A revolution in nanolitre pipetting

In the search for potential drug candidates, enormous numbers of compounds are screened for biological activity. With continuous pressure to reduce assay cost, Tom Edwards, of TTP LabTech, reviews an innovative nanolitre pipetting system that minimises the use of reagents and consumables

In primary and secondary drug screening, huge libraries of compounds are tested for biological activity. These compounds are typically very costly to produce and are stored as master collections in dimethyl sulphoxide (DMSO), which reduces the breakdown of many compounds. The concentration at which they are stored, however, is usually several orders of magnitude higher than the concentration at which they are required for screening.

Because of this, and because DMSO itself in significant concentration interferes with assays, particularly those using live cells, compound sets are usually diluted before addition to the assay.

This requires the production of intermediate 'daughter plates' of compounds – replicas of the compound library but in microtitre plates – which have been diluted so that volumes that are suitable for existing liquid handling systems can be added to the assay at suitable concentrations of both compound and DMSO.

This is a particularly wasteful process as it involves the preparation of a greater quantity of diluted compound than will be used in assays and these daughter plates typically have a short life, since many compounds degrade in aqueous solution, which adds to assay cost. Furthermore, the costs of preparing the daughter plates in terms of consumables – plates, tips, reagents etc – and preparation time, is significant.

If, on the other hand, a very small volume of the compound from the master store in DMSO were added directly to the assay, suitable concentrations can be reached in a single step. For example, addition of 50nl aliquots of compound in DMSO to 50ml assay volumes gives an effective dilution of 1/1000 – sufficiently low DMSO concentration for live cell-based screens.

Mosquito, a new nanolitre pipetting station (figure 1) from TTP LabTech, based in Royston, UK, enables the preparation of screening plates direct from master compound libraries without the intermediate dilution steps. By eliminating the use of consumables for dilution and with no compound lost in the discarded daughter plate, direct addition of the compound offers substantial cost benefits.

The accuracy of the preparation is also improved as there is only a single pipetting step rather than a minimum of two steps, and the process can be performed more quickly with fewer intermediate steps to track.

Direct addition of compound into low volume assays is only just becoming possible with the introduction of nanolitre dispensers and until the launch of the mosquito system, there was no instrument that could also guarantee the extremely low cross-contamination threshold required.

As the potency of compounds may be several orders of magnitude higher than the assay concentration, a tiny amount of a potent compound accidentally occurring in another assay well, or compound storage well, would give a false positive in the screen. Cross contamination of the order <10^-4 may, therefore, be problematic. Since mosquito uses disposable micropipettes, cross-contamination is eliminated, as is the need for time-consuming and expensive solvent wash cycles.

In essence, the system makes it possible to take sample compounds from the precious master stores without any worry of contamination, and to do so at a useful and cost-effective volumetric performance and gross throughput.

At the core of mosquito is the miniature syringe-like disposable micropipette made of a stainless steel piston in a close-fitting polyethylene barrel (figure 2), giving it the ability to pierce many plate sealing films. The micropipettes can handle solvents and viscous liquids as well as suspensions of beads or cells and with a diameter of only 0.8mm they are suitable for use with 1536 well plates as well as the larger wells of 96 and 384.

Another feature of the mosquito system is that the micropipettes are supplied as a continuous-feed bandolier to the pipetting head (figure 3).

For easy and accurate handling, the micropipettes are attached to a strong polyester backing tape, which is driven by sprocket holes. Reels are available with bandoliers of up to 35,000 micropipettes – enough to perform 1:1 transfers in more than 90 x 384-well plates. The reel can be replaced in a couple of minutes, so instrument utilisation is maximised.

In the pipetting head (figure 4) 16 tips are operated simultaneously to aspirate and dispense samples to and from the microtitre plates. Once used, tips are disengaged and passed on out of the head to a waste container, ensuring that any contamination is kept away from the assay plate area. Where cross-contamination is not an issue, a set of tips may be used for multiple transfers.

For preparing assay plates with compound volumes of 50-1200nl direct from compound library plates, mosquito can typically create a 384-well plate in less than three minutes, with a coefficient of variance (CV) lower than 8% at 100nl and around 10% at 50nl.

With capacity for making four identical copies of assay plates at a time from a compound plate, mosquito can prepare around 24 x 384 plates –or nearly 10,000 samples – an hour, giving the high throughput required in drug screening.

In addition to drug screening, there are applications including Polymerase Chain Reaction (PCR) assembly, where reduced reagent and DNA sample usage saves cost and can improve speed and accuracy.

Mosquito has also been used to load a wide range of novel and existing devices, such as MALDI-targets, laboratory chips and microfluidics systems, where precision at low volumes is paramount.

It could also be used to prepare drops of material for screening crystallising conditions in projects involving structure elucidation.

With the increased focus on assay miniaturisation, process streamlining and process quality in drug screening, systems such as mosquito offer an innovative, fast and simple solution for improved compound handling.