Team:Pasteur Paris/Demonstrate
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Demonstrate
Systematic Evolution of Ligands by EXponential enrichment (SELEX) - Selection of specific aptamers
The Whole-Cell SELEX method was performed during the summer in order to produce DNA oligonucleotides, commonly referred as aptamers, that specifically bind to target ligands on either E. faecium or S. aureus strains. After optimizing the most important steps of the SELEX procedure (amplification by PCR, digestion using lambda exonuclease, and purification with phenol/chloroform extraction), we performed 10 successive rounds on both E. faecium and S. aureus strains. A very large DNA library was used at the beginning, and at the end of the SELEX procedure, aptamer candidates were sent to be sequenced. We managed to get two specific sequences for E. faecium. The binding affinity and the specificity of these aptamers were characterized through electrochemical measurements using a voltmeter and the strain-specific electrodes. Because no aptamer has yet been published for E. faecium, these two sequences represent a huge success for our team. Unfortunately, no specific sequence could be found for S.aureus.
Computational modeling of aptamer find by SELEX method
Our two aptamers specific for E.faecium were sequenced by Eurofins Genomics. We used Mfold website to model aptamer folding. In accordance with ionic conditions and temperature of our biological samples ([Na+]=140mM, T=25°C) we obtained modeling foldings. In order to complete this simulation, it would be interesting to compare our aptamer folding with analogue DNA aptamers folding.
Biobrick BBa_K3176020
Binding the aptamers to carbon nanotube electrodes
In order to detect bacteria, we first had to connect two parts together: the highly specific aptamers and the nanotube-coated electrodes. To ensure this step was done correctly, we used an electrochemical analysis technique called cyclic voltammetry. The results demonstrated that we have indeed modified the electrodes, though perhaps not in the way we had hoped; however, later experiments proved the binding was sufficient to use the electrodes like we wanted.
Selectively detecting pathogens with aptamers/carbon-nanotube electrodes
Using a voltmeter and the strain-specific electrodes, we have proven that we can detect a signal in the presence of bacteria. We have also demonstrated that the electrodes are only specific to one strain among those available to us.
Entrepreneurship
We have built a real business model to highlight how our company will create and deliver value. To be able to carry it out, we had to lead numerous research and interviews in order to meet a genuine need, requiring an analysis of our "customer segments". A market and a benchmark analysis completed these studies. We were able to assess the strengths and weaknesses of our project in order to be able to carry it out successfully. We were inspired by the Agile methodology by integrating potential users into our approach and making short iterations to minimize the risk of failure. We demonstrated that we are able to be financially responsible by finding grants to ensure this project, through oral presentations to investors. In addition, we have made a real financial projection over three years to show the viability of our project.
Wiki
We have created our wiki from scratch. Indeed we have learn html, css and javascript codes during the summer. We managed to make all our animations functional and to establish a graphic consistency in our pages.
Product design
In order to develop a new diagnostic device suitable for users, we performed several product design steps.
We have been looking for potential users of our tool, so we interviewed Dr Pedro Clauteaux (who coordinated medical activities in secluded areas of the Amazon rainforest and Venezuelan highlands), Dr David Lebeaux (physician and researcher at European Hospital Georges Pompidou) and Sylvie Renard-Duboie (Deputy Director General of French Ministry of Health). In the light of these three eclectic interviews, we planned to develop a device which is waterproof and robust (to avoid false results), lightweight and small (easily transportable) and does not need complex equipment to prepare samples (meaning it can be used without medical laboratory). Moreover, our device need to be suitable for biological samples which can contain pathogens.
We planned to use polyetheretherketone (PEEK) millifluidic system because this kind of system is small, robust, waterproof, lightweight and suitable for biological samples. Details about this PEEK millifluidic system were discussed with the SMC company (leader company in automatic system). Thanks to SMC engineers’ expertise and keeping up with our constraints, Claire Dumont, our design coach, made sketches of our device. We worked together with her to find the best sketches compared with diagnosis device available on the market.
We designed the millifluidic path inside the device from the sample injection site to the waste cartridge. To ensure it worked, we studied and chose, in collaboration with SMC, the different components to be added to the system in order to automate it, such as pumps and electro-valves. We couldn’t order these parts from Japan. However, we printed a 1:1 scale model with a 3D printer and photosensitive SLA resin. Furthermore, we designed and laser-etched our own proof of concept millifluidic device out of PMMA, containing the electrode. Thanks to Claire Dumont we learned how to use McNeel Rhino 3D. This software was very useful for 3D modeling and 3D printing.
Design of our device