Debunking the Myth: Validation of Fluorescein for Testing the Precision of Nanoliter Dispensing

Acoustic energy can precisely and accurately eject a droplet of liquid from a reservoir, enabling delivery of picoliter and nanoliter volumes (Ellson, R.; Mutz, M.; Browning, B.; Lee, L.; Miller, M.; Papen, R. Transfer of low nanoliter volumes between microplates using focused acoustics—automation considerations. Journal of the Association for Laboratory Automation 2003 , 8(5) , 29–34). Acoustic droplet ejection has been shown to be extremely precise (coefficients of variation <2%) over a wide range of dispensed volumes (Ellson, R.; Mutz, M.; Browning, B.; Lee, L.; Miller, M.; Papen, R. Transfer of low nanoliter volumes between microplates using focused acoustics—automation considerations. Journal of the Association for Laboratory Automation 2003 , 8(5) , 29–34). However, measuring the performance of low-volume fluid transfers can be difficult because the data are often masked by variability in bulk dispensers and fluorescence readers used as part of the overall measurement process (Petersen, J.; Nguyen, J. Comparison of absorbance and fluorescence methods for determining liquid dispensing precision. Journal of the Association for Laboratory Automation 2005 , 10(2) , 82–87; Rhode, H.; Schulze, M.; Renard, S.; Zimmerman, P.; Moore, T.; Cumme, G.; Horn, A. An improved method for checking HTS/uHTS liquid handling systems. Journal of Biomolecular Screening 2004 , 9 , 726–733). The fluorophore used must also be stable so that thermal bleaching and photobleaching do not contribute additional variability to the measurements. This study assesses the suitability of fluorescein to measure the precision of fluid transfers of 2.5-nL DMSO droplets. The short-term and long-term stabilities of fluorescein are first qualified using a reference standard. Next, we determine the noise contribution of the filler and reader. Lastly, data are presented for the precision of 5- and 50-nL fluid transfers using this fluorescein measurement process.