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Two-Dimensional Algal Collection and Assembly by Combining AC-Dielectrophoresis with Fluorescence Detection for Contaminant-Induced Oxidative Stress Sensing.

Siebman C, Velev OD, Slaveykova VI - Biosensors (Basel) (2015)

Bottom Line: An alternative current (AC) dielectrophoretic lab-on-chip setup was evaluated as a rapid tool of capture and assembly of microalga Chlamydomonas reinhardtii in two-dimensional (2D) close-packed arrays.The results showed significant increase of the cellular ROS when C. reinhardtii was exposed to high concentrations of methylmercury, CuO-NPs, and 10⁻⁵ M Cu.Overall, this study demonstrates the potential of combining AC-dielectrophoretically assembled two-dimensional algal structures with cell metabolic analysis using fluorescence staining, as a rapid analytical tool for probing the effect of contaminants in highly impacted environment.

View Article: PubMed Central - PubMed

Affiliation: Environmental Biogeochemistry and Ecotoxicology, Institute F.-A. Forel, Earth and Environmental Science, Faculty of Sciences, University of Geneva, 10 route de Suisse, Versoix CH-1290, Switzerland. coralie.suscillon@unige.ch.

ABSTRACT
An alternative current (AC) dielectrophoretic lab-on-chip setup was evaluated as a rapid tool of capture and assembly of microalga Chlamydomonas reinhardtii in two-dimensional (2D) close-packed arrays. An electric field of 100 V·cm⁻¹, 100 Hz applied for 30 min was found optimal to collect and assemble the algae into single-layer structures of closely packed cells without inducing cellular oxidative stress. Combined with oxidative stress specific staining and fluorescence microscopy detection, the capability of using the 2D whole-cell assembly on-chip to follow the reactive oxygen species (ROS) production and oxidative stress during short-term exposure to several environmental contaminants, including mercury, methylmercury, copper, copper oxide nanoparticles (CuO-NPs), and diuron was explored. The results showed significant increase of the cellular ROS when C. reinhardtii was exposed to high concentrations of methylmercury, CuO-NPs, and 10⁻⁵ M Cu. Overall, this study demonstrates the potential of combining AC-dielectrophoretically assembled two-dimensional algal structures with cell metabolic analysis using fluorescence staining, as a rapid analytical tool for probing the effect of contaminants in highly impacted environment.

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Effect of the AC-field duration on fluorescence intensity of 2D cell assemblies. (a) Fluorescence intensity corresponding to the mean corrected CellROX® fluorescent intensity per pixel calculated from the fluorescent images obtained in FITC channel for different modes of the AC-field application. Different letters (from (a) to (d)) indicate the existence or not of the statistically significant difference and were obtained by Student-Newman-Keuls test with p < 0.05; Microscopy images of CellROX® stained 2D-assembly with continuous AC-field (b) and AC-field applied for 30 min (c); FCM cytograms for CellROX® Green stained cells with no AC-field and AC-field applied for 30 min for exposure in the control medium for 30 min (d) and 120 min (e). The initial concentration of algae in suspensions was 5 × 106 cells·mL−1.
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biosensors-05-00319-f004: Effect of the AC-field duration on fluorescence intensity of 2D cell assemblies. (a) Fluorescence intensity corresponding to the mean corrected CellROX® fluorescent intensity per pixel calculated from the fluorescent images obtained in FITC channel for different modes of the AC-field application. Different letters (from (a) to (d)) indicate the existence or not of the statistically significant difference and were obtained by Student-Newman-Keuls test with p < 0.05; Microscopy images of CellROX® stained 2D-assembly with continuous AC-field (b) and AC-field applied for 30 min (c); FCM cytograms for CellROX® Green stained cells with no AC-field and AC-field applied for 30 min for exposure in the control medium for 30 min (d) and 120 min (e). The initial concentration of algae in suspensions was 5 × 106 cells·mL−1.

Mentions: No shift in the fluorescence intensity of CellROX® Green stained cells between AC-field of 100 V·cm−1 applied for 30 min and 120 min and no AC-field was observed in the flow cytograms (Figure 4d,e). For a cell collection duration of 30 min, the combination of field intensity/frequency of 100 V·cm−1/100 Hz has no significant effect on the CF of the CellROX® positive cells indicating no enhancement of intracellular ROS (Figure 4a,c). The CF values were comparable with those determined in the control measurement with AC-field applied, where CF increased from 1.42 ± 0.22 for 30 min to 2.94 ± 0.49 for 120 min. However for the same field intensity/frequency combination, longer DEP duration resulted in CF increase from 1.75 ± 0.38 for 30 min to 14.1 ± 1.1 for 120 min, suggesting significant increase in the algal ROS generation upon longer exposure and collection times. Such increase of ROS generation in the algal cells could be due to the release of heat from the continuous electric field application. Moreover, the electric field could induce undesired motion of internal organelles through the outer cell membrane as shown with the chloroplasts of Eremosphaera viridis [48]. Similarly, DEP treatment of yeast cells for longer than 4 h reduced significantly the number of viable cells by 56.8%–89.7% [49]. Positive DEP has only been suitable for short time (<10 min) trapping of genetically modified E. coli [50]. Overall, the results demonstrate the importance of the careful selection of the AC-field duration to avoid detrimental long-term field effects on cell oxidative status.


Two-Dimensional Algal Collection and Assembly by Combining AC-Dielectrophoresis with Fluorescence Detection for Contaminant-Induced Oxidative Stress Sensing.

Siebman C, Velev OD, Slaveykova VI - Biosensors (Basel) (2015)

Effect of the AC-field duration on fluorescence intensity of 2D cell assemblies. (a) Fluorescence intensity corresponding to the mean corrected CellROX® fluorescent intensity per pixel calculated from the fluorescent images obtained in FITC channel for different modes of the AC-field application. Different letters (from (a) to (d)) indicate the existence or not of the statistically significant difference and were obtained by Student-Newman-Keuls test with p < 0.05; Microscopy images of CellROX® stained 2D-assembly with continuous AC-field (b) and AC-field applied for 30 min (c); FCM cytograms for CellROX® Green stained cells with no AC-field and AC-field applied for 30 min for exposure in the control medium for 30 min (d) and 120 min (e). The initial concentration of algae in suspensions was 5 × 106 cells·mL−1.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493552&req=5

biosensors-05-00319-f004: Effect of the AC-field duration on fluorescence intensity of 2D cell assemblies. (a) Fluorescence intensity corresponding to the mean corrected CellROX® fluorescent intensity per pixel calculated from the fluorescent images obtained in FITC channel for different modes of the AC-field application. Different letters (from (a) to (d)) indicate the existence or not of the statistically significant difference and were obtained by Student-Newman-Keuls test with p < 0.05; Microscopy images of CellROX® stained 2D-assembly with continuous AC-field (b) and AC-field applied for 30 min (c); FCM cytograms for CellROX® Green stained cells with no AC-field and AC-field applied for 30 min for exposure in the control medium for 30 min (d) and 120 min (e). The initial concentration of algae in suspensions was 5 × 106 cells·mL−1.
Mentions: No shift in the fluorescence intensity of CellROX® Green stained cells between AC-field of 100 V·cm−1 applied for 30 min and 120 min and no AC-field was observed in the flow cytograms (Figure 4d,e). For a cell collection duration of 30 min, the combination of field intensity/frequency of 100 V·cm−1/100 Hz has no significant effect on the CF of the CellROX® positive cells indicating no enhancement of intracellular ROS (Figure 4a,c). The CF values were comparable with those determined in the control measurement with AC-field applied, where CF increased from 1.42 ± 0.22 for 30 min to 2.94 ± 0.49 for 120 min. However for the same field intensity/frequency combination, longer DEP duration resulted in CF increase from 1.75 ± 0.38 for 30 min to 14.1 ± 1.1 for 120 min, suggesting significant increase in the algal ROS generation upon longer exposure and collection times. Such increase of ROS generation in the algal cells could be due to the release of heat from the continuous electric field application. Moreover, the electric field could induce undesired motion of internal organelles through the outer cell membrane as shown with the chloroplasts of Eremosphaera viridis [48]. Similarly, DEP treatment of yeast cells for longer than 4 h reduced significantly the number of viable cells by 56.8%–89.7% [49]. Positive DEP has only been suitable for short time (<10 min) trapping of genetically modified E. coli [50]. Overall, the results demonstrate the importance of the careful selection of the AC-field duration to avoid detrimental long-term field effects on cell oxidative status.

Bottom Line: An alternative current (AC) dielectrophoretic lab-on-chip setup was evaluated as a rapid tool of capture and assembly of microalga Chlamydomonas reinhardtii in two-dimensional (2D) close-packed arrays.The results showed significant increase of the cellular ROS when C. reinhardtii was exposed to high concentrations of methylmercury, CuO-NPs, and 10⁻⁵ M Cu.Overall, this study demonstrates the potential of combining AC-dielectrophoretically assembled two-dimensional algal structures with cell metabolic analysis using fluorescence staining, as a rapid analytical tool for probing the effect of contaminants in highly impacted environment.

View Article: PubMed Central - PubMed

Affiliation: Environmental Biogeochemistry and Ecotoxicology, Institute F.-A. Forel, Earth and Environmental Science, Faculty of Sciences, University of Geneva, 10 route de Suisse, Versoix CH-1290, Switzerland. coralie.suscillon@unige.ch.

ABSTRACT
An alternative current (AC) dielectrophoretic lab-on-chip setup was evaluated as a rapid tool of capture and assembly of microalga Chlamydomonas reinhardtii in two-dimensional (2D) close-packed arrays. An electric field of 100 V·cm⁻¹, 100 Hz applied for 30 min was found optimal to collect and assemble the algae into single-layer structures of closely packed cells without inducing cellular oxidative stress. Combined with oxidative stress specific staining and fluorescence microscopy detection, the capability of using the 2D whole-cell assembly on-chip to follow the reactive oxygen species (ROS) production and oxidative stress during short-term exposure to several environmental contaminants, including mercury, methylmercury, copper, copper oxide nanoparticles (CuO-NPs), and diuron was explored. The results showed significant increase of the cellular ROS when C. reinhardtii was exposed to high concentrations of methylmercury, CuO-NPs, and 10⁻⁵ M Cu. Overall, this study demonstrates the potential of combining AC-dielectrophoretically assembled two-dimensional algal structures with cell metabolic analysis using fluorescence staining, as a rapid analytical tool for probing the effect of contaminants in highly impacted environment.

Show MeSH
Related in: MedlinePlus