Revision as of 00:55, 2 November 2017

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 relays on three major building blocks:

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 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.

**Figure 3:** Sandwich immunoassay with Thrombin Aptamers 1 and 2 trigger extension in buffer

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.

In the graph below, we should the titration of the aptamer trigger we performed which show that a the toehold is trigger by this DNA at different concentrations

**Figure 2**: Shaded error graph resuming the results

To see how the levels of absorbance translate to in tube reactions, we prepared tube reactions of the toehold with and without the aptamer trigger extension to assess whether we could see the color change by eye

**Figure 3a**: Aptamer trigger with toehold in lysate reaction incubated for 2 hours att 37°C

**Figure 3b**:toehold without aptamer trigger in lysate reaction incubated for 2 hours att 37°C

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.

**Figure 4**: Toehold A in the iGEM backbone psB1C3

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.

As shown below, we demonstrate that we can design functional toeholds using ToeholdDesigner

**Figure 5a**: Kinetic measurements of the 4 generated toeholds in a usual lysate reaction
Shaded error graphs

**Figure 5b**: End point measurements of the 4 generated toeholds in a usual lysate reaction