Luminescent yeast cells entrapped in hydrogels for estrogenic endocrine disrupting chemical biodetection.
In the construction of luminescent yeast cell based fibre-optic biosensors, we demonstrate a novel approach for estrogenic endocrine disrupting chemical (EDC) biodetection by entrapping genetically modified Saccharomyces cerevisiae cells, containing the estrogen receptor alpha-mediated expression of the luc reporter gene, in hydrogel matrices based on calcium alginate or PVA. In order to insure a significant signal, an optimal immobilization ratio of 1:2 alginate 3% (w/v): 5 x 10(6) [cells/ml], respectively, was used with the highest 17-beta-estradiol (beta-E2) induction factor after 2.5 h of incubation with 10[nM] beta-E2. It was shown that biocompatible alginate beads, 4.27-4.55 x 10(5) [CFU/bead], which were characterized by a detection limit of 0.08[microg l(-1)] and an EC50 of 0.64[microg l(-1)] for beta-E2, retained their viability for luminescence measurements after 1 month of storage at -80 degrees C slow freeze condition, and thus repeated cell cultivations were not required. The assay reproducibility for each tested EDC, represented by the coefficients of variation (CV), ranged from 4.35 to 18.47%. An alternative immobilization method, based on a room temperature partial drying of polyvinyl alcohol (PVA) solution (LentiKat Liquid) and cell suspension mix, was investigated with only a slightly lower detection limit for beta-E2 than that reported with alginate beads. Alginate yeast based hydrogels may also be applicable to the analysis of environmental water samples since the trend of detected estrogenic activities with alginate beads roughly correlated with LC-MS-MS analytical results.
Fine
T
T
Department of Biotechnology Engineering, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel.
Leskinen
P
P
Isobe
T
T
Shiraishi
H
H
Morita
M
M
Marks
R S
RS
Virta
M
M
D001681:Biological Assay / Q000295:instrumentation* / Q000379:methods; D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods; D020099:Coated Materials, Biocompatible / Q000737:chemistry; D004867:Equipment Design; D019544:Equipment Failure Analysis; D004967:Estrogens / Q000032:analysis*; D005336:Fiber Optic Technology / Q000295:instrumentation; D020100:Hydrogels / Q000737:chemistry*; D008163:Luminescent Measurements / Q000295:instrumentation* / Q000379:methods; D012441:Saccharomyces cerevisiae / Q000187:drug effects* / Q000302:isolation & purification*
D020099:Coated Materials, Biocompatible; D004967:Estrogens; D020100:Hydrogels