Resumen
The present invention supplies biosensors based on microalgae for the determination of the presence of toxic compounds in a sample, characterized by the high sensitivity and specificity thereof with respect to the target toxic substance. The biosensors are based on measurement of the photosynthetic activity of algae by means of monitoring of the production of molecular oxygen by the microalgae by using a luminescent compound whose emission depends on the quantity of oxygen in the environment.
Reivindicaciones
1. A specific biosensor for detecting a toxic compound comprising: a) a first population of algae the oxygen production of which decreases in the presence of said compound, b) a second population of algae the oxygen production of which does not vary substantially in the presence of said compound, of the same species as the first population and which has been obtained by means of a process comprising the steps of (i) a first selection of spontaneous mutants viable to said compound by means of exposure to said toxic compound and (ii) a second selection from the mutants obtained in step (i) in the presence of progressively higher concentrations of said toxic compound by means of at least one ratchet cycle c) means for stimulating the photosynthetic activity of the two populations of algae and d) means for detecting the oxygen produced by each of the two populations of algae characterized in that the means for detecting the oxygen produced by each of the populations of algae comprise at least one compound the optical properties of which vary according to the concentration of oxygen in the medium and at least one optoelectric element suitable for detecting the optical properties of said compound. 2. A biosensor according to claim 1, wherein the first population of algae has been obtained from a wild-type algae strain by means of selecting spontaneous mutants showing an increased sensitivity to said toxic compound. 3. A biosensor according to claim 1 or 2, wherein the second population of microalgae is obtained from a wild-type algae strain which has been obtained by means of selecting spontaneous mutants showing an increased sensitivity to said toxic compound. 4. A biosensor according to any of the previous claims, characterized in that each of the populations of algae is immobilized in a support. 5. A biosensor according to claim 4, wherein the support is a three-dimensional matrix. 6. A biosensor according to any of the previous claims, characterized in that the two populations of algae are separated from the aqueous medium in which the measurement is made by means of a porous membrane impermeable to the microalgae but permeable to molecular oxygen. 7. A biosensor according to any of the previous claims, wherein the compound the optical properties of which vary according to the concentration of oxygen in the medium is impregnated in a membrane. 8. A biosensor according to any of the previous claims, wherein the compound the optical properties of which vary in the presence of oxygen in the medium is a complex of a transition metal and at least one diimine type ligand. 9. A biosensor according to claim 8, wherein the transition metal is selected from the group of ruthenium, osmium, rhenium, rhodium, platinum and indium. 10. A biosensor according to any of claims 8 or 9, wherein the diimine-type ligand is selected from the group of 2,2'-bipyridine and 1,10-phenanthroline and derivatives thereof with different degree of substitution. 11. A biosensor according to claim 10, wherein the compound the optical properties of which vary according to the concentration of oxygen is selected from the group of tris(1,10-phenanthroline)ruthenium(II), tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II), tris(2,2'-bipyridine)ruthenium(II), tris(4-phenyl-7-para-hexanolphenyl-1,10-phenanthroline)ruthenium(II) and (5-isothiocyanate-1,10-phenanthroline)bis(2,2'-bipyridine)ruthenium(II). 12. A biosensor according to any of claims 7 to 11, wherein the membrane impregnated with the compound the optical properties of which vary according to the concentration of oxygen in the medium is separated from the cells by an opaque membrane permeable to oxygen. 13. A biosensor according to any of the previous claims additionally comprising means for detecting the fluorescence of the chlorophyll of each of the populations of algae in response to the stimulus of the photosynthetic activity. 14. A biosensor according to any of the previous claims, wherein the species of alga forming the first and the second population of algae isDictyosphaerium chlorelloides . 15. A biosensor according to any of the previous claims, wherein the compound which has been used to select the second population of algae is simazine. 16. Use of a biosensor according to claims 1 to 15 for detecting the presence of a toxic compound in a sample. 17. Use of a biosensor according to claims 1 to 15 for distinguishing the presence of the toxic compound causing a decrease of the oxygen production in the first population of algae from other toxic compounds in a sample. 18. Method for the detection of a toxic compound in a sample comprising the steps of a) contacting the sample with a biosensor according to any of claims 1 to 15 wherein the second population of algae has been selected in the presence of said toxic compound and b) detecting the production of light of each of the populations of cells in the biosensor in the presence of said sample wherein a decrease in the production of light of the first population of cells with respect to the second population of cells indicates that the sample contains the toxic compound against which the cells of the second population have been selected and wherein a decrease in the production of light by the two populations of cells indicates that the sample contains a toxic compound different from the compound against which the first population of cells has been selected. 19. Method according to claim 18, wherein the total amount of algal biomass in each immobilized population in the absence of toxic agent is determined simultaneously and the values of variation in the production of light by the two populations of algae are standardized according to the amount of immobilized algal biomass of each population. 20. Method according to claim 19, wherein the determination of the amount of immobilized algal biomass is carried out by means of detecting the fluorescent emission of each population of algae in response to the stimulus of the photosynthetic activity. 21. A process for obtaining microalgae resistant to a toxic compound comprising the steps of a) a first selection of viable spontaneous mutants in the presence of said toxic compound and b) a second selection from the mutants isolated in step (a) in the presence of progressively higher concentrations of said toxic compound by means of at least one ratchet cycle. 22. A process according to claim 21, wherein the toxic compound obtained in the first and second step is simazine. 23. A process according to claim 21 or 22, wherein the alga which is subjected to selection isDictyosphaerium chlorelloides. 24. An algae strain resistant to a toxic compound which can be obtained by means of a process according to any of claims 21 to 23.
