Periodic Reporting for period 1 - NanoLabels (Labelling of engineered nanomaterials for nanosafety tracing)
Periodo di rendicontazione: 2018-02-01 al 2020-01-31
There has been a notable rise in the development and production of engineered nanomaterials (ENMs) in recent years. However, concerns still remains regarding their potential impact on environmental safety and human health. Despite much research effort devoted to nanosafety studies in the past 15 years, a mechanistic understanding of the action of ENMs remains limited. A particular challenge is the detection of ENMs in complex biological tissues and environmental media, and against high natural background levels of either namoparticulate matter (natural borne nanoparticles) or constituent elements (e.g. Cu, Zn, or Fe). Besides, ENMs are highly dynamic, and prone to transformation (physical or chemical) upon entering the environment or biological tissues. For example, some metal-based NMs (silver, copper, zinc oxide) may dissolve quickly or transform to structurally and/or chemically different phases. These processes further complicate the detection of ENMs.
A common solution for this problem involves the introduction of a tracer in the ENMs (“labelling”). A tracer maybe a fluorescence dye, a foreign element of low natural abundance, or a less-abundant isotope (stable or radioactive) of the same constituent element(s) of the ENM. Labeling with fluorescent dye or exogenous radioactive isotopes, however, possibly modify and change the surface chemistry of ENMs and thus alter their environmental and biological behavior. The labels may also detach from the core ENMs and would thus not replicate the real behavior of ENMs. Using radioisotope labelling is of more limited applicability due to the hazards involved in handling a radioactive substance. Compared with the labeling methods above, stable isotope labeling is safer and more versatile. The tracers may be detected using most commonly highly sensitive ICP-MS analysis (or other techniques that can distinguish isotopes of the same element, e.g. SIMS/nano-SIMS, thus providing very sensitive signals that could distinguish them from endogenous background elements in a variety of samples. Stable isotope labeling has no quenching issue of labels, thus is very suitable for life-cycle monitoring of various products and also conduct trophic transfer experiments.
The objective of NanoLabels is to assign “ownership” or “source” to ENMs using different labelling techniques thereby enabling tracing of them in environment. The project not only helps scientific community to understand fundamental questions in nanosafety, i.e. the biological and environmental behaviour (uptake, translocation, transformation) by improving the tracing ability, but also provide labelling strategy that can be adopted by industry to facilitate applications such as nanosafety assessments before ENMs enter the market and environment, as well as for product authentication and tracking.
A common solution for this problem involves the introduction of a tracer in the ENMs (“labelling”). A tracer maybe a fluorescence dye, a foreign element of low natural abundance, or a less-abundant isotope (stable or radioactive) of the same constituent element(s) of the ENM. Labeling with fluorescent dye or exogenous radioactive isotopes, however, possibly modify and change the surface chemistry of ENMs and thus alter their environmental and biological behavior. The labels may also detach from the core ENMs and would thus not replicate the real behavior of ENMs. Using radioisotope labelling is of more limited applicability due to the hazards involved in handling a radioactive substance. Compared with the labeling methods above, stable isotope labeling is safer and more versatile. The tracers may be detected using most commonly highly sensitive ICP-MS analysis (or other techniques that can distinguish isotopes of the same element, e.g. SIMS/nano-SIMS, thus providing very sensitive signals that could distinguish them from endogenous background elements in a variety of samples. Stable isotope labeling has no quenching issue of labels, thus is very suitable for life-cycle monitoring of various products and also conduct trophic transfer experiments.
The objective of NanoLabels is to assign “ownership” or “source” to ENMs using different labelling techniques thereby enabling tracing of them in environment. The project not only helps scientific community to understand fundamental questions in nanosafety, i.e. the biological and environmental behaviour (uptake, translocation, transformation) by improving the tracing ability, but also provide labelling strategy that can be adopted by industry to facilitate applications such as nanosafety assessments before ENMs enter the market and environment, as well as for product authentication and tracking.
The overall workflow of this project was: 1. Synthesize ENMs and optimise their labelling. 2. Characterise and test the stability of the new materials as well as the labelling. 3. Demonstrate applicability of the labelling using field experiment. The original plan was to synthesize a broad library of labelled ENMs, and given budgetary limitations, it was decided to focus on Fe3O4, ZnO and graphene oxide for labelling using stable isotope enrichment approach. Specifically, we synthesized 57Fe enriched 57Fe3O4 and 68ZnO nanoparticles. Traceability of 57Fe3O4 and 68ZnO in environment have been tested in higher plants. The detection sensitivity of Fe and Zn in environment and plant was remarkably enhanced by isotope enrichment. Labelling of graphene nanomaterials using 13C isotope has also been initiated during the project and will be continued in ongoing work in our group. Combining the labelling approaches developed in this project with that have been developed previously in our group, we formatted a standardized protocol of the labelling strategy of some typical metal and metal oxide nanomaterials and published in Nature Protocols (2019). In total, nine papers were generated under this project, published in Nature Protocols, Small, Environmental Science & Technology, Environmental Science: Nano, Environmental International, Environmental Science & Technology Letters, Journal of Hazardous Materials, Science of the Total Environment and NanoImpact. Results have been also disseminated in several international conference including 13th International Conference On The Environmental Effects Of Nanoparticles And Nanomaterials (ICEENN 2018, September 5-8, Duke University, USA), SOT conference 2018 (March 11-15, San Antonio Texas, USA), China Nano 2019 (July 22-26, 2019, Beijing, China), The 10th National Conference on Environmental Chemistry 2019 (15-19 August, 2019, Tianjin, China).
Stable isotope labelling of ENMs has begun to emerge since 2011, with pioneering contributions from our group. The approaches have been proved to be efficient and highly sensitive for detecting the ENMs at environmental relevant concentrations. Nanolabels further moved this field forward. Three new stable isotope enriched materials, specifically Fe3O4, ZnO, and graphene have been /or currently being developed through this project. Fe, Zn and C are abundant in environment and biological organisms, which means the environmental and biological background concentration of the three elements are high. Quantifying the newly accumulated elements from the Fe-, Zn- and C- based nanomaterials against the background elements in environment and biological organism are challenging. The methodology developed in this project allows detection of the uptake of these nanomaterials in environment even at a very low environmental concentration. For example, we exposed rice plants to 57Fe enriched Fe3O4 nanoparticles and 68ZnO nanoparticles as well as the non-enriched nanoparticles as comparison. Without isotope enrichment, the uptake of Fe and Zn cannot be detected by ICP-MS even at exposure concentration as high as 10000 μg/L). With the isotope enrichment, the detection sensitivity were remarkably enhanced. More precisely, the uptake of the Fe3O4 nanoparticles were detected at 1000 μg/L in root and 100 μg/L in shoot (Figure 1). The traceability was also demonstrated in cellular experiment; uptake of Fe can be detected when the exposure concentration was as low as 10 μg/L. We formatted a protocol of the labelling strategies and published in Nature Protocols.
We expect the labelling strategies can be adopted by toxicologist and environmental scientist for their own study. We also expect that scaling up of the synthesis of stable isotope labelled ENMs could be tested, modified and standardized by industry for safer design of ENMs and for tracing studies of ENMs before entering into the market (playing also a role as quality control and counterfeiting agents).
We expect the labelling strategies can be adopted by toxicologist and environmental scientist for their own study. We also expect that scaling up of the synthesis of stable isotope labelled ENMs could be tested, modified and standardized by industry for safer design of ENMs and for tracing studies of ENMs before entering into the market (playing also a role as quality control and counterfeiting agents).