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Acoustic Droplet Ejection: Bringing Liquid Handling to a New Level

"I like to work on projects and products that are revolutionary as opposed to evolutionary—things that drastically change what scientists can do," says Joe Olechno, Ph.D. of Labcyte Inc. (Sunnyvale, CA, USA), a guest editor of the JALA special issue, Advancing Scientific Innovation with Acoustic Droplet Ejection. "So when I was asked 12 years ago to market an instrument that moves things with sound, and could really make a difference in the lab, I was intrigued and excited."

 

Acoustic droplet ejection (ADE) technology has matured since then, and current applications of the technology now enable "new, far-reaching science," write Olechno and co-guest editors Clive Green of AstraZeneca (Cheshire, UK) and Lynn Rasmussen of Southern Research (Birmingham, AL, USA) in their introduction to the special issue.

"When we first developed the instrument, we were looking at it mainly for high-throughput screening and dose-response experiments," says Olechno. "But people have taken the technology far beyond that."

From Printer to Lab

ADE technology was first described in 1927 by Alfred Loomis, who observed that immersing a high-power acoustic generator in an oil bath would create a mound at the surface that "erupt[ed] oil droplets like a miniature volcano," according to a seminal review of ADE by Richard Ellson of Picoliter Inc. (now Labcyte) and colleagues published in JALA in 2003. In the 1950s and '60s, the ability to localize and focus acoustic energy led to the development of devices that formed "geysers of small droplets," enabling the process to be used in commercial nebulizers. In the 1970s and 1980s, the use of ADE was explored for ink-jet printing and other areas of electronics. In the early 2000s, efforts to adapt the technology for liquid handling in the life sciences "began in earnest," Ellson and colleagues write.

Novel Applications

ADE technology "is now becoming an essential part of many labs," according to the guest editors, who have compiled 22 peer-reviewed articles that illustrate myriad practical uses of ADE in various stages of drug discovery and development, as well as monitoring crystal formation and non-pharmaceutical applications. All 22 of the original scientific reports in this special issue are freely available at JALA Online thanks to a generous sponsorship by Labcyte Inc.

Precision medicine. Two papers address the use of ADE in precision medicine, the focus of a massive U.S. initiative to overturn a "one-size-fits-all" approach to treating disease by taking into consideration people's genes, environments and lifestyles. "Typically, precision medicine means a doctor takes a sample of a patient's cancerous tissue; DNA sequencing is done to determine the types of mutations in the tissue; and based on the mutations and information from previous research—for example, 60 out of 100 patients with these specific mutations responded well to a particular drug regimen—a decision is made to try the regimen," Olechno explains. "That's much better than simply guessing which drugs might work, but it still means there's a 40 percent chance that the regimen won't work, and a patient might endure rounds of chemotherapy while the cancer remains unaffected."

Researchers at Uppsala University in Sweden and, separately, researchers at the Institute for Molecular Medicine in Finland took different routes. Besides DNA sequencing, Kristin Blom and colleagues at Uppsala took primary cells from patients and, leveraging ADE, tested them against 431 (latest number, per Olechno) cancer drugs that had been approved by the U.S. Food and Drug Administration or European Medicines Agency, though not necessarily for a patient's particular cancer. "Using that approach, they might discover that just four of the drugs are active against a particular patient's cancer," Olechno says. "Finding a drug that kills cancer ex vivo doesn't guarantee it will work in vivo, but if it doesn't work ex vivo, then it's almost for sure not going to work in vivo. So, this method can dramatically reduce the number of failed rounds of chemotherapy."

Krister Wennerberg and colleagues in Finland took a similar approach with primary cells in their Drug Sensitivity and Resistance Testing (DSRT) program. Their paper describes how ADE is enabling the DSRT program to " a) generate consistent but configurable assay-ready plates and how this impacts data quality; b) flexibly prepare drug combination testing plates; c) dispense reagents and cells to the assay plates; and d) [perform] ultra-miniaturized follow-up assays on the cells from DSRT plates."

According to the authors of both studies, ADE provides greater accuracy and flexibility than other liquid handling approaches, enabling the development of a reasonably priced chemosensitivity profiling platform.

Drug combinations. Although combination therapy is known to be effective in cancer, HIV and other diseases, practical considerations have precluded screening combinations of drugs early in the discovery process, according to Olechno. "Under normal conditions, trying to screen and test mixtures becomes too huge," he explains. "Even just trying to test five different concentrations of three drugs, A, B and C against each other comes to 125 different combinations, which is already quite big. And researchers often have thousands of drugs to analyze, which makes it practically impossible. But papers by Kevin Cross of Astra Zeneca and colleagues and Grace Ka Yan Chan of Genentech (San Francisco, CA) and colleagues show how ADE is making it easier to do a lot more work with a lot less material."

The paper by Cross et al. describes a recently developed software tool that, in conjunction with ADE technology as part of a compound-management platform, facilitates "the design and ordering of assay-ready compound combination plates and the processing of combinations data from high-content organotypic assays." The paper by Chan et al. shows how ADE "unlocks the potential of high-throughput dose matrix analysis" by enabling assays that test compound synergy, or lack thereof, in a 384-well format. "In the simplest embodiment, synergy can be demonstrated if the combined effect of two drugs is greater than additive effect expected if there was no functional interaction," the authors write. Using their approach, "novel therapeutically effective combinations of marketed and experimental drugs can be identified, providing a basis for further validation with in vivo efficacy models and ultimately clinical trials."

Combating infectious diseases. Rasmussen and colleagues describe the development and use of ADE applications for high-throughput screening of infectious agents. According to the authors, the technology's original calibration for dispensing compounds in DMSO enabled the preparation of assay-ready plates that can be sent to other locations. The plates were used as part of a collaboration between Southern Research and Galveston National Laboratory, University of Texas Medical Branch to screen compounds against Ebola and other biological safety level 4 pathogens. ADE aqueous calibrations, developed over the past several years, "have been instrumental in miniaturizing assays used for infectious disease, such as qPCR, tissue culture infectious dose 50, and bacterial motility, to make them compatible with HTS operations," the authors write. Much of the infectious diseases research conducted using ADE technology in the HTS Center at Southern Research "would have been difficult or impossible to do otherwise," they conclude.

Synthetic biology. Cost savings is a major benefit of ADE, according to Olechno, and two papers in the special issue show how this plays out in the lab. Paulina Kanigowska and colleagues at the University of Edinburgh (UK) describe the first application of ADE in synthetic biology, for DNA synthesis and assembly at the nanoliter scale. The authors write: "We were able to successfully downscale PCRs and the popular one-pot DNA assembly methods, Golden Gate and Gibson assemblies, from the microliter to the nanoliter scale with high assembly efficiency, which effectively cut the reagent cost by 20- to 100-fold."

Similarly, Carol Cain-Hom and colleagues at Genentech demonstrate cost savings and other benefits with the use of an ADE system for ultra-high-throughput screening of genetically engineered animal models. They write that transitioning to ADE for liquid handling led to "extremely low detectable cross contamination in PCR and qPCR despite extensive use, greatly increased data reproducibility (large increases in data quality and Cq consistency), lowered reaction volumes for large cost savings, and nearly a magnitude increase in speed per instrument."

Protein crystallography. Two papers describe the use of ADE for protein crystallography and one describes the system's ability to preserve protein potency throughout the discovery workflow. "Crystallizing a protein isn't easy," Olechno says. "Some proteins, such as rhodopsin and lysozyme, crystallize quickly, but many are difficult to crystallize and it takes a very long time to find the conditions appropriate for a particular protein. Miniaturizing assays is one way to facilitate the process."

Ping Wu of Genentech and colleagues discuss their use of ADE technology in crystallization experiments at the low-nanoliter scale, including aqueous crystallization reagents and ultra-small volumes of crystallization seed stocks, chemical additives and small-molecule ligands. They also developed an automated system that integrates the acoustic liquid handler with a robotic arm, plate centrifuge and other devices to enable unattended high-throughput crystallization experiments.

Daniel Ericson of Brookhaven National Laboratory (Upton, NY) and colleagues describe the system they developed to use ADE not only to harvest crystals but also to detect them and to monitor crystal growth. The current system detects crystals as small as 50 μm, and they expect that, in the future, the technology will permit the detection and monitoring of individual 3 μm crystals.

In other work with proteins, a study by Jacqueline Naylor from Medimmune (Cambridge, UK) and colleagues demonstrates benefits, including improved peptide potency and less peptide adsorption to plasticware, of ADE compared with tip-based approaches for automated peptide dilution.

Mass spectrometry. The special issue also features a paper by Ian Sinclair and colleagues at AstraZeneca (UK) based on the 2015 SLAS Innovation Award-winning presentation by Jonathan Wingfield (additional information is available in the SLAS ELN article, 2015 SLAS Innovation Award Winner Jonathan Wingfield: Bringing Screening with Mass Spectrometry to the Next Level). They showed that the ESI-interface developed to access the acoustically generated spray ensures the system will have wide application from cell metabolites, small-molecule drugs, peptides and other biomolecules. In addition to demonstrating that combining ADE and mass spectrometry enables researchers to analyze as many as three samples per second, the team shows that they can generate ion beams from typical test analytes with characteristic ESI spectra. They believe that the sampling speed may extend mass spectrometry to be more widely used in kinetic studies.

The AstraZeneca team also believes that future development and optimization of both the acoustic mechanisms and transfer interface can only enhance sensitivity, reproducibility and the complexity of matrices from which samples can be detected.

To complement the publication of this special issue and this report, in particular, free access to Wingfield's winning presentation also is available courtesy of Labcyte Inc.

Practical Considerations

Companies considering adding an ADE system to their life sciences R&D capabilities should keep the following points in mind, Olechno advises:

Initial cost. "An ADE system saves money in the long run, but there's a substantial initial cost," Olechno says. Systems start at about $200,000 and go up from there. However, as Wingfield reports, "the savings made by reducing solvent use and plasticware were shown to offset the cost of purchase within two years."

Changing workflows. "It's important to recognize that you can't just replace one system with another," Olechno says. "It's like a car versus a horse. When you buy a car, you'll have lots of advantages over the horse—but one of the disadvantages is that a car doesn't eat grass. Similarly, you can't simply take your old system that's transferring milliliters of solution and put it in a new ADE system and expect it to still transport milliliters; that will take forever and cost too much. You need to change your workflows so that they're compatible with the miniaturized format." The JALA special issue paper by Karen Roberts and colleagues at Astra Zeneca describes how, during the initial implementation of ADE, the company changed its workflows to address the "unique set of challenges" involved in applying the technology to 96-well, 384-well and 1536-well plates.

Plate availability. Another consideration is that "while we can shoot into anything, the plates we shoot out of must be acoustically compatible for highest precision and accuracy. That means, among other things, the bottoms have to be absolutely flat; otherwise, they'll act like a lens and make the sounds go in the wrong direction, which can affect droplets ejection," Olechno explains. Although acoustically compatible plates are only moderately more expensive than traditional multi-well plates, they're available only from a few vendors, as opposed to the hundreds of sources that can provide traditional multi-well plates.

"Readers will find a lot of interesting research in the special issue," Olechno emphasizes. "We're covering a lot of ground. Our hope is that once readers see what's happening with ADE technology, it will open up their eyes to what's currently possible and motivate them to come up with more new applications."

Learn More in the February 2016 JALA Special Issue

In addition to the contributions discussed in this article, the JALA Special Issue on Advancing Scientific Innovation with Acoustic Droplet Ejection features other original reports and technical briefs on a wide range of ADE applications. Free public access to the special issue is generously sponsored by Labcyte Inc. (Sunnyvale, CA, USA), whose ADE equipment is used in 21 of the 22 papers presented in the issue.

As noted earlier, to complement this special issue, free access to a live recording of the 2015 SLAS Innovation Award-winning presentation by Jonathan Wingfield of AstraZeneca (UK) also is available courtesy of Labcyte Inc. The webinar provides context and details for the special issue paper, "Novel Acoustic Loading of a Mass Spectrometer – Towards Next Generation High-Throughput MS Screening," by Sinclair and colleagues, which describes the first-ever coupling of mass spectrometry with ADE to analyze as many as three assays per second. In addition, two podcasts with Olechno and SLAS Podcast Editor David Pechter also will be freely available at JALA Online.

January 22, 2016