Building a cell-free biosensor for protein detection based on aptamers for target recognition and toeholds for signal generation
How does our biosensor work
1. How do we detect the presence of a target protein
To demonstrate the ability of our aptamer pair to bind to their target protein Thrombin we used microfluidics, and measured the fluorescence of the Cy3 probe which is attached to the second aptamer. If high levels of fluorescence are measured, this indicates that Thrombin was bound between the two aptamers.
Our experimental set up was as follows: the biotinylated Thrombin Aptamer 1 was first flown, then human thrombin in
the top half of the chip and finally the Cy3-labeled Thrombin Aptamer 2 trigger extension in the whole chip.
2. How do we generate a colorometric signal upon protein detection
To generate the signal upon the human thrombin detection, we used toehold switches and the aptamer trigger that recognizes the human thrombin.
3. Streamline toehold design by writing a software
Generating new toehold sensors requires in-silico processing. It is a quick step (~5 min) if a tool that pipelines the required processes is available. In the scope of our project we generated our own switches targeting Hepatitis C viral RNA and sucessfully proven that the toeholds are functional, i.e they unfolded only in the prescence of a complementary sequence to allow the translation of the downstream reporter lacZ.
We tested the ToeholdDesigner's output, and chosen the best 4 toeholds for a unique sequence of the Hepatitis C virus we have found using different online resources such as BLAST, in our home-made lysates.
The results are shown below