Moving liquids with sounds

High-throughput screening (HTS) is the essence of drug discovery. Latest developments show new assays, new targets and new readers coupling with state-of-the-art data analysis and information management. Essential to drug development is rapid and efficient ADME/toxicology. Here, new technologies accelerate processes in an attempt to move drugs through the pipeline with increasingly greater efficiency.

In these rapidly evolving environments, the transfer of liquids may seem mundane. Most HTS liquid transfer systems are based either on arrays of eye-dropper-like pipettes or rubber-stamp-like pin-tools. For the past two decades, researchers have designed their assays around the limitations of these devices. However, as the drive to smaller, less costly assays continues, the intimate contact of the transferred fluid and the tool conducting the transfer is becoming the single most important source of error.

When an instrument transfers liquid through a contact-based mechanism, some liquid inevitably sticks to the instrument surfaces. As the absolute volume of liquid is reduced, this miniscule amount becomes an increasingly large and variable percentage of the whole. This variability degrades both precision and accuracy. Not only does this affect the reliability of the transfer but the residue can cross-contaminate other samples. To eliminate the cross-contamination, most HTS groups either use pipette tips for one transfer only or go through extensive washing steps. Disposing tips is costly and generates a large amount of solid waste. Washing pipettes for multiple use also generates significant solid waste and in addition environmentally suspect liquid waste. Cross-contamination and inaccurate, imprecise transfers increase with rising viscosity and when near saturated solutions crystallize or precipitate.

Acoustics in the HTS lab

When placing a glass of water on a working loudspeaker, ripples will form on the surface. With increasing sound, the ripples will eventually shoot droplets of water upward from the surface. Using acoustic droplet ejection (ADE), Labcyte Inc. refines this basic idea into laboratory practicality. A focused pulse of sound ejects a droplet of fluid with extraordinary accuracy and precision. An inverted microwell plate or any other suitable surface positioned over the source solution captures the droplet via surface tension. To dispense a greater volume than the amount contained in a single droplet, the instrument ejects multiple droplets rapidly – as quickly as 500 times per second. In this manner, droplets of samples transfer from source microplates with either 384 or 1536 wells. Samples can be transferred to destinations of 96-, 384-, 1536- or 3456-well plates or onto flat surfaces such as microscope slides or tissue sections. Each source well is addressed individually, so fluid from any source well can be transferred to any destination well or position. Since nothing ever touches the fluid, cross-contamination and clogging are prevented. Likewise, there is no need to wash any component of the system between transfers thus reducing toxic waste. For companies with single-use pipette tips , ADE can save the lab between $200,000 and $500,000 per instrument per year by eliminating tips, washes and waste disposal.

Researchers at Merck-Frosst reported that they used ADE to move assays from 384-well plates to 3456-well plates. They lowered reagent usage by 90 percent, and reduced both the time and space required for assays. Results of the miniaturized assays were shown to be comparable to those in 384-well format.

Increasing the quality of answers

While lower operating cost motivates change, quality may be the ultimate incentive for some organizations to use ADE. Researchers at Bristol-Myers Squibb (BMS) showed that ADE-based systems provided better answers and prevented the loss of strong drug candidates for further development. BMS researchers generated dose-response curves for a collection of compounds using a standard serial dilution technique and direct dispensing with ADE. The serial dilutions implied that many compounds were non-active when they were actually extremely potent. Some compounds, when transferred by ADE, proved active at concentrations that were more than two orders of magnitude lower than when transferred by serial dilutions. Studies at BMS and elsewhere showed that compounds with high logP values were especially prone to this difference. The absorption of compounds from dilute samples by intermediate vessels and pipette tips appears to be the genesis of the problem. Recent results based on mass spectroscopic (MS) data indicates that the concentration of compound in assay wells prepared by serial dilution often contain dramatically less compound than expected. In contrast, MS experiments showed that the measured concentrations of ADE-transferred liquids matched expected concentrations.

ADE in ADME/Tox

ADE for the transfer of low nanoliter and picoliter volumes has significant financial impact in secondary screening including the miniaturization of secondary screening. In addition, ADE is important to a new field, imaging mass spectrometry (IMS). This technique promises dramatically enhanced discovery in proteomics, cancer research, drug metabolism and systems biology. The analytical power of mass spectroscopy may now be linked with the localization of histology. IMS reveals the position of proteins and small molecules in tissues such as animal models and tumor samples. In contrast to 2-D gels from homogenized samples, this approach determines the concentration of specific compounds at each point in the tissue. This data produces a “protein image” of the tissue.

The structures in which particular proteins are found can be pinpointed, even when the protein has not been previously identified and isolated. IMS can track the distribution and metabolism of drugs without requiring radioactively labeled compounds. ADE brings high precision and accuracy to the automation of reagent deposition onto thin tissue samples for IMS. Precise drop-on-drop positioning prevents the migration of reagents and substrates from one portion of the sample to another to ensure an accurate representation of their physical location. Biochemical reactions such as proteolysis can be performed in situ. Flexible droplet timing allows the user to optimize reaction conditions and crystal formation, and enables the user to generate more reproducible and higher quality mass spectral data.

Labcyte recently introduced the Portrait 630 reagent multi-spotter. It uses ADE to transfer droplets of 170 pL onto tissue slices for IMS. As shown at the Vanderbilt University Mass Spectrometry Research Center, ADE provides significantly more sensitive and more reproducible mass spectral data. ADE can be used to find novel drug targets by associating unknown proteins with pathological states. It can also monitor the uptake, distribution, breakdown and elimination of drugs from organisms without resorting to fluorescently- or radioactively-labeled compounds. This allows the simultaneous identification and localization of the native candidate drug and its metabolites, while eliminating the expense of making radioactively labeled analogues.

ADE has removed the liquid handling roadblock to reliable low-volume liquid handling. The Echo series 500 liquid handlers reduce false negatives while reducing costs both through miniaturization of assays and the elimination of tips, plates and washes. The Portrait 630 reagent multi-spotter improves the results of imaging mass spectrometry giving researchers a new tool for finding targets and monitoring drug metabolism. Additional ADE applications include coating micro-devices, making mono-dispersed particles and the micro-miniaturization of assays to the nanoliter level. Over the last two years, the sound of liquid handling has changed from the splash of milliliters to the ultrasonic ping of ADE.