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or multichannel (8, 12, 96 or 384) pipetting have been developed over the years. For a recent review covering state-of-the-art high-speed conventional pipetting devices see Stevens et al. [32]. Conventional pipetting devices usually work on a positive displacement principle. That is, movement of a piston creates a vacuum that liquid is then pulled into. These mechanical pipettors are generally quite accurate and precise when pipetting volumes greater than 1 |L. The loss of accuracy and precision at lower volumes is due to the viscosity of the liquid, which causes adherence to both the pipet tip and the well. As the pipetting volume decreases, the amount of liquid that remains adhered to the tip becomes a large percentage of the total pipetted volume.

An assay run in a 1536-well plate typically has a volume in the range of 3 to 5 |L. Mechanical pipettors are quite good at pipetting volumes down to 1 |L but tend to lose accuracy and precision when pushed below this range. Consequently, these mechanical pipettors can be used for much of the liquid handling in 1536-well plates. However, most of the test compounds are solubi-lized in neat DMSO, and the majority of biological reactions cannot tolerate greater than 1% DMSO. This means that the test compounds must be pipetted into each well in a volume of no more than 50 nL for a 5 |L assay, and these volumes cannot be achieved by mechanical pipettors.

For assays miniaturized to 1536-well formats, pipetting volumes from 10 nL up to a few |L is necessary. In order to achieve these volumes, a switch to noncontact dispensing is preferable. Devices based on either ink-jet or piezoelectric technology have been developed that are capable of accurately and precisely pipetting volumes between 100 pL and 5 |L. These devices dispense liquid as a series of small droplets between 10 picoliters and several nanoliters (Fig. 1) and thus never come in direct contact with the well itself. There are several advantages to this method of dispensing liquid. First, the effect of viscosity is minimized, since the liquid is dispensed as microdroplets in a noncontact fashion. Second, there is no need to exchange or wash tips between dispense cycles, since l>

Figure 1 Microliquid handling using a device based on ink-jet printer technology. The figure displays such a device dispensing 10 nL drops in rapid succession. Devices based on this principle are capable of dispensing volumes from several hundred picoliters up to the microliter range. (Courtesy of Cartesian Technologies, Irvine, CA.)

Figure 1 Microliquid handling using a device based on ink-jet printer technology. The figure displays such a device dispensing 10 nL drops in rapid succession. Devices based on this principle are capable of dispensing volumes from several hundred picoliters up to the microliter range. (Courtesy of Cartesian Technologies, Irvine, CA.)

the tips never come in contact with reagents in any of the wells. Carryover from one well to the next is essentially eliminated.

A technological hurdle had to be overcome in liquid handling in order for miniaturized assays to become practical. Similarly, a conceptual hurdle needed to be overcome in terms of plate design and construction. 96-well plates come in several formats, and one of these has become the standard set by the Society of Biomolecular Screening [33]. These plates are typically constructed of polystyrene or polypropylene, have a round or square shape, and have a volume of between 50 and 300 |L (for screening plates; volumes up to 3 mL for compound storage plates).

Miniaturization creates a number of challenges in plate design and construction. As can be observed from Table 4, a volume decrease from 200 |L in a standard 96-well plate to 4 |L in a 1536-well plate results in a 6.5-fold increase in the surface-area-to-volume ratio and greater than a 7-fold increase in the plastic-area-to-volume ratio. The increased surface-area-to-volume ratio translates into a much greater rate of evaporation from the 1536-well plate (Fig. 2). Consequently, reagent addition to these plates has to be rapid in order to avoid concentration changes via evaporation. Also, during incubations, the plates may need to be kept in a humidified chamber so that the volume in the wells does not change during the reaction.

The plastic-area-to-volume ratio is also a concern, since it is known that many proteins adhere to and denature when in direct contact with certain materials. As a well is miniaturized, the plastic-area-to-volume ratio increases from 0.55 mm2/|L for a 96-well plate to almost 4mm2/|L for a 1536-well plate. This means that if one of the reagents binds to the plastic, the proportion of that reagent bound to the plastic will be much greater in the 1536-well plate than in the 96-well format. This has serious consequences for assay development. For example, if one were looking for a kinase inhibitor and wanted to avoid compounds that would compete with ATP, then one would usually run the reaction at several fold the Km for ATP. However, if the ATP bound nonspecifically to the plastic, then the actual concentration of ATP in solution would be much lower than expected. Consequently, all the inhibitors that may be found in this assay format could potentially be competitive with ATP, exactly the opposite of what was searched for. Since the plastic-area-to-volume ratio is much higher in a 1536-well format than in the 96-well format, it will be imperative that Kms and other enzymatic parameters be determined directly in the new format.

Table 4 Specifications for Various Density Microtiter Plates

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