SLAS

Dry dispensing small volumes with disposable tips

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Authored by: Caliper Life Sciences Inc.

(Originally published as Caliper Life Sciences Application Note # 705.  Reprinted with permisson.) 

Small molecule chemical libraries used in drug discovery research are typically dissolved and stored in dimethyl sulfoxide (DMSO). In order to reduce the final concentration of DMSO during the assay (typically less than 5%) and to minimize compound usage small volumes of target compound are often pre-dispensed into the assay plates. As a result, volumes on the order of 0.5-2.0 μL are transferred to empty plates at the start of an assay. Because the plate is empty, the dispense is considered dry.

This application note explores the use of disposable tips for dry dispensing. Disposable tips have the advantage of being discarded between liquid transfers thus removing any chance of cross contamination. The discarding of tips is also a much faster process than washing and does not need to be validated to prove its effectiveness. Several types of microplates will be explored as will several dispense volumes.

Contents

Materials

Dry Dispensing Figure 1.jpg
Figure 1. Flow diagram of dry dispensing
  • Sciclone ALH equipped with a Low Volume Head and 96 Mandrel Array
  • BMG PheraStar plate reader set to read absorbance at 492nm
  • Thermo Multi-Drop 384 to back fill plates
  • Dimethyl Sulfoxide (JTBaker)
  • Tartrazine (Sigma)
  • De-ionized water (Millipore Milli-Q Gradient A10)

Microplates

  • Corning #3701, #3702, #3640
  • Matrix #4310, #4313
  • Greiner #655096

Tips

Caliper Life Sciences 384 disposable tips (P/N 108848 or 108849)

Experimental

A 6 g/L solution of Tartrazine in DMSO was prepared. An aliquot was placed in a reservoir. The Sciclone was programmed as described in Figure 1.

A calibration curve was generated for each type of plate using a calibrated manual pipette. This curve was used to calculate the actual volume dispensed. All plates were filled to a final volume of 80 μL for the 384-well plates and 150 μL for the 96-well plate with de-ionized water using a Multi-Drop dispenser. The 96-well plate was shaken for 2-3 minutes be¬fore reading on the BMG PheraStar. The 384-well plates were centrifuged for 1 minute at 2000 rpm to remove any bubbles before reading. Data was col¬lected for 3 replicates of 0.5 μL, 1.0 μL and 2.0 μL in each of the 6 plate types.

Dry Dispensing Figure 2.jpg Dry Dispensing Figure 3.jpg
Figure 2. 1 μL inside the disposable tips Figure 2. 1 μL drops on the end of the disposable tips

Results and Discussion

The data in Table 1 shows dry dispensing with disposable tips is possible down to 0.5 μL. Adjusting the head and/or plate locator until the tips and microplate bottom are parallel is required to get uniform dispensing. The solution to be dispensed must contact the surface of the well as uniformly as possible. The procedure described in Figure 1 aspirates a known volume and dispenses the entire contents into the destination well. Adjusting the accuracy of dispenses was accomplished by comparing the results against a plate prepared manually. A calibration plate was created for each plate type explored since the optical properties of each type must be included in the calibration calculations.

Accuracy and precision were influenced by the dispense height, dispense speed, wait time, and withdraw speed. The dispense height was set to 0.1-0.2 mm above the bottom of the plate. This allowed the DMSO to contact the plate on exit of the tip. Touching the plate sometimes created a seal. The back pressure generated was released abruptly on exit, causing a bad dispense. Pressing the tips on the plate bottom can also cause a deformation, squeezing out liquid inconsistently. If the tips are too far away from the bottom, the DMSO can creep up the outside of the tips or can stay attached to the end of the tips. Once the dispense height was optimized, dispense speed and wait time were adjusted. A speed of 2 μL/second with a 10-second wait was found to give the most reproducible dispenses. Withdrawing the tips at 2% speed gave the DMSO a clean break. Dispensing 1.0 μL plus the volume of interested was chosen because it was enough volume to get all liquid out without blowing the pre-air gap out. A blowout caused an inconsistent splashing of the DMSO.

Each plate in this experiment had a different coating. The coating dramatically changed the performance of dispensing. Surface interaction directly influenced the behavior of the DMSO on dispense. The un-coated and medium binding plates performed much better than non-binding and tissue culture treated plates. Corning 3702 and the Greiner plate were not coated. The Matrix 4310 had a medium bind coating. The Corning 3701, 3640, and Matrix 4313 were coated for tissue culture or had a non-binding coating. These plates would not be the ideal choice compared to the other plates in this list for dry dispensing of compounds; however, they must be used for the downstream assays.

Table 1. Dry dispensing with disposable tips
Image:Dry_Dispensing_Table_1.png



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