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Figure 2 Evaporation from microwell plates presents a significant challenge for developing assays in miniaturized formats. The figure shows the evaporation rate of dH2O from a Greiner 1536-well plate (10 ||L volume) and a Vision 1536-well plate (3 ||L volume). The evaporation rate in the Vision plate is approximately 5.6 nL per minute and almost twice as rapid as the evaporation rate, 3.1 nL per minute, in the Greiner 1536-well plate. The more rapid loss of liquid from the Vision plate is a direct function of the greater surface area. Since most HTS assays are complete within a matter of a couple of hours, evaporation may not pose a significant problem. However, should longer incubations be required, incubation can be performed in a humidity-controlled environment. Relative humidity was 42%, and the lab temperature was 24°C.

The shape of the well, as well as the material from which it is constructed, can serve to ameliorate some of these potential problems. For example, the data in Table 4 for 1536-well plates assumes that the well shape is an inverted pyramid. If a standard cylindrical 96-well is miniaturized to the format of a 1536-well then the plastic-area-to-volume ratio is nearly four times larger. This inverted pyramidal shape can also aid in liquid handling.

The inverted pyramidal shape confers two additional advantages in assay miniaturization. First, since the walls separating the wells come to a knife edge, liquid can be ''sprayed'' into the wells much as a spray painter can paint a wall.

Any liquid that hits the apex or intersection between two adjacent wells must, by well design, run into one of the two wells. This lack of interstitial spaces between wells allows reagent to be added very quickly to all the wells on the plate. Oldenburg et al. [31] report that using an ink-jet device they were capable of adding 100 nL of liquid to each of the wells of a 9600-well plate in under 70 seconds with CVs of less than 4% using this method. This ''angled'' well will also be important for mechanical pipetting as well. Since the surface area to be hit by the pipettor is quite small, the angled well will help guide the pipet into the center of the well. Thus, the tolerance required of the disposable pipet tips can be more generous. If one were to miniaturize the shape of the current 96-well plates into a 1536-well format, this would not be the case, and problems will result when the pipet tips hit the flat interwell spaces. Second, as will be discussed further below, the angled well will help increase the image quality when data is collected from these plates by CCD technology.

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