Effect Of Speed Of Withdrawal On Volume Transferred

See more recent speed of withdrawal data at Lab Automation 2001 presentation and comprehensive tests on all pins.

Assay Conditions

FP9, FP1, FP3, FP9H, FP1H, FP3H, FP1S6, FP1S10, FP1S50, FP3S100, FP3S200 and FP3S500 Pins were dipped into 200 ul  FITC in 100% DMSO in a 96 well source plate.

Pins removed from source plate at 15 RPM (slow - .78 cm/sec), 30 RPM (med – 1.56cm/sec), 60 RPM (fast – 3.12 cm) and 140 RPM (very fast - 7.28 cm/sec).

Pins transferred to 200 ul 0.1 M Tris, pH 8.0 in black polystyrene assay plate.

Uncoated Pins

• FP9
• FP1
• FP3

Hydrophobic Pins

Slot Pins (FP1 Pins)

Slot Pins (FP3 Pins)

Conclusion

The speed of pin withdrawal is proportional to the volume transferred on the pins.  Solid pins will transfer ~ 3 larger volumes at very fast withdrawal speeds (7.84 cm/sec) than at the slowest speed.  CV's at all speeds were generally in the acceptable range of under 10% with one or two exceptions.  There were no significant differences between uncoated and hydrophobic coated pins.

Slot pin total volume transferred are less dependant on the speed of withdrawal then solid pins.  The larger the slot, the less the increase in volume with increasing withdrawal speed.  

It is possible to reproducibly adjust the volume delivered with both solid and slot pins by increasing or decreasing the speed of withdrawal.  

  Effect Of Dwell Time On Volume Transferred To A Dry Plate

Assay Conditions

FP3 and FP3H Pins were dipped into 200 ul  FITC in 100% DMSO in 96 well source plate. 

Pins removed from source plate at 15 RPM (slow - .84 cm/sec) speed and spotted into  a dry black polystyrene assay plate.   

The pins were allowed to "dwell" on the dry plate for various time intervals.  Then 200 ul 0.1 M Tris, pH 8.0 was added to each well prior to reading the plate.

Uncoated Pins

Hydrophobic Pins

Conclusions

Optimal dwell time for delivering maximum volume is 1 second.  There were no significant differences between uncoated and hydrophobic coated pins.

  Hanging  Drop Delivery

Assay Conditions

FP9, FP1, FP3, FP9H, FP1H and FP3H Pins were dipped into 200 ul  FITC in 100% DMSO in 96 well source plate. 

Pins were removed from source plate at 15 RPM (slow - .84 cm/sec) speed and blotted off on a dry nylon membrane to remove the hanging drop or the volume in the slot. The remaining volume on the sides of the pins were transferred into  200 ul 0.1 M Tris, pH 8.0 in a black polystyrene assay plate.   Unblotted pins were also transferred into 200 ul 0.1 M Tris, pH 8.0 in a black polystyrene assay plate.   The hanging drop was determined by subtraction of these two results. 

Uncoated Pins

Hydrophobic Pins

Conclusion

Hanging drops are from 1/3 to 1/2 the total volume delivered by the pin.  There were no significant differences between volume transferred of  uncoated and hydrophobic coated pins.  This experiment does not address non-specific binding to the pins.

  Effect Of Variable Volumes/Concentrations In Source Well On Volume
  Transferred

Assay Conditions

FP9, FP1, FP3, FP9H, FP1H, FP3H, FP1S6, FP1S10, FP1S50, FP3S100, FP3S200 and FP3S500 Pins were dipped into  variable volumes from 200 ul to 25 ul  FITC in 100% DMSO in 96 well source plate.  The concentration of the volumes increased proportionally to the decrease in the volume to simulate concentration of samples that have been reduced by evaporation (edge effect drying).     

Pins removed from source plate at 15 RPM (slow - .78 cm/sec) and transferred to 200 ul 0.1 M Tris, pH 8.0 in black polystyrene assay plate.

Uncoated Pins

Hydrophobic Pins

Slot Pins (FP1 Pins)

Slot Pins (FP3 Pins)

Conclusions

There is only a small difference in volume transferred between 200 ul and 125 ul volumes in the wells with both solid and slot pins.  Slot pins are less effected than solid pins.  The most precipitous drop in volume transferred occurred between 75 ul and 25 ul.   

For general high throughput screening applications volume/concentration effects between 200 and 25 ul will at the most have a 2 fold effect on assay results.

For libraries where there is significant drying of the edge wells slot pins would be the pins of choice as they demonstrate the least volume/concentration effect.  

There were no significant DMSO volume differences between uncoated and hydrophobic coated pins.  This experiment was not designed to test for non-specific binding differences between the pins.

Data for a similar volume of source plate well experiment, without increasing the concentration is found on this hyperlink.