Reporter

For this project it was necessary to select a method by which our system could inform the user of a positive test result, that is, we needed a reporter system. After careful consideration, we decided upon an enzyme/catalyst reporter as we believed that using a reporter such as green fluorescent protein (GFP) simply would not provide a strong enough of a signal for a portable device. GFP would provide a constant signal that would not increase in intensity over time. The fluorescent output of GFP would also require the device to have the ability to excite the protein with the appropriate wavelength of light. This would add to both the complexity and cost of the device, making it harder to offer a portable and cheap solution.

We decided that an enzyme with a colour output would be more suitable and easier to implement in the field. Many common enzymes were analyzed for this purpose, however the main point that came from our conversations with industry was that our system would have to be fast in order to fit into their infrastructure. Because of this, the reporter we chose would have to be not only sensitive, but rapid as well. One example is horseradish peroxidase. This enzyme has high enzymatic activity and is commonly used in many biological applications. One problem with its use however is that it cannot be effectively produced within E. coli. There are other common enzymes that do not have this issue such as alkaline phosphatase. These enzymes however are often multimeric which presents an issue with our in vitro strip system. If the enzymes are not able to assemble correctly with the rest of our proteins, the device will be ineffective. We also modelled the activities of some of these common reporters based on literature review in order to aid in informing us of the appropriate reporter for our system. Upon further investigation, we found two candidates: the ampicillin resistance enzyme beta-lactamase and ferritin, a protein which can be chemically modified to become a strong catalyst. Why two different reporters? Characterizing two reporter systems, both of which have their own key advantages, gives us flexibility in our platform technology to meet the different criteria that will exist for different detection systems. In addition, during our characterization, if one reporter does not act as it is meant to, we have the ability to focus on the other reporter for our system. Click the links below to learn more about these two systems:

Prussian-Blue Ferritin

Beta-Lactamase