Towards developing a porous membrane biosensor for the detection of bacteria based on lysis

Details

Supervisors

Gillis, Annika ; Raskin, Jean-Pierre

Faculty

Faculté des bioingénieurs

Degree label

Master [120] : bioingénieur en sciences agronomiques, à finalité spécialisée

Abstract

This master’s project aims at dealing with the growing issue of bacterial infections through food and water consumption as well as responding to the pressure building up on the health care system due to time-consuming bacterial pathogen detection methods. These issues have been addressed during this project by paving the way for the development of a porous membrane biosensor based on lysis. To achieve that, this study has been divided into two main parts, the first being the standardization of bacterial lysis with bacteriophages, using Escherichia coli as the bacterial model. It consisted into three steps which were the screening of phages through environmental samples where only water samples gave positive results (presence of phages). From there, nine phages isolated, and the one with the highest titer was used to compare it with lysozyme lysis which revealed some differences in lysis between the two methods, both visually and quantitatively. Finally, growth curves were done to study the effect of phages throughout 24 hours and revealed that they lyse more bacteria at 28°C than 37°C even though, the latter is the optimum growing temperature of E. coli. Once the standardization tests were done, a green fluorescent protein (GFP) expressing E. coli strain was induced and used to test the bacterial flow on the membranes through fluorescence using two methods: Spot thin-layer chromatography (TLC) and immersion TLC. Different membranes such as a silica gel fluorescent plate and two nitrocellulose membranes (CN95 and CN140) were used to perform these tests. First, PBS and LB Broth were compared as solvent and revealed that LB Broth caused background fluorescence and was therefore abandoned. Then, the flow between the phage and lysozyme lysates were compared and showed that due to bacteria agglomerating in the lysozyme lysate, the latter didn’t show any flow whereas the phage lysate flew. The absence of flow on the nitrocellulose membranes, due to non-specific interactions blocking the bacteria, was quickly noted and to address the issue, a coating was done using bovine serum albumin (BSA), a blocking agent, which helped the bacteria to flow. Antibodies were also used to control the flow of GFP but didn’t reveal anything to the naked eye which is maybe due to the too low concentration in the spots. Finally, the two methods (spot and immersion) were compared and revealed a bigger flow on immersion tests membranes which could be due to the higher concentration of bacteria. In conclusion, this work has shown that phages are good candidates for bacterial lysis as well as for the use in biosensors. It has also permitted to show the features that gave better results, and which can be used in the future to continue the development of this biosensor.