On 25 August at 12:15 Eerik Jõgi will defend his doctoral thesis “Development and applications of E. coli immunosensor” for obtaining the degree of Doctor of Philosophy in Chemistry.
Associate Professor Toonika Rinken, University of Tartu
Senior Researcher Roman Viter, University of Latvia (Latvia)
The quality of water is among the major global problems usually associated with drinking water. However, problems with the physical, chemical, and biological pollution of bathing water are increasing. The biological pollution is commonly assessed using microbiology methods by identifying and quantifying microbial indicator organisms.
The most common indicator species for water analysis is Escherichia coli – gram-negative, rodshaped bacteria generally found in the guts of warm-blooded animals. Most E. coli strains are harmless, but there is also a group of E. coli strains, which are human pathogens Uropathogenic E. coli (UPEC) is the main human urinary tract pathogen. The most common method for E. coli enumeration is still microbiological cultivation. This method is reliable and simple, but the analysis time is long, the sensitivity is quite poor and the cultivation requires special lab conditions. In addition, E. coli can be detected with qPCR.
A good alternative for E. coli indication and enumeration are biosensor-based systems, which can provide short analysis time, high specificity, and sensitivity. Biosensors also offer options for automation and on-site analysis required to meet modern requirements for data collection.
The objective of this thesis was the design and production of an E. coli-specific immunosensor, its testing for potential applications in environmental monitoring and clinical laboratory analysis, and validation of the biosensor results. The proposed E. coli immunosensor integrates the use of polyclonal E. coli antibodies for bio-recognition and single-use microcolumn analysis system for the rapid detection of E. coli from bathing water and urine samples. The immunosensor the detection limit was below 10 cells/ml, and the working range was between 10…108 cells/ml.
In urine, there was no inference other bacterial species present in urine to the biosensor signal, as there is a small probability of the presence of dead and/or fragmented E. coli cells in urine. The E. coli biosensor results were in the same range as those obtained with qPCR and cultivation methods.
The analysis of the biosensor signal in bathing water samples revealed that the signal was strongly affected by dead cells, cell fragments, and different coliforms, which are abundant in natural waters. The proportion of cultivable E. coli cells in the immunosensor entire signal was only about 10%. The signal of non-cultivable E. coli cells (measured by qPCR) formed 30% of the immunosensor signal and the majority of the measured signal, 60%, was most likely generated by different forms of coliform bacteria and E. coli cell fragments.
Using renewable, single-use E. coli immunosensor is an excellent alternative to time consuming microbiological and molecular methods for analyzing complex natural samples. These immunosensors can significantly shorten the time required to determine and quantify E. coli. It could be used for automated analyses, as quick identification of E. coli allows to take timely measures to minimize potential health risks.