Non-invasive continuous monitoring of pro-oxidant effects of engineered nanoparticles on aquatic microorganisms
© The Author(s) 2017
Received: 20 December 2016
Accepted: 22 February 2017
Published: 7 March 2017
Engineered nanomaterials (ENMs) are key drivers for the development of highly sophisticated new technologies. As all new attainments, the rapidly increasing used of ENMs raise concerns about their safety for the environment and humans. There is growing evidence showing that if engineered nanomaterials are released into the environment, there is a possibility that they could cause harm to aquatic microorganisms. Among the divers effects triggering their toxicity the ability of ENMs to generate reactive oxygen species (ROS) capable of oxidizing biomolecules is currently considered a central mechanism of toxicity. Therefore, development of sensitive tools for quantification of the ROS generation and oxidative stress are highly sought. After briefly introducing ENMs-induced ROS generation and oxidative stress in the aquatic microorganisms (AMOs), this overview paper focuses on a new optical biosensor allowing sensitive and dynamic measurements of H2O2 in real-time using multiscattering enhanced absorption spectroscopy. Its principle is based on sensitive absorption measurements of the heme protein cytochrome c whose absorption spectrum alters with the oxidation state of constituent ferrous FeII and ferric FeIII. For biological applications cytochrome c was embedded in porous random media resulting in an extended optical path length through multiple scattering of light, which lowers the limit of detection to a few nM of H2O2. The sensor was also integrated in a microfluidic system containing micro-valves and sieves enabling more complex experimental conditions. To demonstrate its performance, abiotic absorption measurements of low concentrations of dye molecules and 10 nm gold particles were carried out achieving limits of detection in the low nM range. Other biologically relevant reactive oxygen species can be measured at sub-μM concentrations, which was shown for glucose and lactate through enzymatic reactions producing H2O2. In ecotoxicological investigations H2O2 excreted by aquatic microorganisms exposed to various stressors were measured. Pro-oxidant effects of nano-TiO2 and nano-CuO towards green alga Chlamydomonas reinhardtii were explored in various exposure media and under different light illuminations. Dynamics of Cd2+ induced effects on photosynthetic activity, sensitisation and recovery of cells of C. reinhardtii was also studied.
KeywordsEcotoxicity Nanomaterials Reactive oxygen species Oxidative stress Hydrogen peroxide Optical biosensor Multiscattering Absorption spectroscopy
The material revolution engendered by nanotechnological advances in the last decades has not only enabled the development of highly sophisticated fine-tuned materials for new applications but also confronted established risk assessment and regulatory affairs with new challenges: the possible (eco-)toxicological implications of the expected increment of engineered nanomaterials (ENMs) discharged into environmental compartments .
It is postulated that increased levels of ROS and oxidative damage will occur in exposed organisms (despite the presence of basal or enhanced antioxidant defence systems of repair and replacement), which may be linked to some aspect of impaired biological functions at cellular or higher levels of organization . Thus, from the nanoecotoxicological perspective seeking the elucidation of environmental hazards of ENMs, it follows that an in-depth understanding of their toxic mode of action, that is, of normal and ENM-stimulated ROS production as well as antioxidant levels in aquatic organisms is required. This will allow to quantitatively link the presence of ENMs with pro-oxidant processes and to estimate the expected degree by which ENM-stimulated oxidative damage may potentially affect overall health of organism.
Hence, there has been a keen interest in the detection and quantification of ROS in aqueous and biological systems, which is a technically tricky task due to their very low concentration in the pico- to micromolar range and their extremely short-lived nature with half times ranging from nanoseconds to hours . Most conventional ROS sensing methods rely on exogenous probes or resulting endogenous reaction products and molecular biomarkers reflecting oxidative damage and antioxidant status [13, 15–17]; they suffer one major technical drawback—the invasive nature of the detection method itself .
The present article provides an overview of the main findings of the project “Non-invasive continuous monitoring of the interaction between nanoparticles and aquatic microorganisms” within in the framework of the Swiss National Research Program 64 on the Opportunities and Risk of Nanomaterials. The review begins with a brief introduction in the ENMs-induced ROS generation and oxidative stress in the aquatic microorganisms (AMOs) as well as short presentation of the existing detection techniques. The newly developed method for non-invasive quantification of extracellular H2O2 in real-time and monitoring with an unprecedented limit of detection is described, while its capabilities are illustrated by exploring the pro-oxidants effects of the ENMs to AMOs .
ENMs and oxidative stress in aquatic microorganisms
Investigations performed in the mid-90’s led to the conclusion that nanoparticles have the ability to stimulate the generation of reactive oxygen (ROS) and nitrogen species (RNS) at or near the cell surface and to induce oxidative stress [10, 12, 19]. The oxidative stress hypothesis was successfully expanded into nanotoxicology and recognised as a major mechanism for nanoparticle induced effects . Therefore, the impacts of ENMs on the pro-oxidant/antioxidant equilibrium can provide relevant information on their ecotoxicical importance .
Selected examples of ENM-induced oxidative stress or damage in microalgae
Generation of ROS by photocatalysis
TiO2 and UV light
Lake water and MOPS buffer
Generation of intracellular ROS by HA
MES, MOPS, HEPES
Al2O3, SiO2, ZnO and TiO2
ROS may not be the dominant mechanism for algal growth inhibition
C. vulgaris, Dunaliella tertiolecta
Growth medium BG-11
ROS induced lipid peroxidation and a decrease of cell viability
ISO 8692 medium and 4-fold diluted tris-acetate-phosphate medium
Substantial oxidative stress and negligible membrane damage; significant growth inhibition
Coated and uncoated CuO
High salt medium
ROS formation may be the primary toxicity mechanism
Standard US EPA
The oxidative activity is mediated by OH and initiation of lipid peroxidation
High salt growth medium
ROS are responsible for chlorophyll deterioration, significant decrease of PSII primary photochemistry
Various media, lake water
Oxidative stress and damage of membrane integrity
CuO and light
Synthetic fresh water
Chlorophyll bleaching, oxidative stress and membrane damage; CuO and UV-light has synergistic effect
TiO2, CdTe and QDs
CM growth medium
Lipid peroxidation induced by oxidative stress, QDs and TiO2 exhibit different mechanisms
Photoactive ENMs including fullerenes and semiconducting metal oxides, such as TiO2, CuO, CeO2, ZnO and Al2O3, can generate ROS when illuminated [43, 44]. It has been demonstrated that these ENMs, the most prominent being TiO2, can activate molecular oxygen radicals, 1O2 and O2 −, which belong, together with OH·, to the biologically most potent ROS. It is well known that those photoactive particles are primarily active at wavelength in the UV regime (<390 nm) but it has also been demonstrated in several studies that TiO2 is capable to induce oxidative stress in the absence of light.
Overall, environmental contaminants, including ENMs, have the capability to induce generation of ROS in AMOs and, consequently, to alter the cellular redox homeostasis leading to oxidative stress. Oxidative stress occurs as a result of (i) increase in oxidant generation, (ii) reduction of antioxidant protection and (iii) failure to repair oxidative damage .
Towards development of the novel tool for non-invasive monitoring of the pro-oxidant effects of engineered nanomaterials
The above-mentioned oxidative stress “indicators” can provide a useful picture on the cell-ENM interactions. However, they are endpoint-based and qualitative, thus unable to provide quantitative information about the rate and amount of generated ROS. In addition they are often very laborious and fail to provide dynamic and continuous information on specific physiological phenomena happening at the exposed living cells.
Selection of H2O2 detection methods 
Absorbance of H2O2
Optimal reaction in low ionic strength solutions
Xylenolorange + Fe3+
Absorbance of complex
Carried out in acidic acids
Xylenolorange + Ti4+
Absorbance of complex
Carried out in acidic acids
Interference with Mn2+ and Fe3+
Fluorescence of product
Can be oxidised by other ROS
Fluorescence of product
Optimal reaction at pH > 8.5
Fluorescent and chemi-luminescent methods exhibit low LODs in the nM range. However, a major drawback of those methods is their incompatibility with bioorganisms and they are therefore endpoint detection schemes.
Multiscattering enhanced absorption spectroscopy (MEAS)
Sensitive real-time detection of H2O2
Koman et al. presented a detection scheme for sensitive and real-time detection of those metabolites . Taking advantage of the above presented multiscattering approach they were detected with sub-micromolar LODs. Moreover, this enzymatic approach allows real-time measurements of multiple analytes in parallel which offers the possibility to follow the evolution of several metabolites. This feasibility has been demonstrated using the example of parallel detection of glucose and H2O2.
Portable setup and microfluidic chip
Here ENM-induced H2O2 excretion by cells exposed to ENMs was monitored with a recently developed optical biosensor in a portable setup (POSS; portable oxidative stress sensor) specifically designed for field experimentation . In this way, POSS may contribute to the elucidation of ENM-specific pro-oxidant interactions with cells and thus help to narrow the gap between material innovation and sound risk assessment.
Selected applications to probe the pro-oxidant effect of nanoparticles to microalga C. reinhardtii
To demonstrate the performances of the developed sensing tool, the pro-oxidant effects of CuO and TiO2 nanoparticles to green alga C. reinhardtii, a representative model AMO are presented [32, 85] together with measurements of the potential to generate abiotic ROS as well as oxidative stress and membrane damage. These two ENMs were chosen since they have different properties—CuO nanoparticles have a tendency to dissolve, while nano-TiO2 is rather inert; (ii) both have photocatalytic properties; (iii) nano-CuO is with relatively high toxic potential , while nano-TiO2 is moderately toxic; (iv) they are of high environmental relevance given their increasing use in different products.
The nanoparticle-induced cellular pro-oxidant process in C. reinhardtii were studied using the newly developed cytochrome c biosensor for the continuous quantification of extracellular H2O2 and fluorescent probes (CellRoxGreen for oxidative stress and propidium iodide for membrane integrity [32, 41, 87]) in combination with flow cytometry. Both the dynamics of abiotic (ENM only) and biotic (ENM + cells) pro-oxidant processes related to the exposure of C. reinhardtii to nano-CuO and nano-TiO2 are present below.
Chlamydomonas reinhardtii were exposed to CuO nanoparticles in five different media, namely TAP, MOPS, OECD, MES and Geneva lake water  and the biological responses including growth, size increase, chlorophyll autofluorescence, intracellular ROS and membrane damage were quantified.
The nano-TiO2 exposure experiments were performed in MOPS and water sampled from lake of Geneva . The observed pro-oxidant effects were strongly dependent on the exposure concentration and medium. In lake water exposures the proportion of cells affected by oxidative stress increased with the concentration of nano-TiO2, with highest responses obtained for algae exposed to 100 and 200 mg L−1 nano-TiO2. Similarly, membrane damage predominantly occurred in lake water rather than in MOPS. UV light pre-treatment of TiO2 enhanced median intracellular ROS levels in lake water exposure while no significant effect was found in MOPS.
The biotic exposure experiments revealed higher decay rates of the initial peaks at the beginning of the experiments, suggesting a peroxide annihilation by algae.
Overall, our findings showed that (i) irrespective of the medium, agglomerated nano-TiO2 in the micrometer size range produced measurable abiotic H2O2 concentrations in biologically relevant media, which is enhanced by UV irradiation, (ii) c H2O2 undergo decay and are highest in the first 10–20 min of exposure and (iii) the generation of H2O2 and/or the measured H2O2 concentration is a dynamic process modified by the ambient medium as well as nano-TiO2 concentrations and the presence of cells.
Comparison of the extracellular H2O2 measurements and intracellular oxidative stress [32, 82] further showed significant differences between extracellular and intracellular pro-oxidant processes. Indeed, an increase of the intracellular oxidative stress was found under the conditions where no significant increase in extracellular biotic H2O2 was measured. The above observation indicates that extracellular H2O2 measurements cannot directly serve as a predictor of cellular pro-oxidant processes or oxidative stress in C. reinhardtii, however, they provide valuable information about the extracellular dynamics of the most stable ROS in the extracellular medium.
Extracellular H2O2 measurements during altering illumination regimes
Recovery and sensitisation
In contrast to end-point measurements, sensitive and non-invasive continuous H2O2 measurements enable the investigation of recovery and sensitisation. To demonstrate the practicability of such experiments the C. reinhardtii were repeatedly exposed to Cd2+, using a microfluidic configuration as described above . Cd2+ concentrations are typically <10 nM in fresh water. However, higher concentrations of Cd2+ were found in the exposure media containing CdSe quantum dots  or CdTe/CdS .
1st exposure of C. reinhardtii to Cd2+ → H2O2 production
2nd exposure of C. reinhardtii to Cd2+ → increased production rate of H2O2
This shows that exposure to even low concentration of Cd2+ leads to a sensitisation of exposed cells, thus suggesting an adverse impact on the health of microorganisms. In parallel, intracellular ROS was assessed based on the fluorescence intensity of de-esterified H2DFC-DA . At high Cd2+ concentrations (500 nM) intra- and extracellular measurements correlated very well, confirming the suitability of extracellular H2O2 measurements as indicator of cellular stress. However, unlike extracellular H2O2 concentrations, intracellular levels remain stable in the 100 nM exposure, suggesting an efficient ROS/AOX regulation through the cell walls.
Conclusions and outlook
This review paper provides a short overview on nanoparticle toxicity for aquatic microorganisms based on the paradigm of oxidative stress and highlights the recent developments of an optical biosensor based on absorption measurements of cyt c for the sensitive, non-invasive and continuous measurement of H2O2. The use of this new tool for studying the pro-oxidant effects of ENMs to aquatic microorganisms was demonstrated by exposing the representative aquatic microorganism C. reinhardtii to nano-CuO and nano-TiO2 in various exposure media and under different light treatments. Sensitive continuous measurements of extracellular H2O2 provided valuable information on both the potency of the studied nano-CuO and nano-TiO2 to generate ROS as well as on the mechanisms of toxicity. The results were in good agreement with the oxidative stress and membrane damage results obtained under the same conditions using a combination of fluorescent staining with flow cytometry. The developed biosensor allows rapid measurement of the rate and amount of H2O2 measured in the extracellular medium in response to cell exposure to ENMs. Hence, detailed knowledge of the dynamics of H2O2 excretion can provide valuable insights into complex biological responses. The development of the portable setup and the multi-layered microfluidic chip with an integrated optical sensor for the continuous sensitive detection of extracellular H2O2 opens novel avenues for new types of exposure experiments, leading to a better understanding of ROS biology as well as to numerous opportunities for nanoecotoxicological studies. Developing and employing new sensing tools and methods enables conducting experiments under more realistic conditions such as environmental relevant concentrations, aged nanomaterials and simultaneous exposure to various stressors. Furthermore, studying the dynamics of cellular metabolites leads to new insights in the extremely complex adverse outcome pathways.
reactive oxygen species
multiscattering enhanced absorption spectroscopy
optical path length
limit of detection
portable oxidative stress sensor
- cyt c :
OECD standard media
dissolved organic matter
Suwannee River fulvic acid
CHS contributed to the development of the sensor and to the coordination of the experiments, NVM carried out the experiments on AMOs, VK contributed to the development of the sensor and the AMO experiments, VS coordinated the AMO experiments, PB coordinated the characterisation of the nano-particles, OJFM participated in the coordination of the study and manuscript writing, CHS, NvM and VS wrote the manuscript, all authors edited and approved the manuscript. All authors read and approved the final manuscript.
This work was supported by the Swiss National Research Program NRP 64 Project No. 406440-131280/1 of the Swiss National Science Foundation.
The authors declare that they have no competing interests.
Availability of data and materials
Not applicable since it is a review article. Data are available from the original publications.
Consent for publication
We accept the submission conditions.
Swiss National Research Program NRP 64 Project No. 406440-131280/1 of the Swiss National Science Foundation.
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