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NanoSIMS Analysis of Arsenic and Selenium in Cereal Grains. 3 rd year D.Phil Department of Materials – University of Oxford. Katie Moore. Supervisor: Chris Grovenor. Motivation. Why is a materials scientist looking at plants?

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Nanosims analysis of arsenic and selenium in cereal grains

NanoSIMS Analysis of Arsenic and Selenium in Cereal Grains

3rd year D.Phil

Department of Materials – University of Oxford

Katie Moore

Supervisor: Chris Grovenor


Motivation
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.


The arsenic problem
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


The selenium problem
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:


Agricultural solutions
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


Secondary ion mass spectrometry sims
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:

http://www.eaglabs.com/training/tutorials/sims_theory_tutorial/index.php


The nanosims 50
The NanoSIMS 50

Schematic of the NanoSIMS

The Oxford NanoSIMS

Ref: CAMECA, http://www.cameca.fr/doc_en_pdf/ns50_instrumentation_booklet.pdf, Instrumentation booklet, June 2007.


Characteristics of sims
Characteristics of SIMS

  • 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, http://www.cameca.fr/doc_en_pdf/ns50_instrumentation_booklet.pdf, Instrumentation booklet, June 2007.


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


Structure of wheat grain
Structure of Wheat Grain

80µm

Aleurone layer

Starchy

endosperm

Cross section

Embryo


Selenium in wheat grain
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


Selenium in wheat grain1
Selenium in Wheat Grain

Aleurone cell

Starch grains

16O-

12C14N-

12C14N-

16O-

80Se-

80Se-

31P16O-

32S-

Ref: K. L. Moore et al., New Phytol., (185), 2010


Selenium in wheat grain2
Selenium in Wheat Grain

16O-

12C14N-

16O-

32S-

Starch

grain

80Se-

31P16O-

80Se-

32S-

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


Arsenic in rice grain
Arsenic in Rice Grain

Arsenic is localised in the sub-aleurone protein

Ref: K. L. Moore et al., New Phytol., (185), 2010


Rice roots experiment setup
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


Rice roots fe plaque
Rice Roots – Fe Plaque

28Si-

12C14N-

31P-

SE

75As-

56Fe16O-

Sc

Ex

EP

25 µm


Rice roots lsi2 mutant
Rice Roots – Lsi2 mutant

28Si-

12C14N-

31P-

SE

75As-

Xy

En

25 µm

Colour merge: Red = As, Green = CN, Blue = Si


Conclusions
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


Acknowledgements
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


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