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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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Data for Cu-induced oxidative stress in 2D-assemblies of C. reinhardtii. (a) Time course of the mean corrected CellROX® Green fluorescent intensity (CF) per pixel of cells assembled in 2D-arrays by DEP exposed to increasing Cu concentrations; (b) Microscopy images of CellROX® stained 2D-assemblies in the absence and presence of Cu for 30 (b) and (c) 120 min of exposure; Fluorescence images of CellROX® stained cells after 30 min of exposure to 10−5 M Cu (d) no AC-field applied and (e) AC-field of 100 V·cm−1 and 100 Hz; (f) Number of pixels obtained with the 2D-assembly versus corrected fluorescence of CellROX® Green; (g) FCM cytograms for CellROX® Green for 120 min of exposure. Conditions are the same as for 2D-assembly measurements.
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biosensors-05-00319-f005: Data for Cu-induced oxidative stress in 2D-assemblies of C. reinhardtii. (a) Time course of the mean corrected CellROX® Green fluorescent intensity (CF) per pixel of cells assembled in 2D-arrays by DEP exposed to increasing Cu concentrations; (b) Microscopy images of CellROX® stained 2D-assemblies in the absence and presence of Cu for 30 (b) and (c) 120 min of exposure; Fluorescence images of CellROX® stained cells after 30 min of exposure to 10−5 M Cu (d) no AC-field applied and (e) AC-field of 100 V·cm−1 and 100 Hz; (f) Number of pixels obtained with the 2D-assembly versus corrected fluorescence of CellROX® Green; (g) FCM cytograms for CellROX® Green for 120 min of exposure. Conditions are the same as for 2D-assembly measurements.

Mentions: No oxidative stress was found for exposure of C. reinhardtti to Cu concentrations below 10−5 M, where the CF values were comparable with those for unexposed controls (Figure 5a). The above observation suggests that the pro-oxidants in the cell can be efficiently balanced by the antioxidant system of the cell. This suggestion is consistent with the enhanced level of expression of genes involved in the enzymatic antioxidant response in algae exposed to comparable concentrations of Cu for 30 and 120 min [10]. 30 min exposure to 10−5 M Cu resulted with no oxidative stress induction in both 2D-assemblies and FCM measurements, which is in agreement with up-regulation of antioxidant genes. Significant increase of CF of the CellROX® stained cell was observed when 2D-cell arrays were exposed to 10−5 M Cu (Figure 5a–c) for times longer than 60 min, suggesting that the ROS generation overwhelms the antioxidant capacity of the cells at this high exposure concentrations. Indeed no further enhancement of the antioxidant gene expression was determined at 120 min exposure [10]. Furthermore, comparison of the fluorescence of the CellROX® stained cells with and without AC-field revealed high fluorescence background around the cells, likely caused by cells sedimentation and dispersion in different heights of the chamber when no AC-field was applied (Figure 5d). Such strongly interfering background fluorescence interference was not observed when cells were assembled in 2D-array. Thus, DEP cell assembly was proved essential in collecting the microalgae and arranging them into a 2D-arrays. Additionally, the formation of 2D single layer close-packed structures without 3D-stacking facilitated fluorescence measurements by providing a larger cell measurement surface with a higher number of cells in precise microscope focus. Furthermore, the results obtained by the 2D-assembly were validated by FCM measurements of CellROX® stained cells. Similarly to FCM, the results of the DEP assisted 2D-structure characterization were presented as histograms (Figure 5e) demonstrating a shift of the fluorescence profiles of CellROX® Green stained cells between the control and 10−5 M Cu at 120 min of exposure (Figure 5f,g), although the shift with FCM was larger. However the sensitivity of the 2D-assembled structures was lower than FCM as demonstrated by the smaller shift in the fluorescent intensity at Cu exposure. This should be expected given that the FCM offers very high sensitivity that is unmatched by fluorescence microscopy detection platforms.


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)

Data for Cu-induced oxidative stress in 2D-assemblies of C. reinhardtii. (a) Time course of the mean corrected CellROX® Green fluorescent intensity (CF) per pixel of cells assembled in 2D-arrays by DEP exposed to increasing Cu concentrations; (b) Microscopy images of CellROX® stained 2D-assemblies in the absence and presence of Cu for 30 (b) and (c) 120 min of exposure; Fluorescence images of CellROX® stained cells after 30 min of exposure to 10−5 M Cu (d) no AC-field applied and (e) AC-field of 100 V·cm−1 and 100 Hz; (f) Number of pixels obtained with the 2D-assembly versus corrected fluorescence of CellROX® Green; (g) FCM cytograms for CellROX® Green for 120 min of exposure. Conditions are the same as for 2D-assembly measurements.
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biosensors-05-00319-f005: Data for Cu-induced oxidative stress in 2D-assemblies of C. reinhardtii. (a) Time course of the mean corrected CellROX® Green fluorescent intensity (CF) per pixel of cells assembled in 2D-arrays by DEP exposed to increasing Cu concentrations; (b) Microscopy images of CellROX® stained 2D-assemblies in the absence and presence of Cu for 30 (b) and (c) 120 min of exposure; Fluorescence images of CellROX® stained cells after 30 min of exposure to 10−5 M Cu (d) no AC-field applied and (e) AC-field of 100 V·cm−1 and 100 Hz; (f) Number of pixels obtained with the 2D-assembly versus corrected fluorescence of CellROX® Green; (g) FCM cytograms for CellROX® Green for 120 min of exposure. Conditions are the same as for 2D-assembly measurements.
Mentions: No oxidative stress was found for exposure of C. reinhardtti to Cu concentrations below 10−5 M, where the CF values were comparable with those for unexposed controls (Figure 5a). The above observation suggests that the pro-oxidants in the cell can be efficiently balanced by the antioxidant system of the cell. This suggestion is consistent with the enhanced level of expression of genes involved in the enzymatic antioxidant response in algae exposed to comparable concentrations of Cu for 30 and 120 min [10]. 30 min exposure to 10−5 M Cu resulted with no oxidative stress induction in both 2D-assemblies and FCM measurements, which is in agreement with up-regulation of antioxidant genes. Significant increase of CF of the CellROX® stained cell was observed when 2D-cell arrays were exposed to 10−5 M Cu (Figure 5a–c) for times longer than 60 min, suggesting that the ROS generation overwhelms the antioxidant capacity of the cells at this high exposure concentrations. Indeed no further enhancement of the antioxidant gene expression was determined at 120 min exposure [10]. Furthermore, comparison of the fluorescence of the CellROX® stained cells with and without AC-field revealed high fluorescence background around the cells, likely caused by cells sedimentation and dispersion in different heights of the chamber when no AC-field was applied (Figure 5d). Such strongly interfering background fluorescence interference was not observed when cells were assembled in 2D-array. Thus, DEP cell assembly was proved essential in collecting the microalgae and arranging them into a 2D-arrays. Additionally, the formation of 2D single layer close-packed structures without 3D-stacking facilitated fluorescence measurements by providing a larger cell measurement surface with a higher number of cells in precise microscope focus. Furthermore, the results obtained by the 2D-assembly were validated by FCM measurements of CellROX® stained cells. Similarly to FCM, the results of the DEP assisted 2D-structure characterization were presented as histograms (Figure 5e) demonstrating a shift of the fluorescence profiles of CellROX® Green stained cells between the control and 10−5 M Cu at 120 min of exposure (Figure 5f,g), although the shift with FCM was larger. However the sensitivity of the 2D-assembled structures was lower than FCM as demonstrated by the smaller shift in the fluorescent intensity at Cu exposure. This should be expected given that the FCM offers very high sensitivity that is unmatched by fluorescence microscopy detection platforms.

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