featuring the Echo Acoustic Technology
TITLES and AUTHORS
The high throughput direct measurement of substrate-to-product conversion by label-free detection has been considered the “Holy Grail” of drug discovery screening. Mass spectrometry as a detection system has the potential to be part of the ultimate screening solution. However, MS with existing sample introduction modes, despite being widely used in drug discovery typically cannot meet the throughput requirements of HTS. We propose a unique, innovative solution to the problem of throughput by using acoustic droplet ejection (ADE) to transfer femtoliter samples from microliter assays rapidly, precisely and accurately directly into a mass spectrometer. Acoustic technology has been widely used to support compound management activities within the pharmaceutical industry. The speed, accuracy, precision and robustness of acoustic dispensers have been proven. In principle, the integration of an acoustic source with a MS detector would result in a system capable of delivering ~4000 data points per hour into a high sensitivity label-free detector. It would enable sampling from 1536-well plates and reduce the total required assay volume to <5µL. The rapidity of sampling would enable real-time kinetic studies to capture multiple data points within the first minute of initiating a reaction. Together Labcyte Inc, Waters Corp and AstraZeneca have built a prototype acoustic source linked to a mass spectrometer. Droplets in the range of 50-200fL are acoustically ejected directly into the MS through a charge field. The ion beam is detected in the single quad MS where the typical signal has a very sharp attack profile and instant stop when the acoustic spray is turned on and off. This process produces a square wave signal which is simple to integrate for quantitative assays, and generates reliable and reproducible spectra. The system works in both positive and negative ion modes. The process is capable of producing singly or multiply charged species. The ability to load samples into a MS detector at such a high rate from much reduced assay volumes has significant potential not only within drug discovery but other areas of industry. Dynamic fluid analysis, the ability of the acoustic injector to adjust automatically to varying viscosities and surface tensions of the sample, allows the generation of droplets from a wide range of fluids including blood, plasma, cell culture medium, acid digests, and chemical syntheses. In principle, the simplicity of the acoustic source enables it to be fitted to any type of MS detector with an atmospheric pressure interface (single quad, triple quad, ToF), extending the range of applications into the “omics” field.
Epigenetics continues to emerge as an important target class for drug discovery and cancer research. As programs scale to evaluate many new targets related to epigenetic expression, new tools and techniques are required to enable efficient and reproducible highthroughput epigenetic screening. Echo liquid handlers can transfer compounds, samples, and reagents in sub-microliter volumes to high density assay formats using only acoustic energy - no contact or tips required. This eliminates tip costs and reduces the risk of reagent carryover. The PHERAstar FS multi-mode plate reader, with the highest sensitivity and lowest read time of assays in high density plate formats, is a perfect complement to enable an unparalleled solution for cost-effective, high-throughput epigenetic screening. Using the HTRF EPIgeneous™ Methyltransferase kit from Cisbio, we developed a miniaturized methyltransferase assay that can be easily adapted to automation and increased throughput, while maintaining high data quality. HTRF assays are typically performed at volumes of about 20 µL in a 384-well low volume plate. However, with the nanoliter dispense increments of the Echo liquid handler, assay volumes can be reduced significantly while maintaining data quality. In this study we were able to reduce a methyltransferase assay to a 2 µL final volume with excellent results.
Acoustic liquid handling utilizes high-frequency sound waves that are focused on the surface of a fluid to eject nanoliter-scale droplets with high accuracy and precision. The Echo® 525 liquid handler increases the transfer droplet volume 10-fold over our Echo 55X products, which allows for transfer rates of up to 5 μL/s and enables workflows for life science applications that were previously less practical. To enable the transfer of larger volumes of fluid at this faster transfer rate we are releasing a companion consumable, the multi-well Echo Qualified Reservoir (ER). The ER is a 2x3 well, ANSI/SLAS compatible source plate that has a maximum starting volume of 2800 μL of fluid per well and a dead volume of 250 μL per well – a large advantage over our standard 384-well plate (Figure 1).
In this work, we showcase two patented Labcyte technologies, Dynamic Fluid Analysis (DFA) and a high voltage (HV) grid, and describe how they enable the transfer of nano- to milliliter volumes of fluid using acoustic droplet ejection (ADE). We utilize a high speed, side-view camera that is coupled with the Echo to capture droplet dynamics in flight (Figure 2), and show how DFA and the HV grid are critical technologies for larger volume acoustic liquid handling.
The need for characterizing apoptotic processes occurs throughout the drug discovery process – from primary screening to toxicity profiling. Apoptosis is a tightly regulated cell death program that can be executed by cells that are no longer physiologically necessary. It is often triggered as a response to extrinsic factors or inhibited for survival as in the case of many cancer cells. Cell death cascades are complex and dynamic, underscoring the importance of a multi-parametric approach to assess apoptosis. This underscores the need to conduct robust and reliable cellular assays at high densities and with small sample sizes. As such, technological advancements such as High Throughput Flow and low-volume liquid handling have become critical components of methods assessing apoptosis. Using the IntelliCyt iQue Screener HD and MultiCyt® 4-Plex Apoptosis Screening Kit in conjunction with the Labcyte Echo series of liquid handler, we were able to simultaneously detect 4 different apoptosis endpoints in Jurkat cells in both 384- and 1536-well formats.
In this study, Jurkat cells were treated for 24 hours with known apoptosis inducing agents: staurosporine, nocodazole and camptothecin. After treatment, cells were labelled for one hour with a no-wash / single step addition of fluorescent markers for caspase 3/7 activation, Annexin V binding, cell viability, and mitochondrial membrane depolarization. Sub-microliter volumes of compounds and dye were transferred to the 384- and 1536-well cell plates with the Echo 555 liquid handler. The results show equivalent potency estimates for the compounds tested in both plate formats and correlate to previously reported activity for the biomarkers measured.
Epigenetics is emerging as an important target class for drug discovery and cancer research. As programs scale to evaluate many new targets related to epigenetic expression, new tools and techniques are required to enable efficient and reproducible high-throughput epigenetic screening. Echo liquid handlers can transfer compounds, samples, and reagents in sub-microliter volumes to high density assay formats using only acoustic energy - no contact or tips required. This eliminates tip costs and reduces the risk of reagent carryover. The PHERAstar FS multi-mode plate reader, with the highest sensitivity and lowest read time of assays in high density plate formats, is a perfect complement to enable an unparalleled solution for cost-effective, high-throughput epigenetic screening. Using HTRF EPIgeneous™ assay technology from Cisbio, we developed a miniaturized methyltransferase assay that can be easily adapted to automation and increased throughput, while maintaining high data quality.
EPIgeneous assays are typically performed at volumes of about 20 μL in 384-well format. However, with the nanoliter dispense increments of the Echo liquid handler, assay volumes can be reduced significantly while maintaining data quality. In this study we were able to develop a methyltransferase assay in a 5 μL final volume with excellent results and demonstrate further miniaturization opportunities for screening and inhibitor characterization.
Next generation sequencers have evolved to sequence hundreds to thousands of samples in a single run by sequencing pools of DNA libraries that have been labeled with unique barcodes. The ability to efficiently pool and normalize such DNA libraries is now a critical requirement of any NGS library production process. In efforts to improve library product processes, many have learned that efficient utilization of sequencing capacity is realized only if the production and management processes associated with upstream library construction, normalization and pooling are also scalable and cost effective.
In this study, we used the Echo liquid handler to normalize and pool the DNA fragments, quantified the DNA concentrations with the BMG LABTECH PHERAstar FS multimode high throughput reader, and validated the pooled fragments with the Agilent 2200 TapeStation The ability of the Echo liquid handler to normalize and pool the DNA in a single step, coupled with the power and flexibility of the Labcyte Echo® applications software, provides a scalable, robust, high throughput solution for an important upstream step in the NGS library production process.
Traditional DNA assembly methods require reactions of 10-20 microliters per well. The Echo® liquid handler can reduce that volume 10-fold, drastically cutting costs. With the ability to reliably transfer volumes as low as 2.5 nanoliters, Echo systems can extend the useful life of a primer library and eliminate the need to dilute high concentration primers, saving on storage and primer costs, and preventing dilution errors. In addition to miniaturization, the Echo system can rapidly pool oligos or DNA fragments from library plates. Since there is no contact with fluid and no time spent changing or washing tips, the Echo system can transfer each oligo or fragment from any well of a microplate in less than a second. This can save 10-15 hours of time in a high throughput setting. Finally, assay performance is essential. The accuracy and precision of Echo liquid handlers eliminate noise from assay data. This enables better detection of small changes in microbes and other organisms.
Quantitative PCR (qPCR) analysis is a powerful tool for gene expression analysis. Miniaturizing qPCR setup in low-volume, 384-well plates saves reagent costs. Using tipless, touchless acoustic droplet ejection (ADE) technology for low-volume liquid handling eliminates costs of disposable tips and washes. We demonstrate that ADE technology and magnetic feedback control (MFC) reagent dispensing can be combined to miniaturize PCR reactions, yielding reproducible results in low-volume reactions.
Overview of Automated Screening Platforms Quality Control Plates Used in Screening An Example of Screening with Echo 550 Current Status of Echo 550 at BMS
High Throughput Calcium Screening in Human Neutrophils; A Possibility in 1536 Well Format Using FLIPRTETRA