Identifying toxins

Technology to identify the effect of harmful toxins on cell membranes is being developed by European researchers led by a team from Leeds University.

They aim to image the effect on a single layer of the molecules that compose the membranes.

The biological membranes that surround cells and the organelles inside them are made of phospholipid molecules.

These have a tadpole-like structure with a hydrophilic (attracted to water) ‘head’ and a hydrophobic (repelled by water) ‘tail’. In nature, they form bi-layers, lined up with the tails together and the heads on the outside, facing the water-rich environment.

These highly-organised structures are essential to many functions in the body and plants, like respiration, photosynthesis and nerve function.

The structure is important as it maintains the cell integrity; if the organisation of the membrane is lost, the cell is destroyed.

Many compounds in the environment such as DDT are toxic, or, in the case of antibiotics, kill harmful cells, because they disrupt the organisation of the cell membrane.

Phospholipids form a single layer (monolayer) when placed on a droplet of mercury, which allows them to be interrogated electrochemically. This principle is used in toxicity testing, where the effect of a substance on the phospholipid monolayer is examined. The idea originated at the Weizmann Institute of Science, Israel in the 1970s.

Dr Andrew Nelson, a senior lecturer in chemistry at Leeds University, has been developing a sensor based on this principle as a proxy for animal testing.

‘The only way you can get organised phospholipids layers is if you put them on mercury,’ he said. ‘Biological membranes are highly fluid, like two-dimensional liquids, and move about all the time, like the colours on a soap bubble.

‘This is why they’re so effective — they preserve all the proteins and enzymes they contain in a highly fluid, two-dimensional fluid.’

Mercury is very fluid so forms an ideal support to hold these biological monolayer structures and, as it is conducting, it can be interrogated electrically. This combination of properties means it has underpinned the most successful membrane models over the last 10 or 15 years. However, research using this technique has been limited because of the toxicity and fragility of the mercury droplets, and the difficulty in imaging what is happening to the membrane.

Nelson’s team developed a concept, now awaiting a patent, by which small platinum microelectrodes are fabricated on a wafer and tiny quantities of mercury are electrodeposited on the tip of each. A phospholipid monolayer can then be deposited on each of these and interrogated and imaged using atomic force microscopy techniques. Nelson founded a spinout called Organisense last March to manufacture these chips.

A three-year, EPSRC-funded project will study imaging the behaviour of the membranes on the surface. ‘When you expose these lipid membranes on mercury to an electric field, they undergo some very strange phase transition, which has a very high scientific, medical and biological significance,’ said Nelson. ‘We want to image these phase transitions to understand what’s happening and gather more scientific information about them.’

In parallel, Organisense is seeking to commercialise its chip into a smaller, practical, flow-cell sensing device that could be used by a water authority or pharmaceutical organisation to screen for toxicity.

The Leeds research team will collaborate with Guelph University in Canada, which will build on its previous experience of imaging phospholipids monolayers on gold and platinum electrodes. The researchers will also continue work with Uppsala University, Sweden on the interaction of toxic peptides with the membrane system.

Nelson said the main challenge is to image the layers. ‘No one really before imaged the phase transitions of monolayers on mercury before because of the fragility of mercury,’ he said. ‘The fact that we’ve got mercury in a robust configuration will mean that this can now be achieved but imaging the phase transitions of these semi-fluid layers on mercury will still be a significant technical challenge.’

The project will use Atomic Force Microscopy imaging equipment, and possibly scanning tunnelling microscopy, now used at Leeds to examine similar membrane-like systems on glass, or on substrates like quartz of mica.

‘We want to look at these layers at the same time as we apply an electric potential to bring about phase changes and see the effect on structure of the layers,’ said Nelson. ‘The toxic elements being detected modify the phase changes, in the same way as when you add salt to water the freezing point changes. We want to find out why these things happen.’

Berenice Baker