Award details

Nanospheres in the cytosphere: interrogating the intracellular milieu with ion-dot ANSors

Reference	BB/D001307/1
Principal Investigator / Supervisor	Professor Elizabeth Hall
Co-Investigators / Co-Supervisors	Dr Zebo Huang, Professor Alan Tunnacliffe
Institution	University of Cambridge
Department	Institute of Biotechnology
Funding type	Research
Value (£)	410,187
Status	Completed
Type	Research Grant
Start date	01/10/2005
End date	31/03/2009
Duration	42 months

Abstract

The programme will create a family of multi-ion analytical nanosphere sensors (ANSors) between 100-400nm, containing quantum dot (QD) conjugates of chromoionophores or chromogenic ionophores. The nanospheres will be introduced into cells to study the management of ion concentration. The study is primarily focussed on the role of ion concentration in gene expression in both an invertebrate model organism (bdelloid rotifer) and human cells in tissue culture and the hypothesis that intracellular ion concentrations are managed during successful anhydrobiosis of bdelloid rotifers through the composition of other cell components (eg specific proteins). By exploiting the special feature of quantum confinement in QDs to selectively excite the encapsulated conjugate ion-selective reagent, we propose that multi-ion polymer nanospheres can be employed intracellularly to obtain a spatio-temporal ion signature. The ANSors also have potential for future study of other analytes and for spatial targeting within the cell through receptors placed on the ANSor surface.

Summary

Nanobiotechnology is biology and biotechnology on a molecular scale. It allows us to investigate and potentially exploit what is happening to living organisms within the cells they are composed of. The project described in this application is a collaboration between two groups at the Institute of Biotechnology, University of Cambridge, i.e. those of Prof Lisa Hall and Dr Alan Tunnacliffe. It brings together expertise in cutting edge analytical chemistry with the molecular biology of stress resistance. Prof Hall has devised novel methods for nanoscale sensing of ions and other small molecules which are ideal for examining events on a sub-cellular level, i.e. within the living cell itself. This involves placing polymer nanospheres in the cell. Contained in each nanosphere is a whole analytical laboratory for measuring concentrations of ions. By using quantum dots [QDs] we can also select a different colour nanosphere for each ion and so we end up with ion-dots and a whole family of analytical nanosphere sensors [ANSors]. Dr Tunnacliffe is investigating a remarkable phenomenon called anhydrobiosis, or life without water, where certain creatures such as the bdelloid rotifer, a tiny invertebrate animal which lives in temporary freshwater pools, can survive complete desiccation. Crucial to an understanding of how rotifers perform this physiological feat is what happens inside the cells and tissues of the animal. If this remains unknown, the extent of the trauma experienced by the rotifer will also be unknown. This project aims to characterise the intracellular events occurring during the period rotifers are drying out and relate these to possible survival mechanisms, including the deployment of hydrophilic ('water loving') proteins, and the expression of survival genes. Comparison will also be made with what happens during a similar, but less extreme stress situation, when rotifers are exposed to high osmolarity medium, for example, salty water. To do this, ANSors will beintroduced into living rotifers by firing from a gene gun; once embedded inside the animal using this apparatus, the ANSors will provide information on how ion concentrations (particularly sodium and potassium ions) change during drying and rehydration, and during osmotic stress. Specifically, it will be possible to ask whether the rotifer uses hydrophilic proteins to minimise the damaging build-up of salt ions during dehydration, and also whether anti-stress genes are switched on as ion concentrations increase. Once achieved in rotifers, a similar set of experiments will be carried out with human cells growing in culture flasks. Human cells cannot survive drying and therefore will allow an interesting comparison with what happens in rotifers; they do survive moderate osmotic stress, however. In addition, several genes are known which are switched on in human cells subjected to these stresses and introducing ANSors into the cytoplasm of the cell will allow testing of whether increasing ion concentration is linked to their expression, as other researchers have proposed. In the future, it is intended to develope ANSors which will be able to monitor concentrations of small organic molecules important for cell metabolism. These, together with the ion-specific ANSors, will form an important battery of nanoscale tools for investigating normal, stress and disease states of the intracellular environment.

Committee	Closed Committee - Engineering & Biological Systems (EBS)
Research Topics	Technology and Methods Development
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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