The Mina & Everard Goodman Faculty of Life Sciences, The Nanotechnology Institute, Bar-Ilan University
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Glick, Y., Avrahami, D., Michaely, E., Gerber, D. High-throughput Protein Expression Generator Using a Microfluidic Platform. J. Vis. Exp. (66), e3849, doi:10.3791/3849 (2012).
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.
1. Device Fabrication
2. DNA Arraying and Device Alignment
3. Priming and Activating the Device
4. Surface Chemistry
5. Protein Expression
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 °C. The flow layer was initially spin coated (Laurell, USA) at 2,600 rpm for 60 sec and baked at 80 °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 °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 μm, 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 μl) 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 °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.
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 °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 °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.
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.
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.
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 synthesis using mammalian reticulocyte lysates in the presence of microsomal membranes11 provides "natural like" conditions required for membrane proteins. Furthermore, microfluidics allows for protein expression in very low volumes and protein purification is not necessary. These are the most common bottlenecks in conventional methodologies. In fact, further optimization of proteins can be achieved by matching the in vitro cell lysate to the proteins, for example E. coli lysate for bacterial proteins.
Microfluidics allows protein expression in very low volumes. In our specific example the chamber volume is 1 nl. There are two advantages to the low volumes. The obvious one is lower consumption of reagents and ability to work with rare materials (i.e. membrane proteins). Another significant advantage is that concentrating the proteins on the surface with antibodies in such a small volume allows us to achieve relatively high concentrations of proteins for interaction assays, which will increase the assay sensitivity.
The button valves have dual role. First they are used for patterning an antibody under the button in each chamber. This allows us to pull down the proteins only under the buttons, while the rest of the chamber is passivated. Second, the buttons allow for mechanical washing and trapping of the proteins by displacing a volume of liquid underneath when actuated. This washing and trapping increases the overall sensitivity of the platform and allows detection of weak and transient molecular interactions 4.
Finally, the microfluidic device can easily be automated and is capable of making many parallel measurements. Thereby saving on costs as well as time. Our estimate is that the cost per protein experiment using PING is about 2 cents per protein, with current devices ranging up to 10,000 experiments per device 4. This estimate includes fabrication and materials. Thus, PING has the potential to be a robust tool for protein array in general with specific advantages such as for membrane proteins.
No conflicts of interest declared.
This work was supported by Marie Curie international reintegration grant.
|PDMS- SYLGARD 184||Dow Corning USA||ESSEX-DC|
|Epoxy coated glass substrates||CEL Associates USA||VEPO-25C|
|Poly ethylene glycole (PEG)||Sigma-Aldrich||81260|
|Biotinylated-BSA||Pierce, Thermo Scientific||PIR-29130|
|Neutravidin||Pierce, Thermo Scientific||31050|
|C-myc Cy3 antibody||Sigma-Aldrich|
|Control box||Stanford Microfluidics Foundry|
|Mold||Stanford Microfluidics Foundry|
|Pin||New England Small Tubes Corporation|
|Tygon microbore tubing||Tygon||S-54-HL|
|Microarrayer||Bio Robotics||MicroGrid 610|
|Silicone pins||Parallel Synthesis||SMT-S75|