Resumen de Dispositius integrats per la concentració i detecció de bacteris associats a l'aigua
Microbial water quality monitoring aims to protect consumers from diseases caused by pathogens transmitted by ingestion, aspiration (air transmission), or contact with contaminated waters. In the last decades, biosensor technology has been postulated as one of the most promising alternatives to substitute the tedious, bulky, and long-time requiring conventional methods. Nevertheless, most biosensors lack the ability to process large water volumes required for current microbial water quality regulations, which is translated into high detection limits. In this regard, the development of devices able to integrate the concentration of bacteria present in large water volumes, and their detection becomes an issue of relevance.
This thesis presents the development of a device able to use microfiltration membranes as support for both processes, concentration, and detection. The device consists of a flexible concentration platform, which is modified according to the detection needs of the target bacteria. Herein, three different microorganisms have been selected as the work scenarios for the concentration/detection prototype. Escherichia coli has been selected for its extensive use as indicator of fecal pollution, Legionella pneumophila, for its health importance and Shynechocystis sp. as model cyanobacterium for their health and environmental impact.
As is the case of most bacteria, Escherichia coli and Legionella pneumophila require labeling in order to make them detectable. Thus, an on-filter immunoassay has been developed, employing the membrane used for concentration, also as the support for the immunodetection. The biggest drawback of using microfiltration membranes as support for immunodetection is the nonspecific binding of antibodies to the membrane, consequently giving false positives. Thus, different membrane materials, membrane blocking reagents, and antibody washings have been tested to find the best combination for both E. coli and Legionella fast detection.
The immunodetection is carried out using antibodies enzymatically labeled with horseradish peroxidase and 3,3',5,5'-Tetramethylbenzidine (TMB) as substrate. Hence, as TMB is widely used either as colorimetric or redox substrate, it has been demonstrated that the developed on-filter immunoassay can be coupled to both absorbance and chronoamperometric measurements. The whole assay takes 2 h reducing significantly the time needed by conventional methods (2-10 days) and providing a detection limit lower than 10 CFU·mL-1, which could permit achieving the limits established by regulation standards.
The developed device has also been adapted to detection of cyanobacteria. Cyanobacteria contain pigments able to absorb light and emit fluorescence naturally, acting as intrinsic bioreceptors. Moreover, the photosynthetic pigment phycocyanin (PhC) allows to distinguish cyanobacteria from eukaryotic algae. Therefore, the concentration device has been provided with optical elements required for on-filter cyanobacteria detection. A light-emitting diode (LED) to excite PhC and a detector that collects its fluorescence emission have been implemented, in addition to integrating the electronics necessary for their control. The developed system is able to detect cyanobacteria concentrations within the vigilance level established by WHO (<500 cell·mL-1) in less than 10 min even in real samples. Moreover, the use of low cost miniaturized optical components has allowed the design and development of a portable and straightforward prototype suitable for fast and in-situ detection and quantification of cyanobacteria in water samples.
The performance and versatility shown by the microbial detection platform designed and tested in this thesis allows us to envision this new technology as a viable alternative towards a fast and cost-effective detection system for microbial water quality monitoring.
