Resumen de Estratègies d'immobilització bacteriana d'alta densitat per al desenvolupament de biosensors microbians
Microbial biosensors are analytical devices that use microorganisms as recognition elements. Microorganisms are immobilized on the surface of a transducer in such a way that the microorganism-analyte interaction generates a signal (electrochemical, optical, among others) that can be quantified. These microbial biosensors can be applied in the fields of clinical, industrial or environmental diagnosis with the advantage of being portable, simple and inexpensive alternatives to many laboratory-based methods. Unfortunately, development of microbial biosensors has been hindered by important technical limitation related to: (i) poor reproducibility, due to non-reproducible cell immobilization protocols, (ii) low sensitivity, by the difficulty of immobilizing high bacterial concentrations, and (iii) short life-time, due to cell death during immobilization or storage.
This thesis describes the development of two immobilization strategies that allow reproducible confinement of microorganisms at the electrode surface, with high densities and in a reproducible manner, while providing a physiological environment that allows adequate diffusion of nutrients, ensuring the functionality and viability of the trapped microorganisms.
In one of the strategies, (1) microbial cells have been trapped in an alginate-graphite polymeric matrix electrodeposited at the electrode surface using very soft and biocompatible conditions (i.e. room temperature, aqueous medium, neutral pH, etc.). Conductive alginate-coated electrodes are obtained after potentiostatic electrodeposition of graphite-doped alginate samples (up to 4% graphite). The presence of graphite reduces electrode passivation and improves the electrochemical response of alginate-coated sensors. Bacterial entrapment in the conductive matrix is highly efficient (4.4x107 cells per electrode), reproducible (CV < 0.5%) and does not compromise bacterial integrity or activity.
In the second strategy, (2) microorganisms are trapped in polyethersulfone when the polymer, initially dissolved in organic solvents, precipitates in aqueous medium through a process of phase inversion. We have shown that microorganisms can be incorporated during membrane formation and remain viable. With this method, 300 µm PES membranes were reproducibly obtained containing up to 2.3x106 cells per electrode, with an entrapment efficiency of 8.2%, while maintaining acceptable levels of cell integrity or viability.
Both systems have been applied to immobilize E. coli at the surface of screen-printed electrodes to develop biosensors in which microorganisms act as recognition elements. Biosensing has been performed electrochemically through ferricyanide respirometry, with metabolically-active entrapped bacteria reducing ferricyanide in the presence of glucose. The analytical performance of the two amperometric microbial biosensors has been assessed carrying out a toxicity assay using 3,5-dichlorophenol (DCP) as a model toxic compound. In both cases, biosensors provided a concentration-dependent response to DCP with half-maximal effective concentration (EC50) of 3.5 ppm (alginate) and 9.2 ppm (polyethersulfone), well in agreement with reported values. This entrapment methodology is susceptible of mass production and allows easy and repetitive production of robust and sensitive microbial biosensors.
