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We report the development of an automated genetic analyzer for human sample testing based on microfluidic rapid polymerase chain reaction (PCR) with high-resolution melting analysis (HRMA). The integrated DNA microfluidic cartridge was used on a platform designed with a robotic pipettor system that works by sequentially picking up different test solutions from a 384-well plate, mixing them in the tips, and delivering mixed fluids to the DNA cartridge. A novel image feedback flow control system based on a Canon 5D Mark II digital camera was developed for controlling fluid movement through a complex microfluidic branching network without the use of valves. The same camera was used for measuring the high-resolution melt curve of DNA amplicons that were generated in the microfluidic chip. Owing to fast heating and cooling as well as sensitive temperature measurement in the microfluidic channels, the time frame for PCR and HRMA was dramatically reduced from hours to minutes. Preliminary testing results demonstrated that rapid serial PCR and HRMA are possible while still achieving high data quality that is suitable for human sample testing. [ABSTRACT FROM AUTHOR]

A method is described for using custom snap-on lids to protect chemicals in microtiter plates from evaporation and contamination. The lids contain apertures (diameter 1.5, 1.0, or 0.5 mm) through which the chemical building blocks can be transferred. The lid with 0.5 mm apertures was tested using a noncontact acoustic liquid handler; the 1.0 and 1.5 mm lids were tested using two tip-based liquid handlers. All of the lids reduced the rate at which solvents evaporated to room air, and greatly reduced the rate of contamination by water and oxygen from room air. In steady-state measurements, the lids reduced the rate of evaporation of methanol, 1-hexene, and water by 33% to 248%. In cycled experiments, the contamination of aqueous solvent with oxygen was reduced below detectability and the rate at which DMSO engorged atmospheric water was reduced by 81%. Our results demonstrate that the lids preserve the integrity of air-sensitive reagents during the time needed for different types of liquid handlers to perform dispensations. Controlling degradation and evaporation of chemical building blocks exposed to the atmosphere is increasingly useful as the reagent volume is reduced by advances in liquid handling technology, such as acoustic droplet ejection. [ABSTRACT FROM AUTHOR]

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 high-throughput epigenetic screening. Assay miniaturization increases screening throughput and reduces operating costs. Echo liquid handlers can transfer compounds, samples, reagents, and beads in submicroliter 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. In this study, we demonstrate the miniaturization of a methyltransferase assay using Echo liquid handlers and two different assay technologies: AlphaLISA from PerkinElmer and EPIgeneous HTRF from Cisbio. [ABSTRACT FROM AUTHOR]

The move toward label-free screening in drug discovery has increased the demand for mass spectrometry (MS)–based analysis. Here we investigated the approach of coupling acoustic sample deposition (ASD) with laser diode thermal desorption (LDTD)–tandem mass spectrometry (MS/MS). We assessed its use in a cytochrome P450 (CYP) inhibition assay, where a decrease in metabolite formation signifies CYP inhibition. Metabolite levels for 3 CYP isoforms were measured as CYP3A4-1′-OH-midazolam, CYP2D6-dextrorphan, and CYP2C9-4′-OH-diclofenac. After incubation, samples (100 nL) were acoustically deposited onto a stainless steel 384-LazWell plate, then desorbed by an infrared laser directly from the plate surface into the gas phase, ionized by atmospheric pressure chemical ionization (APCI), and analyzed by MS/MS. Using this method, we achieved a sample analysis speed of 2.14 s/well, with bioanalytical performance comparable to the current online solid-phase extraction (SPE)–based MS method. An even faster readout speed was achieved when postreaction sample multiplexing was applied, where three reaction samples, one for each CYP, were transferred into the same well of the LazWell plate. In summary, LDTD coupled with acoustic sample deposition and multiplexing significantly decreased analysis time to 0.7 s/sample, making this MS-based approach feasible to support high-throughput screening (HTS) assays. [ABSTRACT FROM AUTHOR]

For a therapeutic antibody to succeed, it must meet a range of potency, stability, and specificity criteria. Many of these characteristics are conferred by the amino acid sequence of the heavy and light chain variable regions and, for this reason, can be screened for during antibody selection. However, it is important to consider that antibodies satisfying all these criteria may be of low frequency in an immunized animal; for this reason, it is essential to have a mechanism that allows for efficient sampling of the immune repertoire. UCB’s core antibody discovery platform combines high-throughput B cell culture screening and the identification and isolation of single, antigen-specific IgG-secreting B cells through a proprietary technique called the “fluorescent foci” method. Using state-of-the-art automation to facilitate primary screening, extremely efficient interrogation of the natural antibody repertoire is made possible; more than 1 billion immune B cells can now be screened to provide a useful starting point from which to identify the rare therapeutic antibody. This article will describe the design, construction, and commissioning of a bespoke automated screening platform and two examples of how it was used to screen for antibodies against two targets. [ABSTRACT FROM PUBLISHER]

The accurate transfer of biological reagents represents a fundamental step in the drug screening process, and the elimination of carryover is critical for the generation of accurate measurements of biological activity. The introduction of automated liquid robotics into screening laboratories has transformed the drug screening process, enabling accurate and reproducible transfer of liquids to become a high-throughput activity, but has also introduced a new challenge for drug discoverers: to establish screening workflows that limit analyte carryover for the generation of high-quality screening data. The widespread use of pipetting tips on automated liquid handlers often necessitates the use of optimized wash protocols for removing contaminants and frequently requires the use and disposal of large quantities of organic solvents. Furthermore, many chemical and biological reagents are recalcitrant to removal from pipetting tips by treatment with organic solvents. The use of cold atmospheric plasma technology provides an alternative approach for removal of contaminants and offers many advantages over traditional decontamination protocols commonly used during biological screening. This report describes the evaluation of a cold plasma tip-cleaning system for reducing carryover in a range of biological screening assays requiring the transfer of low molecular weight compound, nucleic acid, and bacterial liquid transfers. The validation of this technology for biological screening assays is presented, and the impact of this technology for screening workflows is discussed. [ABSTRACT FROM AUTHOR]

This article is about how to improve the positional dispensing accuracy of low-volume (microliters) drops and to understand the factors affecting them. These same parameters can be investigated to reduce deleterious effects on dispensing performance. Many applications, such as those in immunoassay diagnostics, require accurate volumetric dispensing and are also less tolerant of other phenomena that cause the reagent to be located outside the target area of interest. In this article, we work with liquid inertia. This phenomenon is present in positional dispensing and affects its accuracy. The negative effect of liquid inertia is reduced by changing parameters such as pressure and the diameter of the involved conducts. [ABSTRACT FROM PUBLISHER]

Microsomal prostaglandin E synthase-1 (mPGES-1) is the major enzyme catalyzing the isomerization of prostaglandin (PG) H2 to PGE2. Here we report the development of a robust and practical automated assay in a 384-well format for room temperature screening of mPGES-1 inhibitors with high precision and low reagent consumption. The assay should enable precise structure-activity relationship development. It uses acetonitrile as solvent for PGH2, FeCl2/citrate as stop reagent, and a short reaction time. Combined with high-precision liquid transfer and extensive mixing after addition of reactants, these properties let the assay reach Z′ > 0.7 and high reproducibility of inhibitor IC50 values. Thorough investigation of the quality of mixing in all liquid transfer steps proved crucial for reaching high-precision performance.Abbreviations: mPGES-1 (microsomal prostaglandin E synthase-1); FRET (fluorescence resonance energy transfer); HTRF (homogeneous time-resolved fluorescence); PGH2 (prostaglandin H2); PGE2 (prostaglandin E2); SAR (structure-activity relationship); COX-2 (cyclooxygenase-2); GSH (glutathione); ALP (automated labware positioner) [ABSTRACT FROM PUBLISHER]

Cytochrome P450 (CYP) enzymes are key players in drug metabolism. Therefore, it is essential to understand how these enzymes can be affected by xenobiotics with regards to induction and toxicity to avoid potential drug–drug interactions. Typically, information has been gathered by combining data from multiple experiments, which is time-consuming and labor intensive, and interassay variability may lead to misinterpretation. Monitoring CYP induction and cytotoxicity by xenobiotics using an automated, multiplexed format can decrease workload and increase data confidence. Here the authors demonstrate the ability to monitor CYP1A and CYP3A4 induction, combined with a cytotoxicity measurement, from a single microplate well using cryopreserved human hepatocytes. The assay procedure was automated in a 384-well format, including cell manipulations, compound titration and transfer, and reagent dispensing, using simple robotic instrumentation. EC50 and Emax values were derived for multiple known CYP1A and -3A4 inducers. Induction and toxicological responses in the triplex system were validated based on literature values from conventional single-parameter assays. Validation and pharmacology data confirm that multiplexed cell-based CYP assays can simplify workload, save time and effort, and generate biologically relevant data. [ABSTRACT FROM PUBLISHER]

Implementation of regenerative medicine in the clinical setting requires not only biological inventions, but also the development of reproducible and safe method for cell isolation and expansion. As the currently used manual techniques do not fulfill these requirements, there is a clear need to develop an adequate robotic platform for automated, large-scale production of cells or cell-based products. Here, we demonstrate an automated liquid-handling cell-culture platform that can be used to isolate, expand, and characterize human primary cells (e.g., from intervertebral disc tissue) with results that are comparable to the manual procedure. Specifically, no differences could be observed for cell yield, viability, aggregation rate, growth rate, and phenotype. Importantly, all steps-from the enzymatic isolation of cells through the biopsy to the final quality control-can be performed completely by the automated system because of novel tools that were incorporated into the platform. This automated cell-culture platform can therefore replace entirely manual processes in areas that require high throughput while maintaining stability and safety, such as clinical or industrial settings. [ABSTRACT FROM AUTHOR]

Miniaturizing experimental sample volumes to the nanoliter volume range is one of the most economical ways to perform mid- and high-throughput compound screening experiments. Existing automation platforms for nanoliter fluid handling can be bulky, expensive, and require periodic calibration to provide consistent liquid dispensing. In addition, even with frequent calibration, significant instrument-to-instrument variation in low-volume dispensing can occur between different instrument platforms. Many of these issues can be addressed by the use of PocketTips. PocketTips are tips with a defined internal pocket designed to hold specific nanoliter volumes of compound dissolved in dimethylsulfoxide. Although the overall liquid-handling process with PocketTips uses the aspirate/dispense features of the specific liquid-handling device being used, the dispensed nanoliter volume is solely based on the dimensions of the pocket of the PocketTip and thus, the liquid-handling device itself need not have nanoliter dispensing capabilities. In this report, we demonstrate the performance of PocketTips on different automation platforms. In addition, we used a cell-based ß-lactamase reporter assay system to demonstrate that compound delivery by PocketTips compares favorably with a standard compound addition technique. [ABSTRACT FROM AUTHOR]

Serial dilution of compounds solubilized in dimethylsulfoxide (DMSO) for dose–response curves is a common method for efficacy analysis of potential drug candidates. In general, serial dilution methods are particularly prone to error propagation because each dilution is dependent on the previous concentration. Moreover, assumptions about quality control parameters (i.e., dye linearity) can lead to an erroneous process. Here, an inline performance measurement is sought to improve the precision and accuracy of dilution plates. Sulforhodamine 101 (S101) dye is introduced as the quantitative fluorometric method of choice for DMSO-based systems. Although S101 in DMSO behaves in a nonlinear fashion over its detectable range, we account for this with a direct calibration method that includes every point of the dilution template. This report contains dye selection rationale for the S101 dye and its use in quantifying the performance of 96- and 384-well dilution protocols as tested on five identical instruments. [ABSTRACT FROM AUTHOR]

The determination of inhibitory effects that lead compounds have on cytochrome P450 (CYP) enzymes is an important part of today''s drug discovery process. Assays can be performed early in the discovery process to predict adverse drug–drug interactions caused by CYP inhibition and to minimize the costs associated with terminating candidates in late stage development or worse, removing a drug from the market after launch. For early discovery work, testing substantial numbers of compounds is desirable, thus automated “mix and read” assays are beneficial. Here, we demonstrate the automation of the CYP profiling process using a simple, yet robust robotic platform. Compound titration, as well as transfer of compounds and assay components was performed by the same automated pipetting system. IC50s of small molecule drugs were determined using recombinant CYP enzymes, CYP3A4, -2C9, and -2D6 and luminogenic substrates specific to each. Compounds were profiled against all three enzymes on the same 384-well assay plate. [Copyright &y& Elsevier]

The uridine 5''-diphospho-glucuronosyltransferase (UGT) family of enzymes is involved in the metabolism of various compounds. These enzymes transfer a hydrophilic glucuronic acid moiety to their substrates, rendering them more water soluble and amenable to excretion. The UGTs act on various endogenous substrates, such as bilirubin, 17β-estradiol, and testosterone, and drugs and other xenobiotics. The function of these enzymes is essential for the clearance of drugs and toxicants, and alteration of UGT activity is a potential cause of adverse drug–drug interactions in vivo. This has stimulated an increased interest in the study of UGT function and inhibition, and the desire to profile new drug entities against UGT enzymes, similar to CYP450 profiling. However, certain factors have hindered the development of a robust method for UGT profiling. Current methods for assessing UGT enzyme activity are laborious and involve protein precipitation and/or chromatographic separation steps, which are not amenable to rapid screening applications for UGT inhibitors or substrates. The approach presented here is a bioluminescent assay for measuring UGT enzyme activity and inhibition in vitro. Using flexible, robust instrumentation in a 384-well microplate format, this study highlights the quick and easy assay implementation for estimation of inhibition kinetics with a variety of known and suspected UGT substrates and inhibitors. [Copyright &y& Elsevier]

This study presents the implementation and optimization of 3 cell-based assays on a TECAN Genesis workstation—the Caspase-Glo® 3/7 and sulforhodamine B (SRB) screening assays and the mechanistic Caco-2 permeability protocol—and evaluates their feasibility for automation. During implementation, the dispensing speed to add drug solutions and fixative trichloroacetic acid and the aspiration speed to remove the supernatant immediately after fixation were optimized. Decontamination steps for cleaning the tips and pipetting tubing were also added. The automated Caspase-Glo® 3/7 screen was successfully optimized with Caco-2 cells (Z' 0.7, signal-to-base ratio [S/B] 1.7) but not with DU-145 cells. In contrast, the automated SRB screen was successfully optimized with the DU-145 cells (Z' 0.8, S/B 2.4) but not with the Caco-2 cells (Z' -0.8, S/B 1.4). The automated bidirectional Caco-2 permeability experiments separated successfully low- and high-permeability compounds (Z' 0.8, S/B 84.2) and passive drug permeation from efflux-mediated transport (Z' 0.5, S/B 8.6). Of the assays, the homogeneous Caspase-Glo® 3/7 assay benefits the most from automation, but also the heterogeneous SRB assay and Caco-2 permeability experiments gain advantages from automation. (Journal of Biomolecular Screening 2010:30-41) [ABSTRACT FROM AUTHOR]

Four years ago, the first acoustic droplet ejectors (ADEs) were launched on the market, providing a new generation of highthroughput noncontact liquid handlers that outclassed traditional contact instruments in almost every respect. This introduction of noncontact dispensing has triggered radical changes to the screening/compound management interface. Higher quality is achieved through greater accuracy and precision, whereas lower sample volumes can be used, and 1536 plate formats have become a reliable reality. Prior to the ADE instrument launch, 1536 assay-ready plate preparation was a high-effort enterprise requiring users to spend time developing liquid-handling methods along with daily fine-tuning of instruments to reach the desired level of performance. By overcoming the nanoliter dispensing hurdle and successfully transferring assays to high-density formats, a new dimension for cutting costs has emerged. Once the screening customer has adapted to this new world, the rules of supply can also change, with the traditional automated plate store no longer being necessary when the compound library can be stored in 1536 plates. Processing efficiency recently has been further supported by innovative new automation-friendly solutions such as plate desealers, prolonging the life span of working plate copies. Both cost and waste control have never had a higher profile, and noncontact dispensing contributes to these important areas. In some processes (e.g., when piercing septa), contact dispensing remains the best option, but cost control is still essential, and an innovative solution to minimize DMSO consumption from tip washing has had a big impact on consumable budget without compromising quality. [ABSTRACT FROM AUTHOR]