HTS demands highlight sub-microlitre need
during clinical trial processes has highlighted an emerging trend
in drug R&D that uses smaller and smaller test volumes, helped
by sophisticated automation technologies and reagents.
The concept of using nanomolar concentrations represents significant time and cost savings. 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.
Despite all the coverage, high throughput screening remains a new method even for the pharmaceutical industry. The first commercial 384-well plates were introduced in 1992 and the first commercial 1536-well plates arrived in 1997.
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. One company who thinks it is up to the challenge is Labcyte, a company specialising in low-volume liquid handling.
Speaking to DrugResearcher.com, a senior spokesman for Labcyte said: "Traditional HTS screening takes solutions of compounds dissolved in DMSO at ~10 mM concentration and dilutes an aliquot of that solution, usually about 100:1 with DMSO to 100 micromolar."
He added: "A small aliquot of that solution is then dispensed to an assay plate where the final concentration will be in the nanomolar range. To do this serial dilution requires: 1. pipette tips from source to intermediate plate, 2. intermediate plates, 3. DMSO diluent, 4. DMSO washes of pipette tips, 5. pipette tips from intermediate plate to assay plate, and 6. DMSO wash of pipette tips."
LabCyte has developed a solution in which ultrasound energy is used to propel droplets out of a liquid sample store via a process known as acoustic droplet ejection.
Known as acoustic droplet ejection (ADE), 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.
"With ADE all of these steps are eliminated because nanomolar concentrations in the assay plate can be reached by ejecting directly from the source plate," said the spokesman.
There are a number of other qualities of the technology, which could be of interest to researchers working in drug discovery. As well as the money saved on plastics consumables, diluents and wash solutions, contamination is virtually eliminated.
Since nothing ever touches the solution, there can be no cross-contamination. Note that the technique referred to as "non-contact" actually does touch the sample being transferred. It does not touch the vessel being loaded. But this means that the pipette tips or pins have to be washed extensively to eliminate contamination.
The spokesman added: "There is also the better use of compound stocks. People often talk about the dead volume of a plate, that is, the minimal volume required in the plate. Anything less than this volume cannot be transferred to an assay."
"But, in fact, the greatest loss of compound does not occur in the stock source plate but in the intermediate dilution plate. Usually a microlitre or so is transferred to the intermediate plate and then diluted 100-fold. But only 1 or 2 per cent of this diluted material is used in assays and the rest is discarded. This wasteful step is eliminated with ADE."
Submicrolitre assays are challenging for existing technologies as they require additions of nanolitres - droplets of only a few hundred microns in diameter. One problem is the heterogeneity of cells. In reducing the volume of the wells when doing cell-based assays can lead to artifacts.
Using 96-well plates for analysis is usually done with cell-based analyses. ADE eliminates the problem of the interaction of the cells with the bolus of drug-containing DMSO. Usually when a drug is added to a well containing cells, the heavier DMSO drops through the aqueous medium and hits the cells at high concentration.
Since DMSO can affect the integrity of the cell membranes, this can cause cascades of effects. When using ADE, the DMSO is injected into an inverted plate. The DMSO stays at the air/liquid interface and by the time the plate is turned over to its normal position, the DMSO and the drug have diffused into the bulk medium.
The spokesman added: "Also, there are still not that many labs that have readers for 1536-well or denser formats yet. This will change but it means researchers need to purchase these high-density readers for 3456 or 6144 plates. They also need bulk fillers that are compatible with high-density plates."
These problems have made ADE a technology that is expected to shine in the future. As more and more researchers want to reduce the volumes of assays to save money and decrease storage space and assay steps, the total market for liquid handling in the life sciences is more than $1 billion.
According to Frost and Sullivan, nanovolume portion is growing much more rapidly that the microliter and milliliter handlers.
"We have focused on the high-throughput screening market and its emphasis on DMSO-based solutions. We see these other fluids as applications that will spread ADE far beyond the current HTS customers," the spokesman said.
