Artificial Active Transport for Lab-­‐On-­‐A-­‐Chip Applications – Progress Report Eric Baken, Eric Davied, Grant McElhaney, Andres Medina, and Justin Richardson Introduction: Laboratory equipment has become an integral part of medicine. In the first world, every hospital and clinic utilizes many pieces of large and expensive machinery to diagnose disease. However, this method is very impractical for many applications. When natural disasters occur, it is difficult to transport the all of the equipment needed to where the patients are at. This results in substandard or delayed care that harms the patient. The cost of this equipment can also be prohibitive for its use in the third world. For both these issues, we need to rethink the concept of a healthcare laboratory. The solution comes from a tiny device called a lab-­‐on-­‐a-­‐chip. These devices combine the functionality of many different pieces of laboratory machinery and put them all onto a single microchip. This allows for very specific testing without a laboratory. For instance, lab-­‐on-­‐a-­‐chips have been used to diagnose the strain of malaria that a patient is infected with. Normally, this would require at least two pieces of laboratory equipment to do. Obviously the two pieces of laboratory equipment could be used for more than just determining the strain of malaria that a patient has, but for a specific application, lab-­‐on-­‐a-­‐chips are cheaper and more practical. One of the major functions done in a laboratory is concentrating and isolating substances. This is often required before other tests can be performed. The traditional methods for doing this, on a large scale, dialysis, centrifugation, and chromatography have some difficulty working on a smaller scale. There are some techniques currently used to get around this (microfluidic concentration) but these techniques have some limitations. Instead, this project aims to develop a completely novel technique to concentrate substances utilizing biological proteins instead of physical principles. The goal of our project is to construct directionally oriented microtubule pathways and use biological motor proteins attached to antibodies to transport a substance of interest to a concentrating area. Microtubules are intracellular filaments that play roles in cell division, structural support, and transport. Microtubules are polarized with (+) and (-­‐) ends. We are arranging our microtubules so the (+) ends all face a central concentration area. We are utilizing a motor protein, kinesin, that walks towards the (+) end of the microtubule. Thus by connecting the kinesin to a substance of interest using a specific antibody, we can concentrate substances on a molecular scale from very small samples. To make microtubule pathways, there was a need to have consistent and controlled microtubule growth. Most of the progress this semester has focused on ensuring control of these factors. Methods: Microtubules were polymerized from a 50 μM rhodamine-­‐labeled/unlabeled tubulin (at 1:2 ratio) mixture in the o
presence of GMPCPP for 24 minutes at 37 C. The polymerized mixture was diluted after incubation to 500nM in BRB80 and Paclitaxel (Taxol) buffer. This mixture was referred to as 100% solution. After production of the 100% solution, it was diluted to smaller amounts for better visualization and an oxygen scavenger was added. The oxygen scavenger was made with a solution of BRB80, Glucose, Glucose Oxidase, Catalase, and 2-­‐Mercaptoethanol. This acted to preserve the functioning of the rhodamine (a fluorophore) for longer time intervals by scavenging oxygen free radicals. For visualization, the various solutions were added to a flow cell. This was created by cutting small strips of double sided tape and spacing them at regular intervals on a glass slide. Another smaller glass slide was then added on top to seal the device. This created channels that our solution was added too. For each flow cell, the solution was added and then flushed 5 minutes later to remove superfluous rhodamine tubulin in the bulk that caused noise which made the microtubules hard to visualize. The microtubules were visualized using a fluorescence microscope. This device emitted yellow light which caused the rhodamine bound to the tubulin to fluoresce, making the microtubules visible. All images were taken using 20x or 100x magnification. Results and Discussion: The main result of our experiment was consistent microtubule growth. Microtubules were incubated and successfully grown ten times. From these experiments, some patterns of results were derived. Microtubules at a 24 minute incubation period had an average length of 5 μm and a range from 2-­‐10 μm. The number of microtubules per 48x64 μm area could be precisely controlled by dilution of the “100%” solution and were found to be 40 microtubules for 20%, 50 microtubules for 30%, and 62 microtubules for 50%. Many of the results from these experiments were difficult to interpret due to the problem of photobleaching. Originally, all the images were taken at 20x magnification, however, the lower resolution makes it harder to find the microtubules. So to make it easier, we observed the microtubules at 100x. However, this caused the microtubules to disappear from the surface with less than a minute of observation. It was determined (after considerable experimentation) that the higher magnification the microtubules seemed to make them disappear considerably faster. This forced to develop better protocols to get around this problem but still keep the higher resolution of the higher magnification of 100x. The microtubules seemed to continue to grow after incubation. This was observed by leaving the flow cell in the fluorescence microscope after imaging and observing a couple hours later. This indicates that we can polymerize inside of the flow cell. Next semester, polymerization in the flow cell will be attempted, however, it will be done at a higher temperature to speed the process up. All of these results demonstrate control of microtubule growth and the indication that we can move to the next stage of the concentration device design of using electron beam lithography to make patterns that can electrically align the microtubules after growth. This will be attempted next semester. Conclusion: It is clear that there is a need for small scale concentration devices for lab-­‐on-­‐a-­‐chip applications. This project seeks to address this need. However, the research is in its initial steps. So far, the project has shown consistent growth, concentration, and length control of microtubules which are critical for the generation of pathways of microtubules for the next stage of the project. Next semester, the project will shift towards patterning the surfaces for electrically aligned microtubule pathways and the manipulation of kinesin for the concentration of our solute molecules.