featuring the Echo acoustic technology
TITLES and AUTHORS
Acoustic energy can precisely and accurately eject a droplet of liquid from a reservoir, enabling delivery of picoliter and nanoliter volumes. Acoustic droplet ejection has been shown to be extremely precise (coefﬁcients of variation !2%) over a wide range of dispensed volumes. 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. 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.
Increasing the solute concentration of a solution alters the speed of sound in the solution in addition to changing the fluid viscosity and surface tension. For liquid handling devices that transfer fluids with acoustic energy, the change in sound speed can impact the focus of the acoustic energy and ultimately the accuracy of the transfer volume. To determine the effects of increasing solute concentration on transfer accuracy and precision, we studied the change in sound speed in dimethyl sulfoxide with the addition of low-molecular weight compounds and in phosphate-buffered saline with protein. The impact of compound and protein concentration on transfer precision and accuracy was found to be minimal and ADE can be used to transfer fluids regardless of these additions.
IC50 analyses are typically sample, time, and labor intensive. They commonly require multiple dilution steps and consume significant amounts of sample compound. Aqueous intermediate dilutions of concentrated stock solutions can lead to rapid sample precipitation and the generation of false negatives. Hydrophobic compounds may stick to pipette tips or intermediate dilution vessels, reducing the concentration of the analyte in the dilution and also increasing the possibility of cross contamination. The requirement for multiple serial dilutions in common IC50 analyses causes significant accumulated error. Concentrations of dimethyl sulfoxide (DMSO), the typical solvent used to solubilize compound libraries, as low as 1% in the final assay solution can significantly affect the results of the experiment. Finally, the cost of pipette tips and intermediate dilution vessels, and the frequency of the DMSO washes of tips grows significantly as the number of compounds being analyzed is increased. A system incorporating acoustic droplet ejection of compounds improves IC50 results by reducing the amount of sample used in the analysis to nanoliters, eliminating intermediate aqueous dilutions and accumulated pipetting error, lowering DMSO concentrations in the final assay to below 1%, and reducing costs of consumables (plastics, solvents, and their disposal).
Compounds used in high throughput screening (HTS) are typically dissolved in DMSO. These solutions are stored automation-friendly racks of wells or tubes. DMSO is hygroscopic and quickly absorbs water from the atmosphere. When present in DMSO compound solutions, water can accelerate degradation and precipitation. Understanding DMSO hydration in an HTS compound library can improve storage and screening methods by managing the impact of water on compound stability. A non-destructive, acoustic method compatible with HTS has been developed to measure water content in DMSO solutions. Performance of this acoustic method was compared with an optical technique and found to be in good agreement. The accuracy and precision of acoustic measurements was shown to be under 3% over the tested range of DMSO solutions (0% to 35% water by volume) and insensitive to the presence of HTS compounds at typical storage concentrations. Time course studies of hydration for wells in 384-well and 1536-well microplates were performed. Well geometry, fluid volume, well position and atmospheric conditions were all factors in hydration rate. High rates of hydration were seen in lower-volume fills, higher-density multi-well plates and when there was a large differential between the humidity of the lab and the water content of the DMSO. For example, a 1536-well microplate filled with 2μL of 100% DMSO exposed for one hour to a laboratory environment with ∼40% relative humidity will absorb over 6% water by volume. Understanding DMSO hydration rates as well as the ability to reverse library hydration are important steps towards managing stability and availability of compound libraries.
Acoustic droplet ejection (ADE) gently and precisely aliquots nanoliter and picoliter liquid volumes without any physical contact with the solution being transferred. The technology is very automation-friendly, as it is compatible with conventional microplates. Focused energy from an acoustic transducer induces droplet ejection into an inverted standard microplate. The commercial system transfers low-nanoliter volumes of dimethyl sulfoxide–dissolved compound libraries and thereby enables cell-based assays to be performed in 1536-well plates.