Cell-free protein expression in a microchannel array with passive pumping

Abstract

We report in vitro (cell-free) protein expression in a microfluidic device using passive pumping. The polystyrene device contains 192 microchannels, each of which is connected to two wells positioned in a 384-well microplate format. A larger droplet of an expression solution was placed at one well of each channel while a smaller droplet of a nutrient solution was at the other well. Protein expression took place in the larger droplet and we found the expression yield in the expression solution is enhanced due to the replenishment of the nutrient solution supplied by passive pumping via the channel. The pumping pressure was generated from the difference in the surface tension between two different sized droplets at the two wells. We demonstrated expression of luciferase in the device and the expression yield was measured using luminescence assay. Different experimental conditions were investigated to achieve maximum protein yield with the least amount of reagents. Protein expression yields were found to be dependent on the amount of the nutrient solution pumped, independent of the amount of the expression solution within the experimental conditions studied. A higher feeding frequency or delivery rate of the nutrient solution resulted in higher protein expression yield. The work demonstrated the feasibility of using the microchannel array for protein expression with the following advantages: (1) simultaneous production of the same protein with different conditions to optimize the expression process; (2) simultaneous production of different proteins for high-throughput protein expression with high yield; (3) low reagent cost due to the fact that it consumes 125-800 times less than the amount used in a protein expression instrument commercially available.

A Cell Programmable Assay (CPA) chip

“…The CPA chips can be easily adapted to a variety of common cell culture methods and procedures. Importantly, while PDMS was used to fabricate the devices here, we have previously reported passive pumping in non-elastomeric materials such as polystyrene; 17,25,26 thus, for high volume manufacturing the CPAs could be made out of standard cell culture materials lowering costs and eliminating potential biases from PDMS. 27,28 Although the approach we have taken here can be applied to many applications, care must be taken when translating macroscale protocols for microscale devices.…”

Microfluidic Bioreactors for Cell Culturing: A Review

“…Dead cells can be stained red due to the propidium iodide which does not penetrate the membrane of live cells. Other fluorescence assays that can be carried out in microbioreactors include the coupling of reporter genes to detect and track specific cells events using reporter proteins, such as green fluorescent protein (GFP) [141], luciferase [130] and galactosidase.…”

“…The fabrication of non mechanical pumps is often less complex than mechanical pumps, which makes them suited for low cost mass production and easy to dispose. Several types of passive micropumps have been reported, the most common types include osmotic pressure [126], evaporation [127][128] and, surface tension [129][130]. Electroosmotic flow (EOF) is a popular means of pumping liquids in microfluidic devices and is based on the application of a potential difference across a microchannel to induce the flow of a liquid [131].…”

Cell-Free Protein Synthesis in Miniaturized Array Devices and Effects of Device Orientation

“…These goals have been accomplished in the form of droplets, [21][22][23][24] protein-producing gel, 25 and microfluidic devices. 14,[26][27][28] With the droplets, a pseudo-filtration membrane is formed through oil-in-water emulsions, and the platform offers an ultra-high-throughput screening method because a large number of droplets can be easily and rapidly created. [21][22][23][24] For the protein-producing gel, the isolation of the genetic template and reaction solution is achieved through a hydrogel, and the large surface-area-to-volume ratio of the reaction vessel enhances protein synthesis yield.…”

“…By scaling the devices for additional protein expression units, high-throughput protein synthesis is possible. 14,[26][27][28] In this work, we designed and fabricated two miniaturized array devices for continuous-exchange CFPS and investigated the effect of membrane orientation on protein synthesis yield. Compared with the devices reported previously, 14 the membrane in this work is oriented vertically in reference to the table surface to reduce or eliminate possible membrane clogging (due to possible sedimentation of large molecules such as aggregated proteins or ribosomes).…”