Imaging and quantification of nanoparticle uptake by ICP-MS based techniques

Transcription

1 Date MARINA and NanoValid International Conference Event )tle 29 September 2015 OECD Conference Center 2, rue André Pascal, 5775 Paris Cedex 16, France Imaging and quantification of nanoparticle uptake by ICP-MS based techniques Steffi Böhme Helmholtz Centre for Environmental Research (UFZ) Department Bioanalytical Ecotoxicology 1

2 Nanomaterial testing Release into the environment inevitable, hence the hazard for environmental organisms has to be assessed Little consideration of nanoparticle distribution and internalisation processes Toxicity testing Source: Development of suitable analytical detection, quantification and imaging techniques 2

3 Nanomaterial testing Model test organisms Nanomaterials Danio rerio (zebrafish embryo) (OECD guideline 212, 236, 305) NM Primary par/cle size Coa/ng (c) / stabilizer (s) Zeta poten/al Ag- NP 21 ± 8 nm PVP (c) - 19 mv Au- NP 13 ± 1 nm Sodium citrate (s) - 38 mv 250µm Daphnia magna (OECD guideline 202) CuO- NP nm Tetrasodium pyrophosphate (s) - 64 mv ZnO- NP nm mv Provided as powders or suspensions by project partners 500µm 3

4 Quantification of NM uptake + SEM/EDX Quantification of total uptake amounts Semi- quantitative Imaging NP solution Control (time / concentration- dependent studies) Exposure Washing step Collection of 4-8 individuums / compartments for 1 sample Acid digestion AgNP, CuO- NP digestion with HNO3 (30%) AuNP, ZnO- NP digestion with aqua regia ICP- MS measurement Exposure Washing step Fixation of individuums Sectioning with a cryo- microtome 40 µm thick sections Placement on glass slides LA- ICP- MS measurement 4

5 Semi- quantitative Imaging Laser Ablation Parameters: Laser Ablation (LSX- 500, CETAC) Agarose gel ICP- MS measurement (ELAN- DRCe, Perkin Elmer) 60 % laser energy 50 µm spot diameter 30 sec delay between spots Additional gas stream: Argon Spot raster External Calibration Böhme, S., Stärk, H.- J., Kühnel, D., Reemtsma, T. (2015): Exploring LA- ICP- MS as a quantitative imaging technique to study nanoparticle uptake in Daphnia magna and zebrafish (Danio rerio) embryos. Anal. Bioanal. Chem. 407 (18), Böhme, S., Stärk, H.- J., Meißner, T., Springer, A., Reemtsma, T., Kühnel, D., Busch, W. (2014): Quantification of Al 2 O 3 nanoparticles in human cell lines applying inductively coupled plasma mass spectrometry (neb- ICP- MS, LA- ICP- MS) and flow cytometry- based methods. J. Nanopart. Res. 16,

6 Semi- quantitative Imaging Developing Reference Methods for Nanomaterials Exposure: 100 µg/l AgNP 24 hours Zebrafish embryo (Danio rerio) Daphnia magna Visual image of organism section Das Bild kann nicht angezeigt werden. Dieser Computer verfügt möglicherwe ise über zu LA- ICP- MS 2D- color plot Overlay pg / 50µm spot 250µm 500µm Böhme, S., Stärk, H.- J., Kühnel, D., Reemtsma, T. (2015): Exploring LA- ICP- MS as a quantitative imaging technique to study nanoparticle uptake in Daphnia magna and zebrafish (Danio rerio) embryos. Anal. Bioanal. Chem. 407 (18),

7 Quantification of NM uptake over time Developing Reference Methods for Nanomaterials Ag- NP Au- NP Böhme, S., Baccaro, M., Schmidt, M., Potthoff, A., Stärk, H.- J., Reemtsma, T., Kühnel D.: The chorion of the zebrafish (Danio rerio) embryo as a biological barrier for nanoparticle uptake? (submitted) 7

8 Quantification of NM uptake over time Das Bild kann nicht angezeigt werden. Dieser Computer verfügt möglicherweise über zu wenig Arbeitsspeicher, um das Bild zu öffnen, oder das Bild ist beschädigt. Starten Sie den Computer neu, und öffnen Sie dann erneut die Datei. Wenn weiterhin das rote x angezeigt wird, müssen Sie das Bild möglicherweise löschen und dann erneut einfügen. CuO- NP ZnO- NP Böhme, S., Baccaro, M., Schmidt, M., Potthoff, A., Stärk, H.- J., Reemtsma, T., Kühnel D.: The chorion of the zebrafish (Danio rerio) embryo as a biological barrier for nanoparticle uptake? (submitted) 8

9 Quantification of NM uptake over time = Ratio of theoretical and experimental particle number NM Coverage % ZnO- NP 0.8 ± 1.6 NP Ag- NP 14.8 ± 10.6 CuO- NP 23.7 ± 39.1 r (egg) Au- NP 49.7 ± 14.3 Das Bild kann nicht angezei gt werden. Coverage is dependent on: Time Concentration Nanoparticle characteristics Böhme, S., Baccaro, M., Schmidt, M., Potthoff, A., Stärk, H.- J., Reemtsma, T., Kühnel D.: The chorion of the zebrafish (Danio rerio) embryo as a biological barrier for nanoparticle uptake? (submitted) 9

10 Proposed sequence of NM- organism interaction Passage of NPs and ions through the chorion pores Pore blockage and formation of NP layer Sorption of NPs at the chorion surface NP and ion release into the inner mass of the ZFE by thinning process of the chorion Böhme, S., Baccaro, M., Schmidt, M., Potthoff, A., Stärk, H.- J., Reemtsma, T., Kühnel D.: The chorion of the zebrafish (Danio rerio) embryo as a biological barrier for nanoparticle uptake? (submitted) 10

11 Effect correlation Effect correlation alive sublethal lethal 24h exposure AgNP pg / 50µm spot pg / 50µm spot pg / 50µm spot ng Ag / ZFE ng Ag / ZFE ng Ag / ZFE 15.1 ± ± ± 7.3 Böhme, S., Stärk, H.- J., Reemtsma, T., Kühnel D. (2015): Effect propagation after silver nanoparticle exposure in zebrafish (Danio rerio) embryos: a correlation to internal concentration and distribution patterns. Published online, DOI: /c5en00118h 11

12 Effect correlation Das Bild kann nicht angezeigt werden. Dieser Computer verfügt möglicherweise über zu wenig Arbeitsspeicher, um das Bild zu öffnen, oder das Bild ist beschädigt. Starten Sie den Computer neu, und öffnen Sie dann erneut die Datei. Wenn weiterhin das rote x angezeigt wird, müssen Sie das Bild möglicherweise löschen und dann erneut einfügen. The occurrence of toxicological effects is connected to the internal silver concentration of the organism Böhme, S., Stärk, H.- J., Reemtsma, T., Kühnel D. (2015): Effect propagation after silver nanoparticle exposure in zebrafish (Danio rerio) embryos: a correlation to internal concentration and distribution patterns. Published online, DOI: /c5en00118h 12

13 Summary - Methodology Applications: - Visualisation of elemental/particle distribution patterns - Quantitative investigations on NM uptake - Correlation to the occurrence of toxicological effects Outlook: - Developments in the high resolution field to visualise accumulation in fine tissue structures or for single organs - Method combination with SP- ICP- MS to distingish between ionic and particle forms - Development towards a 3D imaging Ag- NP Au- NP Al 2 O 3 - NP Das Bild kann nicht angezeigt werden. Dieser Computer verfügt möglicherw eise über Das Bild kann nicht angezeigt werden. Dieser Computer verfügt möglicherw Das Bild kann nicht angezeigt werden. Dieser Computer verfügt möglicherw eise über intensity (cps) intensity (cps) intensity (cps) 13

14 Conclusion Quantification and visualization of Ag- NP, Au- NP, CuO- NP and ZnO- NP distribution - Ag- NP / Au- NP / CuO- NP accumulation at or within chorion structures - ZnO- NP higher accumulation for embryonic tissues - comparable results for respective ionic substances accumulation dependent on chemical identity the chorion surface is not completly covered by NPs Sorption sequence for NP- chorion interaction proposed correlation of toxic effects with particle distribution patterns possible 14

15 Thank you for your attention! Josefine Müller Hans- Joachim Stärk Rolf Altenburger Thorsten Reemtsma Dana Kühnel Matthias Schmidt Marta Baccaro Annegret Potthoff Das Bild Da s Bil E- mail contact: steffi.boehme@ufz.de 15