Team:EPFL/Applied Design
Applied Design
aptasense is a biosensor targeting proteins. Based on cell-free synthetic biology, it harnesses the molecular machinery of cells, where parameters can be freely adapted, while avoiding the hassle of utilizing living cells and antibodies.
Our concept couples aptamer based affinity reagents to toehold switches, leading to a quick colorimetric output visible by eye. For a given target protein, aptamers can be found with the SELEX process within one to three months. This is a big improvement compared to producing the traditionally used antibodies from animals – aptamers are a lot cheaper and faster to synthesize, store and employ. The toehold switch triggered by the aptamer then renders the signal visible, making the protein assay possible. Together with the software we provide, functional toehold switches can be created from any given sequence within seconds, the whole process represents a novel approach to protein detection that is highly modular, fast and cost-efficient.
Why diagnostics
The ability to correctly diagnose diseases plays a key role in controlling their spread. However, many developing countries are unable to provide adequate diagnostic services to their populations. For example, according to the World Health Organization, only 64% of new tuberculosis cases are detected each year, hindering efforts to eradicate the disease1. The unavailability of diagnostic tests may be due to lack of funds to properly equip and run enough laboratories or lack of trained personnel1. Furthermore, point-of-care diagnostic tests have so far been ineffective due to problems in distribution logistics, high-costs, or their need for trained personnel to administer the tests. Our project set out to address this issue. From the start, we identified several key design requirements that our product would have to meet. It would need to be affordable to meet the budget constraints of developing countries. It would need to be easily distributable and able to work under a wide range of conditions. Moreover, it would need to be easy and intuitive to use so that only minimal training would be necessary to administer the test.
So far, detecting biomarkers usually requires a laboratory setting. We would like to present a novel approach to this problem and introduce our concept.
Our detection scheme
Antibodies are the most prominent research tool to detect the presence of a protein in a given sample.
However, looking more closely at the established ELISA scheme, one may see the advantages that switching from antibodies to aptamers may bring.
First and foremost, aptamers can target proteins and small molecules in a way antibodies cannot. In fact, the minimal size of the proteins that an antibody can target is 600 Daltons, whereas that of an aptamer is about 60 Daltons.
A second reason to choose aptamers over antibodies is the development process. To generate an antibody, you would need an immune response in an animal model. This is quite a long process that takes about 6 months. In comparison, you could generate aptamers within a couple of months performing a SELEX over an initial DNA library.
For these reasons, we opted out of ELISA in favor of an ELISA derived assay based on aptamers.
In this assay, the target protein would be bound by an aptamer pair in a sandwich assay.
How we address these challenges
We wanted to optimize our product to create a tool as cost-efficient, robust and easy to use as possible.
The different parts of our project address each one of these requirements.
Firstly we kept the costs of our biosensor low by using crude lysates from E. Coli to make our cell-free expression systems (CFES) instead of the PURE system used by Pardee et al2. According to a paper from Sun et al. 3, home-made lysate costs approximatively 0.03$ per μL (depending on the method of cell lysis) instead of 0.79$ for PURExpress.
Then all the aptamers and toehold switches, because they are made of DNA, are stable at room temperature and do not require a cold-chain storage to transport. Additionally the lyophilized cell-free expression systems were shown to also retain activity at room temperature for extended periods2. This makes our sensor easily distributable without the need for special infrastructures, and resistant to varying temperatures across different regions.
Finally, by using a CFES to amplify the sensor signal, there is no need for expensive fluorescence readers or other specialized machinery to read the output. A simple change of color indicates whether the test was positive or negative. The only skills required to administer the test is how to pipette and potentially how to draw a blood sample. Furthermore, while we did not have the opportunity to try it out, we had planned on making the sensor fully self-contained by implementing it on paper, inspired by the paper microfluidic devices developed by the Whitesides group at Harvard4.
Finally, because our sensor is not specific to any protein, it can be used to diagnose a wide range of diseases. In addition the sensor can be retooled for any number of other possible uses. For example it could be used to diagnose crop diseases, another major issue for developing countries. The impact of our biosensor could also extend beyond developing countries with over-the-counter tests to identify microorganisms in your garden, analyze your gut microbiome, or authenticate your wine based on the strain of bacteria it contains. Our sensor also pioneers the idea of distributed bio-sensing, allowing for large scale data acquisition in macro-level studies.
In summary, because our biosensor’s modularity, ease-of-use, low-cost and remarkable flexibility, it can impact a wide spectrum of sectors and make a meaningful difference in tackling major issues such as the proliferation of diseases in developing countries.
References
1. McNerney, Ruth. "Diagnostics for developing countries." Diagnostics 5.2 (2015): 200-209.
2. Pardee, Keith, et al. "Rapid, low-cost detection of Zika virus using programmable biomolecular components." Cell 165.5 (2016): 1255-1266.
3. Sun, Zachary Z., et al. "Protocols for implementing an Escherichia coli based TX-TL cell-free expression system for synthetic biology." Journal of visualized experiments: JoVE 79 (2013).
4. Martinez, Andres W., et al. "Diagnostics for the developing world: microfluidic paper-based analytical devices." (2009): 3-10.
