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Taking assays to the sub-microlitre level

02-Dec-2003

Over the last few years, researchers involved in high-throughput screening have been seeking out the use of their expensive reagents by developing assays that use smaller and smaller volumes, helped by sophisticated automation technologies.

Aside from the cost advantages, using smaller volumes of reagents means that diffusion of compounds can occur more quickly, and this speeds up the time it takes for a reaction to complete.

With a number of off-the-shelf products now offering microlitre handling of samples and reagents used in assays, the challenge now is to see how much lower it is possible to go, according to Roeland Papen, director of business development at Picoliter, a company specialising in low-volume liquid handling that last month merged with LabCyte .

"Submicrolitre assays are challenging for existing technologies as they require additions of nanolitres - droplets of only a few hundred microns in diameter," he told the Labautomation Europe/International Biotech conference in London, last week.

The main problem is that physical factors, such as evaporation, surface tension, wettability, precipitation and adhesion play an enormous role at this tiny scale, and this makes conventional dispensing approaches inadequate. One approach to this has been through engineering - straight disposable tips, precision pins and smooth surfaces (e.g. achieved using coatings) - but the cost of manufacturing consumables built to these specifications tends to offset the advantages of miniaturisation.

Ultrasound solution

Picoliter/LabCyte has developed a novel solution to this problem, in which ultrasound energy is used to propel droplets out of a liquid sample store via a process known as acoustic droplet ejection.

This technology relies on the use of an external transducer to project a burst of acoustic energy up through a liquid. This causes the liquid to form a droplet that is expelled upwards; the lower the frequency, the larger the droplet, and vice versa.

Papen noted that experiments with water have shown that a pulse of energy at 30MHz fires out a droplet of 50 picolitres in an extremely reproducible manner. Because the liquid does not come into contact with anything solid, it is not subject to the forces of surface tension, viscosity etc that affect conventional transfer devices.

In fact, LabCyte has demonstrated that it can produce droplets as small as one-tenth of a picolitre using ADE, but at present there is no application for delivery at this scale. Rival approaches such as inkjet and piezotype technologies have been found to function down to about 10 nanolitres, but can go no lower. LabCyte's first instrument will have an effective range of 5-50 nanolitres.

LabCyte is in the process of developing a system - called the Echo 550 - that can fire these droplets into microwell plates. It has been specifically designed to transfer compound libraries from storage into these plates, providing savings in the amount of compound required. It also does away with the need for disposable tips and washing, and can fill a 384-well plate in as little as 40 seconds.

The fact that the liquid does not come into contact with any solid device other than the well plate with ADE means that it is a very gentle process, ideal for transferring cells for whole cell assays, said Papen.

There are a number of other qualities of the technology which could be of interest to researchers working in drug discovery, he noted. For example, building in a sonogram to monitor the acoustic pulses provides a simple means of cross-checking the amount of liquid dispensed into a well, and this can be also used to test sample integrity.

For example, the amount of sound lost as waves go through a sample gives a measure of the concentration of DMSO, a solvent that is widely-used for storing compounds in libraries. The concentration of DMSO goes down as the sample, becomes contaminated with water, and this can lead to precipitation of the sample out of solution and affect the amount transferred to the well plate.

Finally, LabCyte has shown that if two immiscible liquids are stored one on top of the other, firing a pulse of ultrasound through both layers can lead to the formation of micelles, in effect encapsulating the bottom liquid in a protective shell. This could be particularly useful for peptide assays, he suggested.

Commercialisation plans

Elaine Heron, chief executive of LabCyte, told In-Pharmatechnologist.com that an alpha version of the Echo 550 is already being evaluated at a biotechnology company in the US, and beta versions should be sent out in the January/February 2004 timeframe.

This will be followed with a full commercial launch in March/April, with the initial version of the product set up to conduct high-throughput screening in plates using compounds stored in DMSO/water. Additional modifications will adapt the product fr use with microarrays, she said. The price tag of the initial Echo 550 will be approximately €225,000 per unit.

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