Team:BrownStanfordPrinctn/Demonstrate
Implementing Protein-Based Paper Microfluidics
The implementation of archival printing paper and the TR4520 printer into our paper microfluidic device pipeline yielded positive results for our protein-based microfluidic devices. To test our microfluidic devices, we used water with green food-coloring.
BBa_K3260031 Single Layer Print:
The video above shows one of our tests with printing with protein lysate. The lysate that we loaded into the black ink cartridge contained
The blue regions are plain printer ink, while to the white sections in between the blue and the black outline of the channels are printed protein lysate. We can see on the range of straight, separated channels that the water efficiently wicked up the 250um wide channel and the 200um channel. On the largest “flower” template, the water flowed up the 800um and 900um channels. We also see a successful serendipitous negative control of our device once the water flows past the end of one of the 900um channels and starts penetrating the paper printed with blue ink. This provides a nice contrast with the channel flow and illustrates that while the resolution of the channels isn’t extremely sharp, the hydrophobic protein is certainly performing its function. On the smaller scale flower channels, the water efficiently travels down the 100 - 500 um channels, which is indicative of its functionality on the PDMS-scale resolution.
BBa_K3260031 Double Layer and Negative Control Side by Side:
The video above is another printing lysate test. This test print was part negative control (single layer, but underprinted with normal ink) and part double layer printed
On this test we have a firmer grasp on the resolution that is achieved with this printing method, and it is shown that some bleeding into the hydrophobic region of the device design occurs. However, this is sharply contrasted with the complete lack of resolution and function in the negative control, that was single layer printed with our hydrophobic protein but printed on the opposite side with plain blue ink, which undercuts the functionality of the protein. We can see that the water effectively travels up the channels of widths 200 um, 250 um, 800 um, and 900 um.
All in all, we were able to rationally design, synthesize, print, and prototype a microfluidic system whose channel architecture is based around fusion proteins and paper reagents, not wax or PDMS. We were able to create functional microfluidic channels down to 100um in width, which is extremely significant because this falls comfortably within the range of high resolution PDMS device channels (which have widths of 30 nm to 500 um [2, 3]). Even though our channels did experience a significant degree of bleeding, the functionality of their wicking ability even down to 100um widths speaks to their success as a proof of concept. In addition to this, we were able to produce these prototypes using low cost commercial printers, ink cartridges, and paper reagents. Not only did we successfully produce these prototypes with our original fusion proteins idea (BBa_K3260031), but we also experimented with more elaborate protein designs, yielding a fusion system that can be applicable to our paper microfluidic system, but also an extremely wide range of applications involving the functionalization of cellulose with a binding domain, split GFP domains, and complementary leucine zippers (illustrated in BBa_K3260036 and K3260034). Coming back to the central goal of the subproject, this proof of concept for a protein-based paper microfluidic system opens the door for a whole new range of low cost, easily producible, high resolution devices.
Microfluidic Production and Purification
While some difficulties were encountered using our microfluidic hardware to actually produce and purify doses of medicine, both the hardware designed and purification principles behind it are still valid and work under normal conditions. For example, our expression chip was designed to be able to take 2ml of 3 liquids in and hold them for 3 hours at a 12ul/min flow rate without leakage. The residence time and capacity was confirmed by a flow rate test using a syringe pump which pushed liquid through the chip at 12ul/min and confirmed that our chip would hold liquid for 3 hours and had a capacity of slightly over 2ml. Further, we were able to demonstrate that our chip didn’t leak by running a flow test using food coloring on the chip, which showed that our chip wouldn’t lose reagents. Given that a Cell Free System requires mixing 3 components and allowing several hours for transcription, this means our chip can work to produce medicine using a cell free system.
Further, while some difficulties were encountered running our purification chips, the microfluidic hardware itself is largely sound and functional. The chips simply need to hold chromatography media, which then actually does the purification. We were able to flow test both the IMAC and SEC column, and load media into our SEC. Some difficulties were encountered with media loading the IMAC column, because the beads were slightly too large for the channel height. In the SEC the issue came after loading, as some small beads were able to get through the bead blockers and clog the small outlet channels. While we did not have the ability to go through a 3rd prototype development cycle, these issues did not necessarily need to be solved on the hardware level. While these devices would certainly work if the IMAC column were taller, and the SEC had smaller bead blockers or larger outlet channels, if the IMAC resin were filtered to only contain beads under 100um, or the SEC beads were filtered to only include beads larger than the bead blockers, then the system we have already manufactured would also work. While we weren’t able to run the complete process on our microfluidic chips, we were able to run SEC purification on a larger scale column. We mixed food coloring with chromoprotein that fluoresces under UV, and ran it through a SEC column, collecting 4 fractions. The first two fractions were clear but fluoresce under UV, showing that they contained no food coloring and contained chromoprotein. The second two were orange but dark under UV, showing that they had food coloring but no chromoprotein. This shows that our purification steps for our system are overall sound, and that the only issue with our system is getting media on chips.
