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

NanoSIMS Analysis of Arsenic NanoSIMS Analysis of Arsenic and Selenium in Cereal Grainsand Selenium in Cereal Grains

Supervisor: Chris GrovenorSupervisor: Chris Grovenor

Katie MooreKatie Moore33rdrd year D.Phil year D.Phil

Department of Materials – University of OxfordDepartment of Materials – University of Oxford

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• 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.

Ref: X. Y, Xu et al., Environ. Sci. Technol., 42(15), 2008

• Arsenic is a toxic and carcinogenic element

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 elementDaily selenium intake in the UK is

now about ½ of the reference amount

M. R. Broadley et al., Proc. Nutr. Soc. (65) 2006M. S. Fan et al., Sci. Total Environ. (389), 2008MAFF, Food Surveillance Information Sheet, (126), 1997

Refs:

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)

• 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 Oxford NanoSIMS

Schematic of the NanoSIMS

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

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 GrainAleurone layer

Starchy endosperm

80µm Embryo

Cross section

Selenium in Wheat Grain

Max selenium counts: 4Max CN- counts: 105,000

31P16O- 80Se-

32S-12C14N-16O-

SE

30µm

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

Selenium in Wheat GrainAleurone cell Starch grains

31P16O- 80Se- 32S-

12C14N-16O- 12C14N-16O-

80Se-

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

Selenium in Wheat Grain

High resolution, sub-cellular, localisation of ppm concentrations

Selenium is localised in the protein region around the starch grains

Selenium hotspots are found in the aleurone cells

Starchgrain

31P16O- 80Se- 32S-

16O- 12C14N-16O-

80Se-

32S-

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

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

Fe plaque No Fe plaque

Variables:•Arsenate or arsenite•With or without Fe plaque•Wild type or lsi2 mutant

Hydroponically grown rice plants

Lsi2 transporter

Ref: Zhao, F.J., et al., New Phytol., 181(4), 2009

Rice Roots – Fe Plaque

25 µm

EPEPExEx

ScSc

SESE

12C14N-

56Fe16O-

31P-28Si-

75As-

Rice Roots – Lsi2 mutant12C14N-

75As-

31P-

SE

28Si-

25 µm

XyXy

EnEn

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

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

AcknowledgementsSupervisor: Chris GrovenorNanoSIMS postdoc: Markus Schröder

EPSRC:D.Phil funding

Root Sample Preparation: Barry Martin, Chris Hawes

Collaborators:Fang-jie Zhao, Steve McGrath,Malcolm Hawkesford, Peter Shewry

IOM3:For this opportunity