1. NanoSIMS Analysis of Arsenic and Selenium in Cereal Grains
Katie Moore
3rd year D.Phil
Department of Materials – University of Oxford
Supervisor: Chris Grovenor

2. Motivation Why is a materials scientist looking at plants?
Interdisciplinary collaborations allow critical problems in the life sciences, difficult to solve with traditional analysis techniques, to be explored with established physical science techniques.

3. Arsenic is a toxic and carcinogenic element
The Arsenic Problem
Arsenic contamination of groundwater
Contaminated groundwater is used to irrigate rice paddy fields
Resulting in rice grain containing elevated levels of arsenic
A major problem in Bangladesh, India, China and America.
Arsenic is a toxic and carcinogenic element
Ref: X. Y, Xu et al., Environ. Sci. Technol., 42(15), 2008

4. Daily selenium intake in the UK is now about ½ of the reference amount
The Selenium Problem
Between 0.5 and 1 billion people worldwide may be deficient in selenium including populations in developed countries.
In the UK this is caused by a reduction in the amount of wheat imported from America and a fall in the consumption of cereals
Selenium is an important trace element
Daily selenium intake in the UK is now about ½ of the reference amount
M. R. Broadley et al., Proc. Nutr. Soc. (65) 2006
M. S. Fan et al., Sci. Total Environ. (389), 2008
MAFF, Food Surveillance Information Sheet, (126), 1997
Refs:

5. Agricultural Solutions
To increase Se:
Add a selenium fertiliser to the soil (practiced in Finland)
To decrease As:
Polish the grain to remove the high As parts
Both of these solutions require knowledge of where the trace elements are located in the grain.
Determining where these very low concentrations are located with sub-cellular resolution is a serious analytical challenge
Ref: M. H. Eurola et al., J. Sci. Food Agric., (56), 1991

6. Secondary Ion Mass Spectrometry (SIMS)
Sample is bombarded by positively charged primary ion beam
This results in sputtering of the top few atomic layers and ejection of atoms, ions and clusters
Secondary ions are collected and mass analysed
Image adapted from Ref:

7. Schematic of the NanoSIMS
The Oxford NanoSIMS
Ref: CAMECA, Instrumentation booklet, June 2007.

8. Characteristics of SIMS
High sensitivity (down to ppb for some elements)
Detection of all elements from Hydrogen to Uranium including all isotopes
High mass resolution
NanoSIMS
High lateral resolution (50 nm)
Parallel detection of 5 ionic species
Ref: CAMECA, Instrumentation booklet, June 2007.

9. SIMS Sample Preparation
Sample needs to be flat, conducting, and dry
Bulk chemical analysis (ICP-MS) showed
trace levels of 2.5 ppm arsenic in the rice and 17 ppm selenium in the wheat
Rice samples were
grown at Rothamsted Research
Wheat samples were grown
in a field trial in Nottingham

10. Structure of Wheat Grain
Aleurone layer
80µm
Starchy
endosperm
Cross section
Embryo

11. Selenium in Wheat Grain
16O-
12C14N-
32S-
31P16O-
80Se- SE
30µm
Max CN- counts: 105,000
Max selenium counts: 4
Ref: K. L. Moore et al., New Phytol., (185), 2010

12. Selenium in Wheat Grain
Aleurone cell
Starch grains
16O-
12C14N-
16O-
12C14N-
31P16O-
80Se-
32S-
80Se-
Ref: K. L. Moore et al., New Phytol., (185), 2010

13. Selenium in Wheat Grain
16O-
32S-
16O-
12C14N-
Starch
grain
31P16O-
80Se-
32S-
80Se-
Selenium is localised in the protein region around the starch grains
Selenium hotspots are found in the aleurone cells
High resolution, sub-cellular, localisation of ppm concentrations
Ref: K. L. Moore et al., New Phytol., (185), 2010

14. Arsenic is localised in the sub-aleurone protein
Arsenic in Rice Grain
Arsenic is localised in the sub-aleurone protein
Ref: K. L. Moore et al., New Phytol., (185), 2010

15. Rice Roots – Experiment setup
Variables:
Arsenate or arsenite
With or without Fe plaque
Wild type or lsi2 mutant
Hydroponically grown rice plants
Lsi2 transporter
Fe plaque
No Fe plaque
Ref: Zhao, F.J., et al., New Phytol., 181(4), 2009

16. Rice Roots – Fe Plaque 12C14N- 28Si- 31P- 56Fe16O- 75As- SE Sc Ex EP
25 µm

17. Colour merge: Red = As, Green = CN, Blue = Si
Rice Roots – Lsi2 mutant
12C14N-
28Si-
31P-
75As- SE Xy En
25 µm
Colour merge: Red = As, Green = CN, Blue = Si

18. Conclusions
The NanoSIMS has successfully been used to provide a detailed analysis of the distribution of trace elements selenium and arsenic in wheat and rice respectively and the distribution of As in roots.
Selenium is localised in the protein regions around the wheat starch grains with hotspots in the bran layer
Arsenic is concentrated in the sub-aleurone protein of the rice rather than in the aleurone.
The Fe plaque has a strong adsorption affinity for As
The Lsi2 mutant blocks As uptake in the endodermis
These experiments have demonstrated the unique potential of state-of-the-art SIMS instrumentation to analyse the distribution of ppm levels of important trace elements with sub-cellular resolution

19. Acknowledgements Supervisor: Chris Grovenor
NanoSIMS postdoc: Markus Schröder
Collaborators:
Fang-jie Zhao, Steve McGrath,
Malcolm Hawkesford, Peter Shewry
Root Sample Preparation:
Barry Martin, Chris Hawes
EPSRC:
D.Phil funding
IOM3:
For this opportunity