featuring the Echo Acoustic Technology
TITLES and AUTHORS
Recent advances in X-ray sources are rapidly reducing the sample size and exposure time for protein crystal structure determination. In order to fully exploit the reduced sample requirement for creating protein crystals we present an accurate and precise methodology for transferring nanoliter volumes of these solutions using the Echo® liquid handler.
Typical protein crystallization solutions have large variations in both viscosity and salt concentration and often have detergents or other additives. These solutions exhibit a broad range of rheological and acoustical properties. We show that the wide variety of aqueous-based fluids commonly used by the protein crystallography community is readily transferred with the Echo 555 liquid handler even within the broad viscosity range of 1-15 cP. The Echo liquid handler provides the user with a single calibration covering the gamut of protein crystallography solutions. The instrument uses acoustic energy to rapidly and automatically analyze each crystallographic solution just prior to transfer to optimize nanoliter-scale droplet transfer, responding to the state of the crystallographic solution at that instant in time. Results will be presented demonstrating the high precision and accuracy of transfers of binary mixtures of glycerol/water. Additionally, the Echo liquid handler has been used to transfer protein microcrystals to micromesh mountings and the resulting X-ray structures yield high resolution.
The results illustrate new capabilities of the Echo liquid handler that enable in situ optimization of protein crystallography conditions and volume miniaturization to match recent advancements in laser technology.
Acoustic liquid handling—using sound to move fluids and suspensions—has been widely accepted in the pharmaceutical high-throughput screening community and now has been applied to protein crystallography processes. The technology uses low-energy pulses of sound to eject fluids from an open source eliminating any physical contact with the fluid—no pipette tips, no probes, no "whiskers." Transfer volumes start at nanoliter levels but rapid repetition rates easily allow larger volumes to be transferred. We will show the latest results in five different areas relevant to crystallography: 1. Transfer of a wide range of fluids of varying viscosities and surface tensions, identical to or representative of solutions
typically used in protein crystallization. 2. Formulate microvolumes of intermediate solutions through spoton-spot placement. 3. Transfer of microcrystals for subsequent X-ray analysis or seeding experiments. 4. Non-invasive monitoring of protein crystallization fluids 5. Transfer lipidic cubic phase components to facilitate the self-selfassembly of nanoliter-scale environments for membrane protein crystallization. We believe that acoustic liquid handling provides crystallographers with a new tool of multiple dimensions that can be used in many different crystallographic applications.
As researchers begin to utilize the Echo liquid handler in areas new to automation, a need for a small scale automation platform that is easy to use and implement is critical. The Access laboratory workstation is a novel solution to quickly transform protocols created with the Echo® software applications into fully optimized automation routines. With a compact footprint, the Access system can be configured to integrate the Echo liquid handler with plate handling devices and accessories directly on the bench top. This poster examines the key attributes of the Access workstation allowing researchers to quickly adapt Echo application protocols into routines for fully automated assay plate processing.
The lifetime and stability of antibody stock solutions, antibody conjugate solutions and enzymes are increased by the addition of cryoprotectants such as glycerol, sorbitol or ethylene glycol. Aqueous solutions containing glycerol have historically been problematic to dispense in tip-based liquid handlers. Glycerol increases both the solution viscosity and the tendency for sample to adhere to the inside and outside of the tip. Increased solution viscosity and sample sticking reduces pipetting accuracy and precision, which can be especially significant in nanoliter volume transfers.
Acoustic droplet ejection (ADE) is a completely touchless transfer technology that can precisely and accurately transfer viscous materials. The Echo® 555 liquid handler (Labcyte Inc., Sunnyvale, CA) was used to transfer glycerol stock solutions in the viscosity range from 1 -15 cP (corresponding to 0 -60% weight of glycerol). Nanoliter volume transfers are confirmed with a fluorescent marker dye and a series of standard curves which were created on a Perkin Elmer EnVision microplate reader (PerkinElmer Inc., Waltham, MA). The Echo liquid handler was then used to dispense all of the components of a variety of commercially available high-throughput screening assay kit components, with excellent accuracy and precision.
The results for these applications illustrate the new capabilities of the Echo liquid handler that enable assay setup with flexible plate layout and volume reduction for reagent and consumables savings and higher throughput.
Assay miniaturization is often limited by the ability of automated liquid handlers to accurately address higher density microplates. It is also limited by the quality of the 1536-well microplate itself: increased well density often causes microplates to be less rigid, which in turn presents a challenge for automation. Labcyte has developed an released a new 1536-well, low dead volume microplate composed of cyclic olefin copolymer. The low profile microplate design reduces the dead volume to 1.0 µl, and increases the rate of acoustic transfer up to 30% over previous acoustic 1536-well microplates. The Labcyte® 1536-well microplate is manufactured to stringent rigidity and flatness specifications, ensuring reliable performance with automation. Microplate performance is demonstrated in conjunction with acoustic liquid transfer on the Echo 500 series liquid handlers, with accuracy and precision better than 95 percent.
Assay Miniaturization is limited by microplate quality and the ability of automated liquid handlers to accurately address higher-density microplates. Labcyte has developed and released a 1536-well, low dead volume microplate composed of cyclic olefin copolymer. This work presents the validation work proving the ability to acoustically transfer DMSO from the microplate using the Echo liquid handler. Preliminary validation data on the ability to acoustically transfer various aqueous reagents are also presented.
A totally integrated serial dilution assay plate preparation system that fully uses the high precision nanoliter dispensing capabilities of acoustic liquid handlers has been developed and implemented. The application uses a hybrid of a serial dilution method and a direct dilution method, achieving a wide concentration range for the dilution series, while avoiding additive errors inherent to traditional serial dilution methods. The method allows assay miniaturization, which greatly reduces reagent and consumable costs to the customers. The system is in production at AstraZeneca and has generated high-quality assay ready plates for high-throughput screening and secondary screening since 2005. Further development in recent years has expanded the flexibility of the assay ready plate creation process to meet varied screening requirements.
We will discuss the requirements for assay ready plates for concentration response testing and describe the novel plate creation method in detail with the rigorous validation procedures. Along with method validation data, some real-life screening results will be presented to compare an experiment conducted on compounds prepared using the novel hybrid method and those prepared using a more traditional serial dilution method, which endorses the application of the novel method.
Surfactants and proteins are key components present in aqueous solutions used in biological assays and have historically been problematic to dispense with high-speed tip- or nozzle-based liquid handling instrumentation. In the case of buffered solutions containing surfactants, due to their low surface tension these fluids have a high tendency to leak out of tips or nozzles. Furthermore, buffered solutions containing surfactants often contain bubbles that can become entrained or trapped in the tips or nozzles of standard liquid handling instrumentation. Dissolved protein can also serve to introduce bubbles into traditional aqueous dispensing. Because of these difficulties, there is clearly a need to extend the nozzle-less fluid transfer technology of acoustic droplet ejection (ADE) to robustly handle fluids containing surfactants and proteins.
Acoustic droplet ejection (ADE) is a unique liquid transfer technology that eliminates the use of tips or nozzles and has been proven to dispense more accurately and precisely in the low nanoliter regime with DMSO as compared to tip-based liquid handling. When transferring solutions containing protein using ADE, the interaction of the protein at the fluid-well interface can cause a highly tilted or an irregular meniscus (often compounded by the centrifugation of well plates) which can lead to inaccurate and imprecise dispensing in an acoustic-based liquid handling instrument that was not modified to deal with source well meniscus variation. The work described here extends the capability of the Echo® 555 liquid handler (Labcyte Inc.) which utilizes ADE technology to accurately and precisely dispense buffered solutions containing surfactant or protein or both surfactant and protein. Performance with these difficult solutions is now comparable to that of DMSO and of aqueous buffer solutions containing moderate salt concentrations.
Advances in the miniaturization of thermal cycler technologies have enabled PCR in a higher density format with lower total assay reaction volumes. By utilizing newly developed 1536-well plates (IT-IS International Ltd.) for PCR one can save on reagent costs and increase throughput. However, the liquid handling employed in such low-volume PCR reactions must be robust in accuracy and precision. Using tipless, touchless acoustic droplet ejection (ADE) technology for low-volume liquid handling eliminates the cost of disposable tips and wash cycles. For the ADE we used the Labcyte Echo® 555 liquid handler (Labcyte Inc.) to transfer primers and probes from a 384-well source plate into a 1536-well plate. Sample volumes at 50 nL or 100 nL were transferred from each source well. PCR master mix (Hoffman La Roche) was transferred at 1 ìl per well from a single well source reservoir to an entire 1536-well plate. With the ability to transfer from any well of the source plate to any well of the destination plate, setup can be formatted to a wide array of different combinations of primers and probes with each template DNA. We demonstrate that ADE technology dispenses precisely and accurately into 1536-well PCR plates to miniaturize real-time quantitative PCR (qPCR) gene expression reactions, yielding reproducible results in low-volume reactions.
Comparison between POD handling and traditional liquid handlers Insoluble compound tests on POD and Bravo Compound library screening with POD Advantages of POD
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