The pace of change in the life sciences enabled by improved detection systems demands fundamental changes in liquid handling and sample preparation. While traditional tip-based liquid handlers have historically addressed most liquid-handling needs, they often create obstacles to achieving results at the right cost, and may include cross-contamination, compounds and samples binding to tips, unreliable dispense volumes, time-consuming setup and calibrations, excessive consumption of rare samples, or expensive reagents. These issues go well beyond inconvenience: traditional systems have been shown to skew results in ways that scientists may not be aware of.
Acoustic droplet ejection (ADE) addresses these problems by moving liquid with sound waves. This approach is widely accepted as a state-of-the-art liquid-handling solution for compound management in high-throughput screening. ADE rapidly and precisely transfers liquids with nanoliter resolution upward from a microtiter plate to one that is inverted; surface tension prevents the liquid from dropping back down. The technique is replacing traditional aspiration-and-dispense liquid-handling robots in applications spanning synthetic biology, genotyping, personalized medicine, and next-generation sequencing.
As the reach of ADE has broadened, scientists have demonstrated that the use of sonic energy to move fluids has a number of advantages over liquid handlers that require moving parts such as valves, tubes, and tips. Multiple studies have shown that ADE-based handlers improve precision and data reproducibility, reduce costs and hands-on time, and eliminate consumable waste. This article reviews the limitations of traditional liquid handlers, explains ADE technology, and presents applications for which this approach has improved workflows and downstream results.
Conventional liquid-handling robots allow labs to run multiple experiments or scale to higher-throughput pipelines, typically in 384- or 1536-well plates. The many problems associated with tips or pin tools, however, are magnified as miniaturization is explored as a means to achieve lower costs. Tips introduce too much room for error—they carry over sample residue, causing cross-contamination and ruining downstream results, and clog, contributing to the high rates of downtime. Valves and tubes frequently need repairs or replacement to keep robots running properly. Traditional liquid handlers are also inflexible. Plate-to-plate transfer patterns are fixed, making these machines poorly suited to workflows in which scientists need to be able to adjust on the fly or simply have more options than a standard template for a variety of assembly needs.
Harnessing sound with acoustic droplet ejection
Physicists, chemists, and engineers realized they could harness the power of sound waves to transfer fluids, cells, and DNA, and that it was possible to control this sonic energy so precisely that it would enable highly accurate, nanoscale movement of individual droplets of liquid that could be aggregated to the desired total volume. Because transfer is accomplished with sound waves, there is no contact between the instrument and the samples and reagents it handles. This minimizes the cross-contamination that occurs with tips and, coupled with high precision and accuracy, makes data more reproducible and results more reliable. ADE liquid handlers significantly reduce volumes needed because they precisely transfer nanoliter-sized droplets. Scientists working with ADE systems report decreasing their reaction volumes by an order of magnitude or more, yielding dramatic savings in reagent costs. These miniaturized reactions also make it possible to increase capacity with a single instrument; researchers report that one ADE liquid handler could replace 8–10 traditional robots.1
ADE systems are much more flexible, allowing scientists to create their own transfer patterns based on what is needed for each experiment. By moving the transducer and destination plate relative to each other, rapid fluid transfers from any well to any well are achieved. This allows scientists to build assays or assemble genes simply and rapidly with no technician hands-on time. In addition, the lack of pipet tips eliminates a significant source of laboratory waste.
The use of contact-free systems to handle samples and reagents has brought improvements to many applications. For others, it has allowed entirely new opportunities to scale up throughput or for multiple users to run single experiments. At Genentech (South San Francisco, Calif.), scientists replaced several traditional liquid handlers with an ADE robot for their transgenic model qPCR-based genotyping workflow. Their process is truly industrial-scale, running more than 1 million reactions to generate some 800,000 genotypes every year. The scientists note that switching to an ADE-based liquid handler reduced reagent costs by more than 50%, performed cherry-picking in 80% less time and minimized cross-contamination to significantly improve data reproducibility compared to traditional liquid handlers.1
In personalized medicine, ADE technology has enabled the first truly individualized treatments for cancer patients. Scientists at the Institute for Molecular Medicine Finland in Helsinki have made remarkable strides in understanding how to select the optimal treatment for a patient, and cycling back to determine the mechanism of action to learn why the treatment has a particular effect. They screen primary cancer cells collected from each patient against a large array of potential therapeutics, monitoring drug sensitivity and resistance to choose the best drug or combination of drugs for that patient. ADE has been an essential component of this screening because it offers flexible preparation of testing plates, allows for extremely small reaction volumes and produces consistent plates for optimal downstream data quality.2 Precision medicine is not possible with imprecise liquid handling.
Synthetic biology is another important application. At the University of Edinburgh, researchers were the first to report using ADE technology for nanoliter-scale DNA synthesis and assembly.3 They miniaturized PCR reactions and two widely used assembly methods (Golden Gate and Gibson), finding that they could reduce reagent costs by as much as two orders of magnitude simply by adopting ADE liquid handlers. The team notes that the approach would likely “become an instrumental technology in synthetic biology, in particular in the era of DNA foundries.” Other researchers report a 100-fold reduction in the cost of sequencing genes of interest and a tenfold reduction in gene-assembly time. Miniaturization allowed 100 validation runs using the same volume of reagents previously used for one sequence run.4
While compound screening for drug discovery has become a common use of ADE liquid handlers, scientists at Southern Research Institute (Frederick, Md.) and the University of Alabama at Birmingham demonstrated their use for RNA interference (RNAi) screening.5 Because ADE systems are ideally suited for extremely low-volume reactions, they can be used to reliably dispense siRNAs. The RNAi screening pipeline established by this team outperformed other methods for large-scale siRNA screening. Labs are also using ADE for CRISPR and microbiome applications.
A team from London-based AstraZeneca used ADE technology to achieve the first mass spectrometer acoustic loading interface, enabling the researchers to analyze up to three samples per second.6 The process involved rapidly transferring femtoliter-scale droplets into the mass spectrometer, yielding strong readouts. According to the authors,6 this approach is an important step toward high-throughput, label-free detection of peptides or large proteins.
ADE technology is more than an alternative to traditional liquid handlers. Acoustic drop ejection systems can pave the way for entirely new applications, bringing higher throughput and greater flexibility to many areas of biology.
Mark Fischer-Colbrie is president and chief executive officer, Labcyte, Inc., 1190 Borregas Ave., Sunnyvale, Calif. 94089, U.S.A.; tel.: 408-747-2000; e-mail: [email protected]; www.labcyte.com