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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 electrode material on ROS production of C. reinhardtii after 30 min with no AC-field. (a) Two dimensional dot plots represent forward scatter (FSC) versus fluorescence green signal of CellROX® Green obtained by flow cytometry. Percentages in red indicate the proportion of stressed cells stained with CellROX® Green; (b) Micrographs obtained with fluorescence microscopy represent algae observed with the fluorescent FITC filter allowing the detection of CellROX® Green.
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biosensors-05-00319-f003: Effect of electrode material on ROS production of C. reinhardtii after 30 min with no AC-field. (a) Two dimensional dot plots represent forward scatter (FSC) versus fluorescence green signal of CellROX® Green obtained by flow cytometry. Percentages in red indicate the proportion of stressed cells stained with CellROX® Green; (b) Micrographs obtained with fluorescence microscopy represent algae observed with the fluorescent FITC filter allowing the detection of CellROX® Green.

Mentions: For the correct functioning of the biosensor the electrode and microfluidic chamber materials should not induce oxidative stress in algae. Therefore, the generation of the cellular ROS in algae was compared in the devices with brass needles and stainless steel electrodes (Figure 3). In the set up with stainless steel electrodes, the percentage of cells with enhanced ROS was comparable to the results for cell in the chamber when no AC-field was applied, as demonstrated by FCM. By contrast, 97.3% of the cells were CellROX® positive for 30 min with no AC-field and brass electrodes, showing that these electrodes are unsuitable for biosensing purposes. For comparison, after 30 min into the chamber without electrodes, only 9% of the algal cells showed an increase in ROS production using FCM and no bright green fluorescence was observed by microscopy in the absence of AC-field (Figure 3a). In the positive control obtained by exposing the cells to 5 mM of H2O2 for 1 h, increase of ROS production was induced in 97.6% of the population and all cells showed a bright green fluorescence under fluorescence microscopy. In agreement with FCM results, fluorescence microscopy images revealed cellular assembly bright green fluorescence in the brass electrode setup only, but no signal in the stainless steel electrode setup (Figure 3b). The high oxidative stress observed in the setup with brass electrodes is probably due to the release of Cu and Zn when the suspension of cells was injected to the chamber. Thus, the stainless steel electrodes were best fit for AC-field cell immobilization for biosensing purposes. No effect of the chamber material on the cellular ROS was found by comparing the % of the CellROX® stained cells injected in the microfluidic chamber and in native algal suspension (Figure 3a).


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 electrode material on ROS production of C. reinhardtii after 30 min with no AC-field. (a) Two dimensional dot plots represent forward scatter (FSC) versus fluorescence green signal of CellROX® Green obtained by flow cytometry. Percentages in red indicate the proportion of stressed cells stained with CellROX® Green; (b) Micrographs obtained with fluorescence microscopy represent algae observed with the fluorescent FITC filter allowing the detection of CellROX® Green.
© Copyright Policy
Related In: Results  -  Collection

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

biosensors-05-00319-f003: Effect of electrode material on ROS production of C. reinhardtii after 30 min with no AC-field. (a) Two dimensional dot plots represent forward scatter (FSC) versus fluorescence green signal of CellROX® Green obtained by flow cytometry. Percentages in red indicate the proportion of stressed cells stained with CellROX® Green; (b) Micrographs obtained with fluorescence microscopy represent algae observed with the fluorescent FITC filter allowing the detection of CellROX® Green.
Mentions: For the correct functioning of the biosensor the electrode and microfluidic chamber materials should not induce oxidative stress in algae. Therefore, the generation of the cellular ROS in algae was compared in the devices with brass needles and stainless steel electrodes (Figure 3). In the set up with stainless steel electrodes, the percentage of cells with enhanced ROS was comparable to the results for cell in the chamber when no AC-field was applied, as demonstrated by FCM. By contrast, 97.3% of the cells were CellROX® positive for 30 min with no AC-field and brass electrodes, showing that these electrodes are unsuitable for biosensing purposes. For comparison, after 30 min into the chamber without electrodes, only 9% of the algal cells showed an increase in ROS production using FCM and no bright green fluorescence was observed by microscopy in the absence of AC-field (Figure 3a). In the positive control obtained by exposing the cells to 5 mM of H2O2 for 1 h, increase of ROS production was induced in 97.6% of the population and all cells showed a bright green fluorescence under fluorescence microscopy. In agreement with FCM results, fluorescence microscopy images revealed cellular assembly bright green fluorescence in the brass electrode setup only, but no signal in the stainless steel electrode setup (Figure 3b). The high oxidative stress observed in the setup with brass electrodes is probably due to the release of Cu and Zn when the suspension of cells was injected to the chamber. Thus, the stainless steel electrodes were best fit for AC-field cell immobilization for biosensing purposes. No effect of the chamber material on the cellular ROS was found by comparing the % of the CellROX® stained cells injected in the microfluidic chamber and in native algal suspension (Figure 3a).

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