Microfluidic smart monitoring of Escherichia coli in water – BACTERIEAU

The microfluidic E. coli detection strategy developed is innovative because it is based on competition between saccharides, antibodies immobilised on the sensor (electrode) and bacterial epitopes (Figure A). The procedure involves the prior elimination of non-target bacteria by using glycomimetics (GMs) such as calixarene molecules containing sugar residues. These GMs induce aggregation of non-target bacteria, which are removed by sequential filtration before injection into the microfluidic device. The transduction signal indicative of interactions with the electrode coated with E. coli specific antibodies comes from the change in the interface between the electrode and the filtrate and is quantified by impedancemetry or voltametry.
Thus, different saccharides were synthesised for grafting and their affinity to different bacterial antibodies was evaluated. Work aimed at studying the possibility of sensor regeneration was carried out. Finally, several water pre-treatment methods were tested to ensure the best possible access to E. coli.

The specific detection of functionalized monosaccharides by gold electrodes was demonstrated. Methods for the synthesis of oligosaccharides for grafting onto microfluidic systems were optimised.
Significant aggregations of E. coli were obtained with the fucose ends of the synthesised oligosaccharides as well as with IgG antibodies which are easily grafted onto inert supports. The regeneration of the sensor was validated.
Sample pre-treatment by sequential filtration was optimised by the addition of Calix-gal which reduces residual E. coli entrapment.

The development of the oligosaccharide synthesis pathway has lifted scientific barriers, although further development is still required. In terms of microfluidics, studies to improve the protocols for grafting organic compounds onto the sensor and to limit the phenomena of aggregation of bacteria in flow remain to be carried out. The results obtained on sugar/glycomimetic interactions make it possible to envisage new therapeutic approaches, by working on a better understanding of the interactions between viral particles, chemical substances and host cells.

BACTERIEAU resulted in 8 publications and scientific communications, the latter being limited due to the health context and confidentiality aspects.

Monitoring of fecal pollution and protection of water resources, of drinking and bathing waters (fresh and salt water ecosystems) is one of the cornerstones of environmental protection in Europe. Escherichia coli is considered as one of the best indicator of microbiological quality of water, and has been used to predict the risks of gastro-enteritis for bathers among either marine and freshwaters. Current technologies for water-quality monitoring are based on frequent sampling, time consuming, labor-intensive and require extensive training. Therefore, the development of reliable, portable, rapid and cost effective analytical methods for monitoring microbial numbers in water is a critical public health issue.

BACTERI’EAU aims to develop a new microfluidic and multiparametric device using an innovative electrochemical immunosensor based on carbohydrates for highly specific detection of E. coli in water to monitor in situ and in real time (30 minutes) bacteriological quality of water. Two sensors will be developed and integrated on the chip: one targeting total E. coli and the other clinically-relevant pathogenic E. coli (Shiga toxin-producing E. coli). This solution will be easy of use, mobile or in-line for recurrent analysis (monitoring), allow low cost analysis and have a sensitivity below 100 CFU/100 mL (as required by the regulation for bathing water) allowing quantification between 100 and 2000 CFU/100mL.

BACTERI’EAU will thus allow answering agrifood, environmental (e.g. recreative water, shellfish) and industrial processes (picking up environmental water) monitoring key issues.

BACTERI’EAU is public-private collaborative project associating two academic teams and two SME with needed skills and know-how on organic synthesis, electrochemical sensing, surface modifications, microfluidic, microbiology, and an end-users committee. This project directly addresses the challenge #1 of the ANR call 2016, axis 3 “Health - Environment”.