Flexible, Parameter-Controlled Nanoliter Pipetting on a Tecan xyz Platform

Cost pressure and rising throughput requirements are important drivers for assay miniaturization. Typical examples for the trend “smaller is better” are found in BioChip applications and in High Throughput Screening (HTS), which is evolving from the 96-well standard to high-density microplates with 384, 864, 1536 or more wells. These applications require the automated pipetting of liquids in the submicroliter volume range, a difficult task for traditional automated liquid handling systems based on syringe pumps. Tecan developed a new device for the accurate pipetting of volumes in the nanoliter range. Based on ink-jet printer technology, this device allows the exact control of the volume of the ejected droplets via a set of parameters. The integration of this new technology into Tecan's flexible xyz-platforms allows an easy use of this powerful technology for several applications. Results such as volume range, accuracy and precision are discussed.     

Automated High Throughput Screening of Fluorescent Imaging Plate Reader (FLIPR) Assays Using Assay Platform™ Integrated with Activity Base for ‘on the fly’ Compound Reconfirmation

Advances in the field of automation have meant hitherto complex manual cell-based assays can now be automated. These improvements have brought significant enhancements in throughput, data fidelity and consistency, and allowed a reallocation of constrained resources.Building upon these improvements, we have linked our automated cell-based screening system, Assay Platform™, to Activity Base (IDBS), a software package designed to automate the analysis of HTS data. Customisation of this package has resulted in software that can identify ‘active’ compounds and re-pick them ‘on the fly’ from the original compound plates for triplicate re-testing without operator intervention.Based on an operator initially defining ‘normal’ parameters for assay activity in Activity Base, combined with an automated quality control software module that checks data fidelity, wells containing ‘active’ compounds can be re-picked and re-tested at the end of an automated screening run. Automating cell-based assays has significantly improved productivity, and, with the synergism of Activity Base, has given us greater power to complete each screening run and report ‘active’ compounds to Chemistry more rapidly. This article presents our approach to the automation of cell-based Fluorescent Imaging Plate Reader (FLIPR) screening together with automated active re-test confirmation using Activity Base.     

Monitoring of HTS compound library quality via a high-resolution image acquisition and processing instrument

This report presents the high-resolution image acquisition and processing instrument for compound management applications (HIAPI-CM). The HIAPI-CM combines imaging spectroscopy and machine-vision analysis to perform rapid assessment of high-throughput screening (HTS) compound library quality. It has been customized to detect and classify typical artifacts found in HTS compound library microtiter plates (MTPs). These artifacts include (1) insufficient volume of liquid compound sample, (2) compound precipitation, and (3) colored compounds that interfere with HTS assay detection format readout. The HIAPI-CM is also configured to automatically query and compare its analysis results to data stored in a LIMS or corporate database, aiding in the detection of compound registration errors. To demonstrate its capabilities, several compound plates (n=5760 wells total) containing different artifacts were measured via automated HIAPI-CM analysis, and results compared with those obtained by manual (visual) inspection. In all cases, the instrument demonstrated high fidelity (99.8% empty wells; 100.1% filled wells; 94.4% for partially filled wells; 94.0% for wells containing colored compounds), and in the case of precipitate detection, the HIAPI-CM results significantly exceeded the fidelity of visual observations (220.0%). As described, the HIAPI-CM allows for noninvasive, nondestructive MTP assessment with a diagnostic throughput of about 1min per plate, reducing analytical expenses and improving the quality and stewardship of HTS compound libraries.     

Miniaturization of an Enzyme Assay (β-Galactosidase) in the 384- and 1536-Well Plate Format

Miniaturization is one way to realize today's demands in the drug discovery process by moving from the standard 96-well plate to higher density microplate formats. In this article we describe the adaptation of a fluorescence-based enzyme assay to the challenges of the 384- and 1536-well plate format. The liquid-handling was realized by the automated micropipettor CyBi-Well™ 96/384/1536* (CyBio AG - formerly JENOPTIK Bioinstruments Gmbh - Jena, Germany). On the basis of optimized liquid-handling parameters pipetting routines were established to perform an enzyme assay (β-galactosidase) in the microplate formats of higher density. Finally, the experimental results were compared to those obtained in the well-established 96-well format.

In the enzyme assay, the bioconversion of the substrate Fluorescein-di-(β-D-galactopyranoside) (FDG), occurred as a linear function of the β-galactosidase concentration comparably in all three assay formats. We conclude that miniaturization using the higher density 384- and 1536-well plate formats is advantageous as the next evolutionary step in HTS, especially using enzyme assays. A careful individual adaptation procedure for each microplate format and assay at the basis of the optimized liquid-handling parameters is essential. CyBi-Well™ 96/384/1536 proves to be a powerful tool for a careful adaptation of the liquid-handling procedures of biological assays especially also in the 384- and 1536-well formats.     

Heraeus cytomat® 6000 Series Incubators and Storage Systems from Kendro Laboratory Products

There are a number of cases in high throughput screening systems where a controlled environment is desired. These include incubation periods for cellbased assays, incubation for protein detection assays such as ELISA or fluorescence assays, and branching assays for mRNA detection. In addition, as the density of wells in microplates increases and well volumes become smaller, evaporation becomes a concern in all assays.The cytomat® 6000 is a robot accessible, automated CO₂ incubator used for cell-based High Throughput Screening systems. The incubator provides superior environmental conditions, due to the unique access door at the back of the instrument, the PlateShuttle. This small access opening insures that the environment inside the system (temperature, humidity and CO₂) is undisturbed as microplates are accessed.The system provides high speed, random (bar-coded) access to all microplate formats for 24, 96, 384 and 1536 well microplates (up to 261-microplate capacity). Other cytomat configurations offer refrigerated and low humidity environments.     

SOS–A sample ordering system for delivering “assay-ready” compound plates for drug screening

Many bottlenecks in drug discovery have been addressed with the advent of new assay and instrument technologies. However, storing and processing chemical compounds for screening remains a challenge for many drug discovery laboratories. Although automated storage and retrieval systems are commercially available for medium to large collections of chemical samples, these samples are usually stored at a central site and are not readily accessible to satellite research labs.Drug discovery relies on the rapid testing of new chemical compounds in relevant biological assays. Therefore, newly synthesized compounds must be readily available in various formats to biologists performing screening assays. Until recently, our compounds were distributed in screw cap vials to assayists who would then manually transfer and dilute each sample in an “assay-ready” compound plate for screening. The vials would then be managed by the individuals in an ad hoc manner.To relieve the assayist from searching for compounds and preparing their own assay-ready compound plates, a newly customized compound storage system with an ordering software application was implemented at our research facility that eliminates these bottlenecks. The system stores and retrieves compounds in 1 mL-mini-tubes or microtiter plates, facilitates compound searching by identifier or structure, orders compounds at varying concentrations in specified wells on 96- or 384-well plates, requests the addition of controls (vehicle or reference compounds), etc. The orders are automatically processed and delivered to the assayist the following day for screening. An overview of our system will demonstrate that we minimize compound waste and ensure compound integrity and availability.     

Automated High Throughput Purification of BigDye™ Terminator Fluorescent DNA Sequencing Reactions Using Wizard® MagneSil™ Paramagnetic Particles

We describe a reagent system and robotic methods for the purification of BigDye™ Terminator sequencing reactions prior to automated fluorescent sequence analysis. The methods use MagneSil™ paramagnetic particles to isolate sequencing extension products from unincorporated dye-labeled terminators and exchanges sequencer loading solution for reaction buffer. Processed samples give usable data that is greater than 98% accurate from primer plus 5–15 bases to over 700 bases. Typical Phred greater than 20 quality scores range from 600 to over 700 bases. This process has been adapted to a number of liquid handling robotic platforms in both 96- and 384-well formats. One method using a single POD Beckman Biomek® FX can process up to four plates in approximately 40 minutes.     

The μFill: A New 96-/384- Well Microplate Reagent Dispenser for HTS and Drug Discovery

Today's biomedical research requires instrumentation that is both functional and versatile. While high throughput screening (HTS) and drug discovery laboratories require instrumentation that can be automated, pilot assay laboratories may not necessarily need total automation. Towards that end, Bio-Tek has developed the μFill, a 96-/384- well microplate reagent dispenser capable of running stand-alone or computer-controlled as part of a robotics system. The μFill is compatible with both 96- and 384-well microplates, and using a specially designed adaptor can also dispense to deep-well microplates. It is capable of dispensing from 10-3000 μl for 96- well plates and 5-1500 μl for 384-well plates in 1-μl increments. The μFill can dispense 20 μl into a 96-well plate in four seconds and into a 384-well microplate in 12 seconds. The microprocessor-controlled syringe pump is based on a tested, low-maintenance design that requires no calibration, yet provides a high degree of accuracy and precision. The accuracy with an 80 μl dispense is within 1 μl with a percentage CV of less than 2%, and with a dispense volume of 20 μl, the percentage CV is still less than 5%. For those needing to dispense organic solvents or sterile aqueous solutions, a model that is autoclavable and has increased solvent resistance is also available. The programming allows for the control of flow rates from 225 μl/well/sec, for dispensing to cell cultures, and to 1000 μl/well/sec for rapid and vigorous reagent dispensing. The flexible software provides complete programming capabilities from the keypad. For more complete automation, robotics interfaces can be developed using ActiveX® software commands. The μFill's size, with a 14 x 14-inch footprint and a height of seven inches, allows it to be used almost anywhere.     

A 384-Well, Fully-Automatable, Superparamagnetic-Particle Based Dye Terminator Removal Kit

We have developed a method for the automated purification of DNA sequencing reactions using the RapXtract™ 384 Dye Terminator Removal Kit and the Quadra 3™ Workstation. The process enables purification of 384 reactions in five minutes, significantly impacting the through-put potential of sequencing laboratories. The RapXtract technology utilizes superparamagnetic particles, (i.e., particles that are not themselves magnetic but that respond to a magnetic field) and eliminates the need for centrifugation, vacuum filtration, or modified primers. The Quadra 3 Workstation is a 384-channel liquid handling system, fitted with a retractable magnetic nest designed to incorporate a 384 magnetic separator. The combined technologies result in reduced variability associated with manual methods for sequencing reaction purification.     

High Throughput Drug Profiling

With the development of high throughput screening (HTS) during the last two decades new technologies have gained access to chemistry and biology laboratories. The use of both, laboratory robotics and automated workstations has greatly increased the number of chemical entities that are synthesized for and tested against new targets. While a decade ago a daily throughput of about 1,000 compounds was considered sufficient, nowadays screening laboratories aim to achieve 100 times as many samples in the same period of time. Combinatorial chemistry vice versa has increased comparably the daily output and HTS has become an important success factor during early lead finding. Nearly all drug discovery research projects in pharmaceutical industry employ HTS screening assays as initial steps to discover the chemical leads. These compounds provide the structural basis for further medicinal chemistry activities that focuses on optimization of the lead with respect to the activity and selectivity profile in order to identify the development candidate.     

A system for dispensing sub-milligram doses of active pharmaceutical powders for early stage solubility assays

At early stages of drug development, the solubility of compounds is an important screening criterion. However, because scientists lack the automated tools needed to perform comprehensive early stage solubility studies, they are only able to perform a small number of experiments by hand, thus exploring only a fraction of the potential formulation design space. To allow a larger formulation design space to be explored at relatively early stages of pharmaceutical development (when 100 mg of a prototype compound is available), TransForm has created the AquaSFinX^™ and SFinX^™ micro-solubility platforms. In this article, a novel solid deposition system is described which is an enabling component of TransForm's solubility platforms. Given 100 mg of a starting material, the deposition system can dispense over six hundred 50-μg plugs of powder into 384-well or 1,536-well plates with well openings as small as 1.5 mm. Currently no commercial powder deposition system can provide this functionality. Including the time required to characterize and run the system, the dispense times range from around 2 minutes per dose for small runs involving 100 mg of starting material, to 18 seconds per dose for larger runs involving a gram or more of starting material.     

Robotic liquid handling and automation in epigenetics

Automated liquid-handling robots and high-throughput screening (HTS) are widely used in the pharmaceutical industry for the screening of large compound libraries, small molecules for activity against disease-relevant target pathways, or proteins. HTS robots capable of low-volume dispensing reduce assay setup times and provide highly accurate and reproducible dispensing, minimizing variation between sample replicates and eliminating the potential for manual error. Low-volume automated nanoliter dispensers ensure accuracy of pipetting within volume ranges that are difficult to achieve manually. In addition, they have the ability to potentially expand the range of screening conditions from often limited amounts of valuable sample, as well as reduce the usage of expensive reagents. The ability to accurately dispense lower volumes provides the potential to achieve a greater amount of information than could be otherwise achieved using manual dispensing technology. With the emergence of the field of epigenetics, an increasing number of drug discovery companies are beginning to screen compound libraries against a range of epigenetic targets. This review discusses the potential for the use of low-volume liquid handling robots, for molecular biological applications such as quantitative PCR and epigenetics.     

Automated real-time hit picking, retesting, and reflux testing

Advances in automation have enabled screening methods, such as enzymatic assays and ELISAs, to be performed in a high-throughput fashion. Instruments can perform each of the steps more rapidly and with fewer errors than humans, however, in many cases, humans still perform the key steps of integrating the instruments and Laboratory Information Management Systems (LIMS). Typically, the screening process involves multiple steps for sample testing, manual data entry and design of protocols to handle the positive samples, and final processing of positives. Fully integrated systems such as Protedyne's BioCube^™ System combine liquid handling, LIMS, plate washing, and plate reading in a device that is controlled via a single user interface. As an example, we present the results of automating plate reading and retesting of positive samples in a model protocol based on ELISA testing. By performing an automated initial screening of results, our system enables positive samples to be retested at higher stringency or sent to a technician for corroboration of results. The screening process then becomes a single protocol that starts with a sample set and results in a data set of tested and retested/reflux tested positives as well as re-arrayed plates of positive samples that are ready for additional downstream processing.     

Automated in situ hybridisation using Tecan robotics: a kick-start for Functional Genomics

High throughput parallel organic synthesis is now a common practice in discovery chemistry research. Argonaut's modular reaction cassette technology is aimed at parallel synthesis of focused small molecule libraries. The Trident™ automated platform has been designed to perform high throughput organic synthesis using this modular reaction cassette under truly inert conditions and in a robust, reproducible manner. This enables one to generate high quality libraries of small molecules and to access chemistry not normally amenable to automation.Recently we have introduced a Sample Processing Station (SPS), which enhances the upstream and downstream sample handling capabilities of the Trident™ platform. Enhanced software capabilities also provide the Trident™ synthesizer with powerful reaction development and optimization capabilities. Application of this technology in the synthesis of a small library of 1,2-diarylbenzimidazoles using solution-phase chemistry is presented. A number of product purification and isolation protocols using polymer-bound scavengers are discussed.     

GigaMatrix: an ultra high-throughput tool for accessing biodiversity

Recombinant approaches for tapping into the biodiversity present in nature for the discovery of novel enzymes and biosynthetic pathways can result in large gene libraries. Likewise, laboratory evolution techniques can result in large but potentially valuable libraries. Thorough screening of these libraries requires ultra high-throughput methods. The GigaMatrix^™ screening platform addresses this opportunity using reusable high-density plates with 100,000 to 1,000,000 through-hole wells in a microplate footprint. In addition to throughputs of over 10⁷ wells per day, the platform offers a significant reduction in reagent use and waste, has fully integrated automated “cherry picking,” and uses no complicated dispensing equipment. Wells containing putative hits from targeted fluorescent liquid phase assays are revealed by a fluorescent imaging system. Vision-guided robotics are utilized to recover hits by accessing individual 200 μm and smaller wells with a disposable sterile needle. The GigaMatrix platform has proven to be an effective and efficient tool for screening gene libraries for both discovery and evolution applications.     

Corning® 384 Well Low Volume Microplate Performance in Miniaturized Assays

In response to the ever-increasing demand for microplates that facilitate assay miniaturization without compromising assay integrity, Corning's new 384 well Low Volume (384 LV) microplates were evaluated using a homogenous, fluorescence polarization protease assay. Fluorescently labeled casein substrate was incubated over time with dilutions of protease at working volumes that range from 1 to 20 μL and reagent concentrations that range from 0.125 to 50 pg/μL. Four types of plates were evaluated:

• Corning 384 well LV black medium bind

• Corning 384 well LV black non-binding surface (NBS™)

• Corning 1536 well black medium bind

• Competitor 384 well small volume

Protease activity was detected as a decrease in polarized fluorescence resulting from substrate degradation over time. Following assay miniaturization to 20 μL working volumes and protease concentration reduction from 25 to 0.25 pg/μL, Corning 384 well LV medium bind and NBS coated assay plates maintained superior signal to noise ratios compared to competitor's 384 well small volume assay plates. At even lower 10 μL working volumes, Corning 384 well LV medium bind assay plates maintained signal to noise ratios comparable to the competitor's assay plates. However as the reagent concentrations were further reduced to 2.5 pg/μL, Corning 384 well LV medium bind and NBS coated assay plates had significantly greater signal to noise ratios compared to the competitor's assay plates. As working volumes were reduced even further to 5 μL and 1 μL, Corning 384 well LV medium bind and NBS coated assay plates had significantly greater signal to noise ratios than the competitor's 384 well small volume assay plates. Furthermore, at 1 μL working volumes, Corning 384 well LV NBS coated assay plates had significantly greater signal to noise ratios than the competitor's assay plates and standard 1536 well assay plates.These results suggest that Corning 384 well LV medium bind and NBS coated assay plates facilitate assay miniaturization while maintaining assay integrity. Furthermore, as working volumes were significantly reduced to those typically found in 1536 deep well and standard 1536 well assay plates (5 and 1 μL, respectively), Corning's 384 well LV NBS coated assay plates provided superior assay performance.     

Managing the Workflow of a High-Throughput Organic Synthesis Laboratory: A Marriage of Automation and Information Management Technologies

The production of high quality, individually characterized libraries of compounds requires a welldeveloped strategy, along with robust, userfriendly data and automation systems to support this strategy. We developed a workflow process to improve the efficiency of producing greater than 50,000 individual compounds per year. Our workflow process eliminates bottlenecks in traditional procedures and thus ensures a steady, predictable compound production rate. Initially, virtual library creation allowed for the determination of the physio-chemical properties of libraries as well as the ability to calculate the needed amounts of reagents and solvents for a production campaign. At this point, we registered all samples into our library database. Production of libraries began with loading the appropriate linkers onto resins, followed by complete characterization of the resin-linker intermediate. The resins were then loaded into IRORI MiniKans™ and sorted using the IRORI Autosort™ 10Kx sorting workstation. Chemistry was then performed using standard laboratory glassware. Additional sorting and chemistry steps were then performed until the compound was ready for cleavage off of the resin. The MiniKans were then sorted into Bohdan MiniBlocks™ and treated with the appropriate cleavage cocktail. Collection into 48-position racks was followed by removal of cleavage solution through vacuum centrifugation. The concentrate was then dissolved in a solvent mixture that allowed for standard liquid handling automation to create 96 well plates for analysis by high throughput flow inject NMR and LC/MS. Compounds > 80% pure by LC/MS and presenting a > 80% overall yield, as determined by NMR, were automatically flagged “acceptable” in the database. Compounds that failed to pass these analytical criteria were flagged “fail” in the database and were examined for trends that could be corrected in future library production. Finally, acceptable compounds were reformatted from the original source plates into master plates.     

A Fully Automated High-throughput Nucleic Acid Purification System Using Silica Coated Magnetic Bead Technology

The design of a fully automated high-throughput system for the purification of sequencing templates is described. Hardware, software, and chemistries have been optimized to suit the needs of high-throughput laboratories involved in genome sequencing projects. Using this system, up to 5760 samples (60 x 96-well plates) can be purified in less than 16 hours during a single unattended run. The system can also be configured to perform sequencing reaction setup for all 60 plates following template purification, extending total run times to < 24 hours. Final sequencing reactions are prepared in 384- well microplates.     

The Planning and Establishment of a Sample Preparation Laboratory for Drug Discovery

Nature has always been a productive source of new drugs. With the advent of HTS, it has now become possible to rapidly screen large sample collections. In addition to seeking greater diversity from natural products sources (microorganisms, plants, etc.), fractionation of the crude extracts prior to screening is becoming a more important part of our efforts.As sample preparation protocols become more involved, automation can help achieve and maintain a desired sample throughput. To address the needs of our screening program, two robotic systems were designed.The first system processes crude extracts all the way to 96-well plates, containing solutions suitable for screening in biological and biochemical assays. The system can dissolve crude extracts, fractionate them on SPE cartridges, dry and weigh each fraction, redissolve them to a known concentration, and prepare mother plates. The second system replicates mother plates into a number of daughter plates.     

High Throughput Methods for Gene Identification, Cloning and Functional Genomics Using the GeneTAC™ G Robotics Workstation

Using a single robotic platform, the GeneTAC™ G³, we have automated most of the processes involved in the cloning and characterisation of novel disease causing genes by addressing the following; firstly, identifying the BACs of interest and making shotgun libraries. Secondly, automating the set up of sequencing reactions using methodology that eliminates the need for DNA preparation of 384 clones. Thirdly, generating sublibraries using selective re-arraying of library clones to enable the determination of the entire genomic sequence of the gene. Fourthly, determining gene function by combination of differential screening and mini Northerns using microarrays printed using the GeneTAC™ G³ system and hybridised using the GeneTAC™ HybStation (Genomics Solutions, Ann Arbor, USA).