Building a cell-free biosensor for protein detection based on aptamers for target recognition and toeholds for signal generation

How does our biosensor work

Figure 1: Scheme of our biosensor concept

Our system relies on three major foundations:

- Aptamer pair detects the presence of a protein in a sample
- Aptamer can trigger the toehold
- Translation of the downstream reporter for signal generation in our home-made cell-free lysate

1. How do we detect the presence of a target protein

Figure 2: Protein detection scheme using ELISA derived sandwich-based assay

To demonstrate the capability of our aptamer pair to bind to their target protein, Thrombin, we used microfluidic assays, 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 setup was as follows: the biotinylated aptamer 1 was flown first, binding to the surface of the chip. Then, Thrombin (target protein) was flown only in the top half of the chip and finally the fluorescently labeled aptamer 2 with trigger extension in the top and bottom half of the chip (Fig. 3a). For details on how to interpret the read-out and how the experiment was performed, visit this page.

Figure 3a & 3b: Sandwich assay with Thrombin aptamers 1 and 2 trigger extension in buffer
Right side : Fluorescent read-out

2. How do we generate a colorometric signal upon protein detection?

To generate the signal upon the detection of Human α-Thrombin we used toehold switches and the aptamer extended with trigger that recognizes Thrombin.

Figure 4: Toehold opens up after aptamer with trigger sequence extension anneals, beta-galactosidase is expressed

In the graph below, a titration of the aptamer extended with the trigger sequence shows that the toehold is triggered by this DNA at different concentrations. For more details on experimental setup and trigger modularity, visit this page.

Figure 5: Aptamer trigger titration in cell lysate

To see how the levels of absorbance translate to reactions in tubes, we prepared tube reactions of the toehold with and without the aptamer with trigger extension to assess whether we could see the color change by eye.
Figure 6a: Aptamer trigger with toehold in lysate reaction incubated for 2 hours at 37°C
Figure 6b: Toehold without aptamer trigger in lysate reaction incubated for 2 hours at 37°C

3. Streamline toehold design by developing a software

Generating new toehold sensors requires in-silico processing. It is a quick (~5 min) step if a tool that streamlines the required processes is available. In the scope of our project we generated our own toehold switches targeting Hepatitis C viral RNA and successfully proved that the toeholds are functional, i.e they unfold only in the presence of a complementary sequence to allow the translation of the downstream reporter LacZ.

Figure 8: Toehold A in the iGEM backbone psB1C3

We tested the Toehold Designer's output, and chose 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.

As shown below, we demonstrate that we can design functional toeholds using Toehold Designer.
Figure 9a: Kinetic measurements of the 4 generated toeholds in a usual lysate reaction
Figure 9b: Endpoint measurements of the 4 generated toeholds in a usual lysate reaction