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Project Inspiration and Description
DIANE
Diagnosis is Now Easier
Diane is one of the most
important figures in the Greek-Roman pantheon. Goddess of hunting,
war and night, she lives in the woods surrounded by nymphs.
While Diane, praised for her dexterity and vivacity, travels the
forests in search of prey, the DIANE device precisely seeks the
bacteria responsible for an infection.
Today, someone dies of sepsis every 5 seconds in the world [1]. Sepsis is a clinical syndrome of organ malfunction potentially fatal provoked by a deregulated host response to the infection. Its current diagnosis relies on hemoculture, a slow process (24 to 48 hours), while every hour spent to detect which strain is responsible for the infection greatly enhances the risks of neurological and muscular complications and drastically reduces the chances of survival.
CURRENT DIAGNOSTIC METHODS
24h - 48h
In this context, we decided to create DIANE :
DIANE is a swift diagnosis device which aims at allowing the physicians to get almost immediate results of the body fluid sample analysis (blood, urine, saliva, cerebrospinal fluid etc) and to quickly provide the proper treatment to the patient.
Time is a crucial element. As advised by the international guidelines for the management of sepsis and septic shock of the 2016 Surviving Sepsis Campaign [2], physicians often have to act even before getting the results of the analysis and launch a broad empirical antibiotic treatment based on hypotheses regarding the origin of the infection. This can lead to an inadequate and thus inefficient treatment, a loss of time and can eventually result in the diminution of the patient’s chances of survival. Moreover, bring wrong or no specific treatment contributes to antimicrobial resistance (AMR). This is the most problematic as it would become the most deadly cause in 2050, ahead of cancer, with more than 10 million deaths worldwide.
In that way, DIANE would be a major tool against sepsis, but not only. More broadly, it could be used for any situation suggesting a bacterial infection with the advantages of being almost instantaneous, specific and selective. A rapid diagnosis would allow to provide the proper antibiotic treatment immediately and therefore to enhance the chances of survival, to reduce the complications due to the infection, as well as contributing to the fight against antimicrobial resistance, which is one of the biggest current threats for the global public health.
The diagnostic method we developed relies on an electrochemical detection of bacteria using aptamers, single-strand RNA or DNA capable of binding to a specific ligand, connected to carbon nanotubes electrodes.
We chose to work with DNA aptamers for their stability. They are resistant to room temperature (around 25°C) and not as easy to degrade as RNA strands. Aptamers were designed to specifically bind a bacteria.
To this end, we performed the SELEX (Systematic Evolution of Ligands By Exponential Enrichment) method [3]. SELEX is based on several rounds of oligonucleotide selection, in order to become more and more selective with the element we want to target.
The starting library is composed by 10^15 80 base-pair random oligonucleotides. This library is put in solution with one strain of bacteria, in order to make possible the specific interactions between them. Non-binding aptamers are then removed from the solution while binding aptamer stay with the bacteria. This cycle is repeated around ten times to finally obtain the aptamers showing the best affinity with the target.
We performed the SELEX on Staphylococcus aureus and Enterococcus faecium, two pathogens that cause important hospital-acquired infection. They are, above all, on the World Health Organization priority pathogens list as they require a particular attention, and R&D efforts [4].
The protocol we developed and used is of course applicable to other bacterial strains. We will also be able to develop aptamers and electrodes for numerous other pathogens, such as viruses and parasites, since the method relies on recognition of specific patterns on any frame.
Once one aptamer is selected, it is bonded to a carbon nanotubes electrode. Carbon nanotubes electrodes are highly sensitive as they allow to detect 1 CFU in 5 mL [5]. Each electrode will therefore correspond to the detection of one pathogen.
In a diagnostic case, the contaminated sample will be introduced into the device and carried out towards the analysis area. Bacteria will specifically bind to their selected aptamer, inducing a conformational change of their shape. This modification in the electrode’s environment will trigger a change in potential, thus leading to an electrical signal. This is how we can report the presence of pathogens in a biological sample
Simplified operating principle for specific bacteria detection using a regular electrode and another one with our aptamers bound to carbon nanotubes
Our job does not settle for making a electrod-aptamers system capable of detecting and quantifying specific bacteria but it also consists in the creation of an actual prototype capable of indicating which strain is responsible for the infection to a physician, taking into account the user scenario, security, and ethical responsibility linked to the device. We also aim at creating this device in such a way that it could be used in remote locations during humanitarian missions.
More specifically, its size allows it to be transportable, and carried out by hand. It is designed with several specific areas from the introduction of the sample by the physician to the outlet, inside a waste cartridge. An automated millifluidic circuit allow the transport of the sample towards the analysis zone which contains the electrodes coated with aptamers. This area is directly connected to the electrical circuit within an arduino microcontroller. After each use, an automated washing system is launched in order to avoid any kind of contamination. Our technology is compatible with small volumes of samples. All the fluids are therefore collected into the waste cartridge which contains a super absorbent polymer, allowing an easiest waste treatment process.
References
[1]https://www.pasteur.fr/fr/centre-medical/fiches-maladies/sepsis-septicemie
[2] Rhodes, A., Evans, L.E., Alhazzani, W. et al., “Surviving Sepsis Campaign: International Guidelines for Management of Sepsis and Septic Shock: 2016”, Intensive Care Med (2017) 43: 304
[3] Kwame Sefah, Dihua Shangguan, Xiangling Xiong, Meghan B O’Donoghue & Weihong Tan, “Development of DNA aptamers using Cell-SELEX”, Nature Protocols, 2010, vol 5, pages 1169–1185
[4] “Global priority list of antibiotic-resistant bacteria to guide research, discovery, and development of new antibiotics”
[5] Gustavo A. Zelada-Guillén et al., “Immediate Detection of Living Bacteria at Ultralow Concentrations Using a Carbon Nanotube Based Potentiometric Aptasensor”, Angew. Chem. Int. Ed., 2009, vol 48, pages 7334 –7337 Protocols, 2010, vol 5, pages 1169–1185