Difference between revisions of "Team:Pasteur Paris/test/special prizes"

To add a small stone to this objective, we decided to contribute the "good custody" of antibiotics with the creation of a new diagnostics device (DIANE) to detect pathogens in blood with selectivity, rapidity and sensitivity. To achieve this ambitious goal, we had to develop in parallel: the aptamers against members of a WHO list of pathogens, the carbon nanotube-based electrodes for their detection, the millifluidic analysis system which serves respectively to introduce and remove sample into and from the detection cavity of the device, the context dependant usage scenario, the electronic system which displays the results and address ethical and regulatory issues that may arise from the use of DIANE.

To develop a device as sensitive as possible we decided to design its core around an aptamer-based carbon nanotube electrode. Indeed, this kind of electrode is extremely responsive as it can react to as little as 1 bacterium per 5 ml of solution. First, we needed to find a selective set of aptamers for two of the pathogens we decided to work on: Staphylococcus aureus and Enterococcus faecium. These aptamer sets were identified by the whole-cell SELEX approach (wiki1 link). Afterwards aptamers were attached to carbon nanotube electrodes chemically (wiki2 link). Thanks to their 3D structure, the aptamers can recognize selectively bacterial membrane proteins (surface or trans) or other molecules (lipopolysaccharides). When the aptamer, linked on the carbon nanotube electrode, binds to another molecule on the surface of the cell, the aptamer undergoes conformational changes. This alteration in geometry leads to a change in electrostatic interactions (charge attraction/repulsion) with ions, which in turn induces a potential difference along the electrode. It is this potential difference that we want to measure.

We wanted to create a device small enough to be easily portable (for remote regions, humanitarian areas and urban “point of care” testing). Therefore, we plan to use a millifluidic system which constitutes the path for the biological sample into the detection cavity. In fact, the aptamer-based carbon nanotube electrodes are in the detection cavity protected from external contaminants and shock. The millifluidic system is made of polyether ether ketone (PEEK) which is chemically resistant, has a very low friction coefficient, and is approved by the FDA.

The circuit is designed without nooks and crannies to avoid static pressure zones and therefore bacterial adherence with the formation of a biofilm. After analysis, the liquid biological sample is expelled into a disposable recovery bag. The biological sample is moved across the millifluidic circuit thanks to a miniature pump incorporated into the device. We worked with millifluidic engineers from SMC company (www.smc-france.fr) and with an industrial designer (ex-iGEMer) to optimize the external casing of the device and the milli-fluidic system.

The potential difference readout is processed by electronic system layer. The electrical signal induced by aptamer conformational changing is recorded through a voltage gate/port on an Arduino board.

The software code we included directs the Arduino board to compare this potential against a calibrated reference potential, to perform an analogic to numeric conversion and to display the measurements results on an LCD screen.

To make the measurement as reliable as possible, the Arduino board has been set up to do an average on 5000 points before printing a value so that high frequency noices are eliminated. The setup will allow to generate a result within 5-6 minutes.

We expect our device will be able to improve access to treatment for all thanks to its speed, precision, portability, robustness and its ease-of-use for non-expert users. Through this new rapid diagnostics tool we hope to reduce unnecessary use of antimicrobials by providing essential information on the exact type of pathogen to medical personnel, which in turn this will contribute to cutting down the selection pressure on pathogens and limit antibiotic resistance.