Servizio Comunitario di Informazione in materia di Ricerca e Sviluppo - CORDIS

Hydrogel beads for cell immobilisation

The first stage of developing the immobilized yeast cells biosensor was to assess and optimized the condition that effect of the luminescence intensity that emitted by the immobilized yeast cells. The aspects of cell density in the hydrogel beads, the time that require for the cells induction and luminescence kinetics were examined.

The evaluation was carrying out both for alginate and PVA hydrogel beads. It was found that the best signal to noise ratio was obtained at a volume ratio of 1:2 alginate 3% (w/v): 5X106 [cells/ml]. The luminescence kinetics reflects a decrease of the luminescencentic signal during the incubation time.

The low ß-E2 induction factors at higher incubation times could be explained by the increasing basal luminescence background effect of the non-induced cells. As a result, 2.5 h of ß-E2 bead induction was set as the optimal induction time for further applications.

The characterization of the Yeast cell immobilization in PVA based hydrogels shows different pattern of induction by ß-E2 in comparing to alginate immobilize beads. The optimum induction time was longer (between 2.5-5.5 h), the luminescence intensity response was higher and also the sensitivity as reflected in the experiments of ß-E2 dose-response calibration curves.

The higher luminescence intensity reported by PVA hydrogels than that of alginate beads, could possibly be linked to the difference in viability of immobilized cells and gel porosity. Despite of the higher sensitivity and stability that shows the PVA hydrogel beads, since it is impossible to count the yeast colonies that trapped in the PVA, we chose to work with the alginate hydrogel yeast beads.

It has been previously reported that the addition of CoA to the luciferin solution increases the luminescence intensity and lowers the rapid inhibition of light production (25) in invitro conditions. We did not observe in changes of the luminescence intensity as results of the presence of CoA in our experimental system.

These results could possibly be explained by the non-specific effect of CoA on the non-ß-E2 induced control bead samples, which increased the basal luminescence background. It is also possible that CoA affects the metabolism of the yeast cells and thus causes changes in the luminescence via indirect ways, which need to be investigated.

The option to store the hydrogel yeast biosensor in low temperature for long periods is a remarkable advantage. it is allow us to perform the experiment at shorter time without growing the continually yeast culture, also it provided uniformly of the yeast beads biosensors.

The experiments of response to estrogenic EDCs (synthetic, endogenous and the phytoestrogens estrogenic chemicals), which were assessed, observed high sensitive response of the alginate immobilized yeast cells. This result leads us to monitoring of environmental samples.

The environmental samples demonstrate the ability of the immobilized yeast cells biosensor to serve as a tool for detection the presence of EDCs contamination in liquid samples - water or extracts of different sources.

The luminescent yeast based hydrogel bioassay should be applicable to the analysis of environmental water samples, as a primary screening tool, and as the biological and chemical immobilisation parts in the construction of fibre-optic biosensors.
In conclusion, we have demonstrated that it is possible to assess the estrogenic activity of a sample by measuring the reported luminescence with calcium alginate and PVA immobilized yeast cells.

The advantages of the developed bioassay are:
- Rapidity: the bioassay was characterized by a total period time of 2.5 h.
- Shelf life: long term storage of the yeast cells.
- Biocompatibility: hydrogels form a protective environment for the entrapped yeast cells protecting from contamination, thus allows the user to work under non-sterile conditions.
- Simplicity: the bioassay is simple to perform, the hydrogels are stable and easy to handle.
- Cost: inexpensive method in comparing to chemical analysis methods like LC-MS-MS which requires an expensive instrument and well-trained personnel.

In addition, we have shown that luminescent yeast based hydrogel bioassay should be applicable to the analysis of environmental water samples, as a primary screening tool, and as the biological and chemical immobilisation parts in the construction of fibre-optic biosensors.

The ability of long run storage, easy and cost effective preparation, no need of continuous cell culture cultivation, and only 2.5h of assay time make the immobilized estrogen inducible yeast based hydrogel a promising alternative to the current used recombinant yeast reporter gene assay of cell suspension.

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Michael JAKUSCH
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