device. Connect the free side of the tube to the manual manifold and open the air pressure flow (5 psi). </li>
 <li>Load 40 &mu;l of Biotinylated-BSA (1 &mu;g/&mu;l) in a new tube and flow approximately half of it for 20 min through the device, the BSA will bind to the epoxy surface. </li>
 <li>Use Hepes (50 mM) for washing unreacted substrate between each of the different surface chemistry steps. </li>
 <li>Flow 25 &mu;l Strepavidin (0.5 &mu;g/&mu;l) for 20 min on top of the biotinylated-BSA.</li>
 <li>Wash with Hepes for 5 min. </li>
 <li>Close the 'button' valve and flow the rest of the biotinylated-BSA (as described above), passivating the surface surrounding the button. </li>
 <li>Wash with Hepes for 5 min. </li>
 <li>Release the 'button' valve and flow 30 &mu;l of penta-His-biotin (0.16 &mu;g/&mu;l) for 20 min. The antibody will bind to the exposed strepavidin; specifically to the area under the 'button' creating an anti His-tag array. </li>
</ol>
5. Protein Expression
<ol>
 <li>Express the proteins on the device using rabbit reticulocyte quick coupled transcription and translation reaction. Protein expression from the spotted synthetic genes on the device creates an array of proteins ready to use. For example, one such use is for a protein binding screen. </li>
 <li>Open the 'neck' valves and flow rabbit reticulocyte quick coupled transcription and translation reaction through the device into the DNA chamber. Next, close the 'sandwich' valves and separate each gene from its environment. Incubate the device on a hot plate for 2.5 hr at 30 &deg;C. Expressed proteins will diffuse from the DNA chamber to the reaction chamber spontaneously (neck valve is open) and bind to the anti-His antibody under the 'button' valve immobilizing the protein through its C-terminus tag. </li>
 <li>Label the proteins with C-myc Cy3 antibody. The antibody will bind to its corresponding epitope located at the protein N-terminus. </li>
 <li>Determine protein expression levels with a microarray scanner (LS Reloaded, Tecan) using a 532 nm laser and 575 nm emission filter. </li>
</ol>
6. Representative Results
1. Device Fabrication
A graphic design for the device was created by AutoCAD based on our experimental needs. The design was printed on a transparency film by a high-resolution image setter. This transparency serves as a photomask in contact photolithography. A surface micromachining technique was used to create 3-D templates on a silicon wafer determined by the patterns inscribed on the masks used. 
The microfluidic device was fabricated on silicone mold casting silicone elastomer polydimethylsiloxane (PDMS, SYLGARD 184, Dow Corning, USA) (Figure 1). Each device consists of two aligned PDMS layers, the flow and the control layer. The mold was first exposed to chlorotrimethylsilane (TMCS, Aldrich) vapor for 10 min to promote elastomer release after the baking steps. A mixture of silicone based elastomer and curing agent was prepared in two different ratio 5:1 and 20:1 for the control and flow molds, respectively. The control layer was degassed and baked for 30 min at 80 &deg;C. The flow layer was initially spin coated (Laurell, USA) at 2,600 rpm for 60 sec and baked at 80 &deg;C for 30 min. The control layer was separated from its mold and control channel access holes were punched. Next, the flow and control layers were aligned manually under a stereoscope and baked for 2 hr at 80 &deg;C. The two layer device was peeled from the flow mold and flow channels access holes were punched.
2. Device Description
 The device capacity can range from 500 up to 10,000 unit cells (Figure 2). The device consists of two layers; flow layer (grey) and control layer (color). The control layer contain a variety of valves with different function. Each unit cell, in the flow layer, is composed of one DNA and one reaction chamber and is controlled by three types of micromechanical valves; 'neck', 'button', and 'sandwich' (Figure 2A). A spotted 'synthetic gene' deposited within the DNA chamber is blocked from the reaction chamber by the 'neck' valve (green). Trapping and mechanical washing of surface bound proteins in the reaction chamber is performed by the 'button' valve (blue). The 'sandwich' valve enables each reaction to occur in its own unit cell (red). The address valves can split the device up to 8 independent sections, for different condition assay. In addition, the control layer contain input valves that enable the flow of selected fluid into the flow layer. The PDMS control channels are full with DDW. When a valve is actuated the pressure increase (15 psi) results in the expansion of the PDMS. In places where the membrane is thin enough (i.e. crossing of control and flow lines) this is sufficient to completely block the flow line. Average unit cell height is 10 &mu;m, and average unit cell volume is less than 1 nl. 
3. Proteins Expression and Detection
 Proteins were expressed on the device using rabbit reticulocyte quick coupled transcription and translation reaction (Promega). The expression of the proteins created an array of proteins ready for use in a binding screen (Figure 3). An expression mix (12.5 &mu;l) was loaded into the device and then flooded into the DNA chambers by opening the 'neck' valves. Next, the 'sandwich' valves were closed leaving each unit cell separated from its neighboring cells and the device was incubated on a hot plate for 2.5 hr at 30 &deg;C. Expressed proteins diffused through the gene chamber into the reaction chamber, where their C-terminus His tag could bind the anti-His antibody (Qiagen) localized under the 'button' valve immobilizing the protein to the surface. Proteins were labeled with a C-myc Cy3 (Sigma), which bound to its corresponding epitope located at the protein N-terminus and labeled it. Protein expression levels were determined with a microarray scanner (LS Reloaded, Tecan) using a 532 nm laser and 575 nm filter. The resulting protein array consists of varying levels of protein expression. Usually, about 20% of a gene library fail to express to detectable levels. No correlation with protein size was observed 3. Background levels were determined using chambers that were not spotted with DNA. Thus, signals from the corresponding protein chambers are due to either noise or non-specific adsorption of the labeling antibodies.
<img src="/files/ftp_upload/3849/3849fig1.jpg" alt="Figure 1" /> 
Figure 1. Chip Fabrication. (A) Molds were treated with TMCS vapors for 10 min. (B) PDMS is casted on control and flow silicon molds. (C) Both control and flow molds are baked in 80 &deg;C for 30 min. (D) The control layer peeled of the mold, cut to size and control inlets are punched. (E) The control layer is aligned to the flow layer under a stereoscope and then baked in 80 &deg;C for 2 hr. In parallel to the PDMS device fabrication, a series of synthetic genes are microarrayed onto epoxy coated glass substrates (CEL Associates). (F) The device is then aligned to the DNA microarray trapping the 'synthetic genes' within the DNA chambers.
<img src="/files/ftp_upload/3849/3849fig2.jpg" alt="Figure 2" /> 
Figure 2. A Photo of the PING Device. (A) The device consist of two aligned PDMS layers (control and flow). The flow layer contains parallel DNA and reaction chambers (grey) controlled by micromechanical valves ("button", "sandwich" and "neck") in the control layer. (B) The layers are aligned to a printed microarray, which programs each DNA chamber with a single spot. This eliminates any potential contamination.
<img src="/files/ftp_upload/3849/3849fig3.jpg" alt="Figure 3" /> 
Figure 3. Fluorescent Images of a Protein Array Created with a Microfluidic Chip. The printed spot genes were expressed to proteins within the device (in the DNA chamber) and were pulled down to the surface (below the "button" valve in the reaction chamber) through their C-terminus His tag. Protein expression was detected using their C-myc N-terminus tag and a specific fluorescent antibody. Different signal intensities indicate different protein expression levels. Un-spotted chambers served as controls for background levels.

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No conflicts of interest declared.

In this paper we present a method for generation protein arrays in high-throughput using a microfluidic platform. The array generation is based on microarray printing of DNA templates and in vitro protein expression from the DNA within the microfluidic device. 
Our novel microfluidic platform has several important advantages over currently used methods, which make it a promising and general tool for proteomics. One advantage is with membrane-bound proteins. In vitro protein s...

<table border="1">
 <tbody>
 <tr>
 <td>Reagent/Equipment</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>PDMS- SYLGARD 184</td>
 <td>Dow Corning USA</td>
 <td>ESSEX-DC</td>
 </tr>
 <tr>
 <td>Chlorotrimethylsilane (TMCS </td>
 <td>Sigma-Aldrich </td>
 <td>C72854 </td>
 </tr>
 <tr>
 <td>Epoxy coated glass substrates </td>
 <td>CEL Associates USA</td>
 <td>VEPO-25C</td>
 </tr>
 <tr>
 <td>Poly ethylene glycole (PEG)</td>
 <td>Sigma-Aldrich</td>
 <td>81260</td>
 </tr>
 <tr>
 <td>D-trehalose dihydrate</td>
 <td>Sigma-Aldrich</td>
 <td>T9531</td>
 </tr>
 <tr>
 <td>Biotinylated-BSA</td>
 <td>Pierce </td>
 <td>PIR-29130</td>
 </tr>
 <tr>
 <td>Neutravidin </td>
 <td>Pierce </td>
 <td>31050</td>
 </tr>
 <tr>
 <td>penta-His-biotin</td>
 <td>Qiagen</td>
 <td>34440</td>
 </tr>
 <tr>
 <td>Hepes</td>
 <td><a href="http://www.bioind.com/htmls/article.aspx?c0=741&amp;bsp=13384" target="_blank">Biological 				Industries</a></td>
 <td>03-025-1B</td>
 </tr>
 <tr>
 <td>TNT-T7</td>
 <td>Promega</td>
 <td>L5540</td>
 </tr>
 <tr>
 <td>C-myc Cy3 antibody</td>
 <td>Sigma -Aldrich</td>
 <td></td>
 </tr>
 <tr>
 <td>Control box</td>
 <td>Stanford Microfluidics Foundry</td>
 <td></td>
 </tr>
 <tr>
 <td>Mold</td>
 <td>Stanford Microfluidics Foundry</td>
 <td></td>
 </tr>
 <tr>
 <td>Pin</td>
 <td>New England Small Tubes Corporation</td>
 <td></td>
 </tr>
 <tr>
 <td>Tygon microbore tubing</td>
 <td>Tygon</td>
 <td>S-54-HL</td>
 </tr>
 <tr>
 <td>Microarrayer</td>
 <td>Bio Robotics</td>
 <td>MicroGrid 610</td>
 </tr>
 <tr>
 <td>Silicone pins</td>
 <td>Parallel Synthesis</td>
 <td>SMT-S75</td>
 </tr>
 </tbody>
</table>

high-throughput protein expression generator using a microfluidic platform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays1. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein 4, protein-RNA5 or protein-DNA6 interactions.
The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins7, DNA8, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.

Watch this Scientific Journal Video about High-throughput Protein Expression Generator Using a Microfluidic Platform at JoVE.com

High-throughput Protein Expression Generator Using a Microfluidic Platform

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

high-throughput-protein-expression-generator-using-microfluidic

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JoVE Journal

Bioengineering

11.6K Views.  Bar-Ilan University. Presentiamo un approccio microfluidica per l'espressione di proteine ​​array. Il dispositivo consiste di migliaia di camere di reazione controllati da micro-valvole meccaniche. Il dispositivo microfluidica è accoppiato ad un microarray stampata libreria genetica. Questi geni sono poi trascritta e tradotta on-chip, risultante in un array proteico pronto all'uso sperimentale.

Video: High-throughput Protein Expression Generator Using a Microfluidic Platform

CometChip: A High-throughput 96-Well Platform for Measuring DNA Damage in Microarrayed Human Cells

DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular metabolites. Ironically, at high doses DNA damaging agents are also used to treat cancer. The ability to quantify DNA damage responses is thus critical in the public health, pharmaceutical and clinical domains. Here, we describe a novel platform that exploits microfabrication techniques to pattern cells in a fixed microarray. The &#8216;CometChip&#8217; is based upon the well-established single cell gel electrophoresis assay (a.k.a. the comet assay), which estimates the level of DNA damage by evaluating the extent of DNA migration through a matrix in an electrical field. The type of damage measured by this assay includes abasic sites, crosslinks, and strand breaks. Instead of being randomly dispersed in agarose in the traditional assay, cells are captured into an agarose microwell array by gravity. The platform also expands from the size of a standard microscope slide to a 96-well format, enabling parallel processing. Here we describe the protocols of using the chip to evaluate DNA damage caused by known genotoxic agents and the cellular repair response followed after exposure. Through the integration of biological and engineering principles, this method potentiates robust and sensitive measurements of DNA damage in human cells and provides the necessary throughput for genotoxicity testing, drug development, epidemiological studies and clinical assays.

We describe here a platform that allows comet assay detection of DNA damage with unprecedented throughput. The device patterns mammalian cells into a microarray and enables parallel processing of 96 samples. The approach facilitates analysis of base level DNA damage, exposure-induced DNA damage and DNA repair kinetics.

DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular ...

High Throughput Microfluidic Rapid and Low Cost Prototyping Packaging Methods

In this work, 3 different packaging and assembly techniques are presented. They can be classified into two categories: one-time use and reusable packaging techniques.
The one-time use packaging technique employs UV-based and temperature curing epoxies to connect microtubes to access holes, wire-bonding for integrated circuit connections, and silver epoxy for electrical connections. This method is based on a robust assembly technique that can support relatively high pressure close to 1 psi and does not need any support to strengthen the microfluidic architecture.
Reusable packaging techniques consist of PDMS-based microtube interconnectors and anisotropic adhesive films for electrical connections. These devices are more sensitive and fragile. Consequently, Plexiglas support is added to the microfluidic structure to improve the electrical contact when anisotropic adhesive films are used, and also to strengthen the microfluidic architecture. In addition, a micromanipulator is needed to maintain tubes while using a thin PDMS layer to connect them to the access holes. Different PDMS layer thicknesses, ranging from 0.45-3 mm, are tested to compare the best adherence versus injection rates. Applied injection rates are varied from 50-300 &#956;l/hr for 0.45-3 mm PDMS layers, respectively. These techniques are mainly applicable for low-pressure applications. However, they can be extended for high-pressure ones through plasma-oxygen process to permanently seal the PDMS to glass substrates. The main advantage of this technique, besides the fact that it is reusable, consists of keeping the device observable when the microchannel length is very short (in the range of 3 mm or lower).

In this article we describe different techniques for microfluidic rapid prototyping platforms. The proposed techniques are based on ultraviolet (UV) sensitive and temperature curing epoxies, polydimethylsiloxane (PDMS) based tubing, wire-bonding, and anisotropic adhesive films. The assembling procedures presented are developed for both one-time use devices as well as reusable microfluidic systems.

In this work, 3 different packaging and assembly techniques are presented. They can be classified into two categories: one-time use and reusable packaging ...

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected within in vivo oral biofilms. Furthermore, a system that uses natural human saliva as the nutrient source, instead of artificial media, is particularly desirable in order to support the expression of cellular and biofilm-specific properties that mimic the in vivo communities. We describe a method for the development of multi-species oral biofilms that are comparable, with respect to species composition, to supragingival dental plaque, under conditions similar to the human oral cavity. Specifically, this methods article will describe how a commercially available microfluidic system can be adapted to facilitate the development of multi-species oral biofilms derived from and grown within pooled saliva. Furthermore, a description of how the system can be used in conjunction with a confocal laser scanning microscope to generate 3-D biofilm reconstructions for architectural and viability analyses will be presented. Given the broad diversity of microorganisms that grow within biofilms in the microfluidic system (including Streptococcus, Neisseria, Veillonella, Gemella, and Porphyromonas), a protocol will also be presented describing how to harvest the biofilm cells for further subculture&#160;or DNA extraction and analysis. The limits of both the microfluidic biofilm system and the current state-of-the-art data analyses will be addressed. Ultimately, it is envisioned that this article will provide a baseline technique that will improve the study of oral biofilms and aid in the development of additional technologies that can be integrated with the microfluidic platform.

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

The goal of this methods paper is to describe the use of a microfluidic system for the development of multi-species biofilms that contain species typically identified in human supragingival dental plaque.&#160;Methods to describe biofilm architecture, biofilm viability, and an approach to harvest biofilm for culture-dependent or culture-independent analyses are highlighted.

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

A High-throughput Cell Microarray Platform for Correlative Analysis of Cell Differentiation and Traction Forces

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly controlled, high-throughput engineered system for measuring the impact of arrayed biochemical signals on cell differentiation. Recent efforts using cell microarrays have demonstrated their utility for combinatorial studies in which many microenvironmental factors are presented in parallel. However, these efforts have focused primarily on investigating the effects of biochemical cues on cell responses. Here, we present a cell microarray platform with tunable material properties for evaluating both cell differentiation by immunofluorescence and biomechanical cell&#8211;substrate interactions by traction force microscopy. To do so, we have developed two different formats utilizing polyacrylamide hydrogels of varying Young&#39;s modulus fabricated on either microscope slides or glass-bottom Petri dishes. We provide best practices and troubleshooting for the fabrication of microarrays on these hydrogel substrates, the subsequent cell culture on microarrays, and the acquisition of data. This platform is well-suited for use in investigations of biological processes for which both biochemical (e.g., extracellular matrix composition) and biophysical (e.g., substrate stiffness) cues may play significant, intersecting roles.

Cell differentiation is regulated by a host of microenvironmental factors, including both matrix composition and substrate material properties. We describe here a technique utilizing cell microarrays in conjunction with traction force microscopy to evaluate both cell differentiation and biomechanical cell&#8211;substrate interactions as a function of microenvironmental context.

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly ...

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved useful as a convenient live imaging platform providing capabilities for precise control of experimental conditions in real time. In this article, we demonstrate live imaging of individual worms employing WormSpa, a previously-published custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes. To illustrate the capability, we performed proof-of-principle experiments in which worms were infected in the device with pathogenic bacteria, and the dynamics of expression of immune response genes and egg laying were monitored continuously in individual animals. The simple design and operation of this device make it suitable for users with no previous experience with microfluidic-based experiments. We propose that this approach will be useful for many researchers interested in longitudinal observations of biological processes under well-defined conditions.

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In this article, we demonstrate live imaging of individual worms employing a custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes.

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved ...

A Multilayer Microfluidic Platform for the Conduction of Prolonged Cell-Free Gene Expression

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic regulatory circuits. The analysis of synthetic genetic regulatory networks in vivo is time consuming and suffers from a lack of environmental control, with exogenous synthetic components interacting with host processes resulting in undesired behavior. To overcome these issues, cell-free characterization of novel circuitry is becoming more prevalent. In vitro transcription and translation (IVTT) mixtures allow the regulation of the experimental environment and can be optimized for each unique system. The protocols presented here detail the fabrication of a multilayer microfluidic device that can be utilized to sustain IVTT reactions for prolonged durations. In contrast to batch reactions, where resources are depleted over time and (by-) products accumulate, the use of microfluidic devices allows the replenishment of resources as well as the removal of reaction products. In this manner, the cellular environment is emulated by maintaining an out-of-equilibrium environment in which the dynamic behavior of gene circuits can be investigated over extended periods of time. To fully exploit the multilayer microfluidic device, hardware and software have been integrated to automate the IVTT reactions. By combining IVTT reactions with the microfluidic platform presented here, it becomes possible to comprehensively analyze complex network behaviors, furthering our understanding of the mechanisms that regulate cellular processes.

The fabrication process of a PDMS-based, multilayer, microfluidic device that allows in vitro transcription and translation (IVTT) reactions to be performed over prolonged periods is described. Furthermore, a comprehensive overview of the hardware and software required to automate and maintain these reactions for prolonged durations is provided.

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic ...

A Microfluidic Platform for Stimulating Chondrocytes with Dynamic Compression

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth plate cartilage architecture and results in growth modulation of long bones of children. To determine the role of compressive stress in bone growth, we created a microfluidic device actuated by pneumatic pressure, to dynamically (or statically) compress growth plate chondrocytes embedded in alginate hydrogel cylinders. In this article, we describe detailed methods for fabricating and characterizing this device. The advantages of our protocol are: 1) Five different magnitudes of compressive stress can be generated on five technical replicates in a single platform, 2) It is easy to visualize cell morphology via a conventional light microscope, 3) Cells can be rapidly isolated from the device after compression to facilitate downstream assays, and 4) The platform can be applied to study mechanobiology of any cell type that can grow in hydrogels.

This article provides detailed methods for fabricating and characterizing a pneumatically actuating microfluidic device for chondrocyte compression.

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth ...

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. The tomato-P. syringae pv. tomato pathosystem is widely used to dissect the genetic basis of plant innate responses and disease resistance. While disease was successfully managed for many decades through the introduction of the Pto/Prf gene cluster from Solanum pimpinellifolium into cultivated tomato, race 1 strains of P. syringae have evolved to overcome resistance conferred by the Pto/Prf gene cluster and occur worldwide.
Wild tomato species are important reservoirs of natural diversity in pathogen recognition, because they evolved in diverse environments with different pathogen pressures. In typical screens for disease resistance in wild tomato, adult plants are used, which can limit the number of plants that can be screened due to their extended growth time and greater growth space requirements. We developed a method to screen 10-day-old tomato seedlings for resistance, which minimizes plant growth time and growth chamber space, allows a rapid turnover of plants, and allows large sample sizes to be tested. Seedling outcomes of survival or death can be treated as discrete phenotypes or on a resistance scale defined by amount of new growth in surviving seedlings after flooding. This method has been optimized to screen 10-day-old tomato seedlings for resistance to two P. syringae strains and can easily be adapted to other P. syringae strains.

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

The seedling flood assay facilitates rapid screening of wild tomato accessions for the resistance to the Pseudomonas syringae bacterium. This assay, used in conjunction with the seedling bacterial growth assay, can assist in further characterizing the underlying resistance to the bacterium, and can be used to screen mapping populations to determine the genetic basis of resistance.

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. ...

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Subtitles

High-throughput Protein Expression Generator Using a Microfluidic Platform