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High-throughput screening: new technology for the 21st century
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High-throughput screening: new technology for the 21st century Robert P Hertzberg* and Andrew J Pope† New technologies in high-throughput screening have significantly increased throughput and reduced assay volumes. Key advances over the past few years include new fluorescence methods, detection platforms and liquid-handling technologies. Screening 100,000 samples per day in miniaturized assay volumes will soon become routine. Furthermore, new technologies are now being applied to information-rich cellbased assays, and this is beginning to remove one of the key bottlenecks downstream from primary screening.
What were (and are) the major stumbling blocks that had to (and have to) be overcome to achieve these goals? We can sum it up in three major categories:
Addresses *Molecular Screening Technologies, SmithKline Beecham Pharmaceuticals, 709 Swedeland Road, King of Prussia, PA 19406, USA; e-mail: [email protected] † Molecular Interactions & New Assay Technologies, SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW, UK; e-mail: [email protected]
In this review, we discuss several successful strategies in fluorescence and radiometric detection that are compatible with miniaturized uHTS, and new methods of liquid handling that are being developed to meet these challenges. While developing appropriate bioware and hardware is difficult, building reliable software to control processes and extract knowledge from millions of data points is perhaps the most challenging aspect of uHTS — and this problem remains to be solved.
Current Opinion in Chemical Biology 2000, 4:445–451 1367-5931/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved. Abbreviations CCD charge-coupled device FA fluorescence anisotropy FCS fluorescence correlation spectroscopy FIDA fluorescence intensity distribution analysis GPCR G-protein-coupled receptor HTS high-throughput screening SPA scintillation proximity assay TRET time-resolved energy transfer uHTS ultraHTS
Introduction Advances in molecular biology, human genetics and functional genomics continue to produce increasing numbers of molecular targets available for therapeutic intervention. This, coupled with major increases in compound collections produced by combinatorial technologies, has fueled an important need for improvements in high-throughput screening (HTS) capabilities. As a result, HTS technologies have undergone a revolution in the latter half of the 1990s. Today, most pharmaceutical companies use HTS as the primary engine driving lead discovery. The increased reliance on HTS labs meant that screening had to change. Assay systems and robotics that were capable of screening thousands of compounds per day in the mid-1990s had to evolve into ultraHTS (uHTS) methods capable of 100,000 assays per day, or more. This explosion in assay throughput meant that reagent production had to be scaled to meet the challenge — or else the HTS labs would have to miniaturize assays to meet the reagent suppliers halfway. Fortunately, many of the technological advances required to reach the goals of miniaturized uHTS have been established and are on their way toward routine use.
1. Assay methods and detection (bioware). 2. Liquid handling and robotics (hardware). 3. Process flow and information management (software).
Homogeneous fluorescence methods It is now clear that fluorescence-based techniques are likely to be amongst the most important detection approaches used for HTS in the future, given the industry-wide drive to simplify, miniaturize and speed up assays. Fluorescence techniques are well suited to uHTS because they give very high sensitivity, which allows fairly straightforward miniaturization. This is illustrated by the fact that simple fluorescence intensity measurements have been successfully applied in an ultra-miniaturized format [1]. However, HTS assays based upon fluorescence intensity measurements are mainly restricted to fluorogenic enzyme substrates (for example, see [2]). A more powerful aspect of other, more complex, fluorescence readouts is their ability to yield information on fluorophore environment, which allows predictive design for a wide range of target types [3••,4]. Two factors have made fluorescence readouts more widely used in HTS/uHTS: the development of new microtiter plate readers [5,6•]; and, to a lesser extent, the development of new dyes and molecular reagents. The latest instrumentation enables routine measurement of a range of fluorescence readouts in 1536-well plate formats [4,5,6•]. One such technique is fluorescence anisotropy (FA; also known as polarization), which yields information on molecular rotation, which is related to mass. Anisotropy can be used to measure bimolecular association events within a specific range, as determined by the fluorophore lifetime [3••]. Many examples of HTS applications of FA have now been reported, including ligand−receptor binding [7,8] and enzyme assays [8,9•]. A number of groups have also demonstrated that FA measurements work well in
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1536-well plates when using high-sensitivity plate readers (for example, see [5,9•,10]). Another important fluorescence readout is time-resolved energy transfer (TRET) [3••]. This is a dual labeling approach that is based upon long-range energy transfer between fluorescent Ln3+ complexes and a suitable resonance energy acceptor. Packard/CysBio (Meriden, CT) [11,12] and PerkinElmer/Wallac Oy (Turku, Finland) [13,14] market reagents and instrumentation for this technique. These approaches have an advantage in that time-gating of the long-lived donor and acceptor signals gives high sensitivity by reducing background. A large number of HTS assays have now been configured using TRET (for example, see [15–17]), including the successful miniaturization of the technique to 1536-well plates [16,17]. Given the large distance for effective energy transfer (~90 Å [3••]), TRET is highly suited to measurements of protein−protein interactions (for example, see [15]). Recently, two-alternative approaches with similar capabilities have been developed by Packard/Biosignal in AlphaScreen [18] and bioluminescence energy transfer [19]. In addition to the readouts described above, there have also been recent advances in the development of microplate instrumentation for other, previously inaccessible, fluorescence methods such as lifetime (and lifetime discriminated) measurements (LJL Biosystems FLARe) [10,20,21•]; and two-photon excitation [22]. The FLARe system from LJL (Sunnyvale, CA) uses phase modulation approaches to measure changes in probe lifetimes that may occur via environmental effects or energy transfer. FLARe can also use knowledge of the probe lifetime to discriminate signal from background, which has been shown to be highly useful in eliminating interference from test compounds [23•]. One source of frustration to those involved in the development of fluorescence approaches is the lack of identification of fundamentally new chemical classes of dyes. However, successful modifications (to improve brightness, photostability, physical properties, etc.) to existing dye chemical families have been achieved, most notably by Molecular Probes (e.g. Alexa dyes [24•]) and Amerhsam (Cy dyes [25]). The use of semi-conductor nano-crystals as fluorescent tags [26,27] is intriguing, particularly because these systems seem to be of highly tunable wavelength, allowing the prospect of multiplexed or very high efficiency energy transfer applications. However, these probes have not yet been used in HTS applications so it is too early to tell how their performance is likely to compare with more conventional fluorophores. One area of fluorescence measurements that has developed very strongly in terms of fundamental approaches is that of single molecule fluctuation-based measurements. One attraction of these methods for HTS/uHTS is their
high information content and intrinsic sensitivity to miniaturization [3••,4]. All are performed using confocal optics in which the observation volume is extremely small (~1 fl) and very few molecules are present in the confocal volume optics at any one time, so the output fluorescence signal fluctuates with time as molecules enter and exit. The classical form of confocal fluctuation spectroscopy, known as fluorescence correlation spectroscopy (FCS) has now been demonstrated to be a viable approach to HTS for a wide range of therapeutic targets (for example, see [28,29•]). FCS can be used to detect binding interactions via changes in translational diffusion rates or, where there is a significant change in brightness during a binding or catalytic reaction, can be configured in a way that is not dependent upon changes in diffusion rates (i.e. mass) [29•]. It is only during the past few years that advances in optics, electronics and computation tools have made FCS a viable proposition for use in routine HTS applications. These same advances have led to a renaissance in the whole area of single molecule fluctuation methods, driven in particular by a number of academic labs and by Evotec Biosystems (Hamburg, Germany) [30], who are developing an integrated miniaturized screening system based upon this detection technology [31•]. Several new methods for analyzing fluctuation data have now been reported, including methods to determine molecular brightness (fluorescence intensity distribution analysis; FIDA [32••] or related methods [33–35]). These methods can be used to configure assays in a variety of ways; molecular brightness can change either by environment or energy transfer, or via changes in the stoichiometry of fluorophore molecules present on a single particle. There has been significant progress, using a number of approaches, to extend these methods by simultaneously analyzing data from more than one output channel. For example, Evotec Biosystems recently showed that combining FIDA with molecular anisotropy (so-called 2D-FIDA) resulted in more robust measurements of kinase activity than when either technique was used individually [31•]. In addition, a number of approaches have been developed that measure the coincidence of two discrete fluors within the same particle [36–38].
Miniaturized radiometric readouts While fluorescence assay technologies are growing in importance, the predicted demise of radiometric assays as an important facet of HTS labs has not yet occurred. Current estimates from various surveys of HTS laboratories indicate that radiometric assays presently constitute between 20% and 50% of all screens performed [39]. Although we expect the fraction of radiometric assays to decrease over the coming years, this technology is unlikely to disappear completely. In the early 1990s, several advances in radiometric assay technology were introduced including scintillation proximity assay (SPA) (Amersham Pharmacia Biotech) and
High-throughput screening Hertzberg and Pope
FlashPlates™ (NEN Life Science Products, Boston, MA). With these approaches, the target of interest is immobilized onto a solid support (e.g. SPA beads or FlashPlate™ surface) that contains a scintillant. When a radiolabeled molecule binds to the target molecule, the radioisotope is brought in close proximity to the solid support, and energy transfer between the emitted beta particle and the scintillant results in the emission of light. Radioisotope remaining free in solution is too distant from the scintillant and the beta particle dissipates energy into the aqueous environment. Thus, scintillation proximity technologies facilitate a homogeneous approach to radiometric assays [25,40,41]. SPA has been used in a wide variety of applications and it is a standard technique in HTS labs. The technology has been applied to kinases [42••,43••], nucleic-acid-processing enzymes [44•], other enzymes [45] and is widely used for ligand−receptor interactions [46,47,48•]. FlashPlate™ technology is similar to SPA but the solid surface is a microtiter plate rather than a bead. Recent FlashPlate™ applications include the detection of cAMP levels [49] and ligand−receptor interactions [50]. Of course, radiometric assays have several disadvantages including safety, limited reagent stability, relatively long read-times and little intrinsic information on the isotope environment. However, new technologies are now emerging to address the issue of read-time and assay miniaturization. The most important of these are the emergence of imaging platereaders such as Leadseeker™ (Amersham Pharmacia) [6•,25] and CLIPR™ (Molecular Devices, Sunnyvale CA) [51]. These instruments consist of high sensitivity CCD (charge-coupled device) cameras and special lenses capable of rapidly flat-field imaging an entire microtiter plate. These systems are capable of reading 1536-well plates (or plates of even higher density) and can rapidly quantify the light emitted by each microtiter well. In addition to sensitivity and speed, imaging platereaders demonstrate further advantages such as low backgrounds and low variability. While the Leadseeker™ and CLIPR™ can read SPA, the former can also read fluorescence and both have the capability of imaging luminescence with high sensitivity. Additional fluorescence imaging platereaders are available such as the ViewLux™ (manufactured by PerkinElmer/Wallac Oy) [13]. Imaging platereaders are likely to become increasingly important relative to point-detection devices in the coming years, given the desire to achieve higher measurement speeds and therefore throughput. In addition to the emergence of imaging platereaders, new SPA beads have recently been introduced by Amersham Pharmacia Biotech [25]. These beads emit light at 615 nm, in contrast to traditional SPA beads, which emit around 420 nm. This longer wavelength is well-suited to the response of the CCD camera in the Leadseeker™ imager. Furthermore, light emission at 615 nm may result in fewer
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problems with color quench by test compounds, which tend to be yellow and red, although this has yet to be definitively shown.
Cell-based assay technologies Advances in technology and instrumentation for cell-based assays have occurred over the past few years. Among these is the emergence of HTS-compatible technology to measure G-protein-coupled receptor (GPCR) [52] and ion channel function [53•], confocal imaging platforms for rapid cellular and sub-cellular imaging, and the continued development of reporter gene technology. The FLIPR™ (Molecular Devices) is a fluorescence imaging platereader with integrated liquid handling that facilitates the simultaneous fluorescence imaging of 384 samples to measure intracellular calcium mobilization in real time [52]. Historically, these measurements have only been possible on single cuvette fluorimeters and by microscopic imaging. FLIPR™ has been used to identify cognate ligands for orphan GPCRs [54,55], to characterize GPCR pharmacology [56] and to screen compound libraries [57•]. The instrument is also capable of measuring ion channel function by coupling the activity of a target channel to a voltage-gated calcium channel [58]. An alternative and promising HTS technology for ion channels is based on voltage-sensitive fluorescence resonance energy transfer (VIPR™; Aurora Biosciences, La Jolla, CA) [53•,59]. Although kinetic platereaders facilitate cell-based functional screens, they are currently limited to 96/384-well plates and are somewhat labor-intensive. In contrast, cellbased reporter gene screens require fewer cells, are easier to automate and can be performed in 1536-well plates. Recent descriptions of miniaturized reporter gene readouts include luciferase [60] and secreted alkaline phosphate [61]. A novel and sensitive beta-lactamase reporter system has been described that allows the clonal selection of living cells and is amenable to miniaturized HTS [62]. Finally, a Cre recombinase reporter system links signal transduction to DNA recombination and results in a permanent readout of gene expression [63•]. Human GPCRs can be screened in yeast to find agonists and antagonists by taking advantage of the pheromone signaling pathway [64]. Although this technology can offer the advantage of a null background for the expression of human receptors, there is only a moderate correlation between ligand activation in yeast and mammalian cells (R Hertzberg, unpublished data). A yeast-based transcription assay for the human progesterone receptor has recently been performed in 1536-well plates [65]. In addition, receptor assays based on cell darkening can be performed in frog melanophores [66]. Cyclic AMP measurements in cell extracts can be performed with SPA, FlashPlate™ [49], fluorescence
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polarization [10,41] or AlphaScreen™ technologies [18]. A novel fluorescent indicator for cyclic AMP in living cells involves tagging protein kinase A with green fluorescent protein mutants [67•]. Recently, laser scanning imaging systems have been developed to measure cellular and sub-cellular quantitation of fluorescence in whole cells. These systems have the capability of bringing low throughput biological studies with high information content into the world of HTS. One of the most advanced systems is the ArrayScan™ (Cellomics, Pittsburgh, PA) which has been used to measure GPCR internalization [68,69] as well as a range of other applications [70]. Other imaging systems have been used to measure ligand-receptor binding in whole cells [71,72]. Current instrumentation does not combine sufficient resolution and speed to allow uHTS-level throughput for true sub-cellular imaging. However, a number of approaches are being developed for high speed cellular imaging (for example, see [73]), which look likely to enable the use of this approach for first-line uHTS in the future.
Technology for assay miniaturization Several advances in liquid handling and microplates have occurred in the past few years, and true miniaturized uHTS is getting closer to being a regular feature of the modern HTS laboratory. The 1536-well plate is now a reality, and although only a few full screening campaigns have been performed in this format, enough proof-of-concept work on assay readouts (e.g. [2,4,5,6•,9•,12,16,17,28, 29•,31•,60,61,65,74]) has been done to show that miniaturization is here to stay. However, an equally important aspect of assay miniaturization is liquid handling. In fact, it is the development of robust technology in this area, particularly for high speed low volume reformatting of test compounds, that is now largely limiting uHTS implementation. Conventional syringe pump pipetters are now capable of delivering volumes as low as 100 nl with touch-off dispensing [75]. Syringe-solenoid technology, a non-contact method that can dispense droplets in the low nanoliter to low microliter range, is well-suited for ‘bulk’ dispensing biological reagents [76]. Piezoelectric dispensers can create drops in the picoliter range and dispense thousands of drops per second [30,59,74]. These devices are well-suited to compound reformatting and will be important tools for miniaturized uHTS, although problems such as clogging and gas bubbles can occur. Assay vessels are also undergoing a revolution. Openwell formats still predominate, but 96- and 384-well plates have now evolved into 1536- [31•,77,78] and 3456well formats [74]. Such carriers probably represent the ultimate development of the equivalent of test tubes. Beyond this are closed ‘lab-chip’ microfluidic systems such as those being developed by Caliper
(Mountainview, CA) [79,80] and ACLARA Biosciences (Mountainview, CA) [81]. These systems offer potential advantages for ultra miniaturization, because they are not prone to evaporation problems and are compatible with sub-microliter volumes. These systems also have some unique properties, as illustrated by the development of rapid separation-based assays in microfluidic systems [82•]. The development of microfluidic systems and microsystems-technology in general is a very rapidly moving area in which important advances will impact HTS in the future. For example, the use of new materials has recently enabled the construction of devices containing monolithic pumps and valves [83•]. However, there are a number of issues with these types of systems that will take time to resolve, such as how they interface with very large chemical libraries. Although these systems are very promising, many in the HTS industry believe that the 1536-well plate will continue as the standard uHTS medium at least until the middle of this decade.
Conclusions HTS technologies have undergone a revolution and the field shows no signs of slowing down. Fluorescence methods such as intensity, anisotropy and time-resolved energy transfer are now routine in HTS labs. Newer techniques such as fluorescence lifetime measurements, FCS and FIDA are showing promise and will be important screening formats in the next few years. Major advances in HTS hardware have also been made. State-of-the-art detection platforms demonstrate significant improvements in sensitivity and throughput. In particular, imaging platereaders show great promise in reducing assay read-times and facilitating miniaturization. Methods are available to perform information-rich cell-based assays with throughputs approaching those of uHTS. New liquid-handling methods are finally coming on line that allow the dispensement of compounds and biological reagents in volumes consistent with miniaturized assay formats. Lab-chip microfluidic systems are on the horizon; these have the promise to drive assay volumes down even further and provide unique separation capabilities. Now that most of the technologies have been developed, the main hurdles to solve are problems of implementation. Applying these exciting new technologies to routine HTS and keeping these systems running on a day-to-day basis will continue to be a challenge. Nevertheless, in a short period of time, the promise of uHTS capabilities will be fully realized and we can look forward to producing higher quality leads to fuel drug discovery.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest •• of outstanding interest 1.
Oldenburg KR, Zhang JH, Chen T, Maffia A, Blom KF, Combs AP, Chung TDY: Assay miniaturization for ultra-high throughput
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screening of combinatorial and discrete compound libraries: a 9600-well (0.2 microliter) assay system. J Biomol Screen 1998, 3:55-62. 2.
Abriola L, Chin M, Fuerst P, Schweitzer R, Sills MA: Digital imaging as a detection method for a fluorescent protease assay in 96-well and miniaturized assay plate formats. J Biomol Screen 1999, 4:121-128.
3. ••
Pope AJ, Haupts U, Moore KJ: Homogeneous fluorescence readouts for miniaturized high-throughput screening; theory and practice. Drug Discov Today 1999, 4:350-362. A review of the theoretical background to homogeneous fluorescence-based methods currently used in HTS. Includes relevant theory and simulations plus practical examples. 4.
Haupts U, Ruediger M, Pope AJ: Macroscopic versus microscopic fluorescence techniques in (ultra)-high-throughput screening. Drug Discov Today 2000, 1(suppl):3-9.
5.
Sportsman JR, Leytes L: Miniaturization of homogeneous assays using fluorescence polarization. Drug Discov Today 2000, 1(suppl):27-32.
6. Ramm P: Imaging systems in assay screening. Drug Discov Today • 1999, 4:401-410. Review of CCD-based systems for rapid measurement of scintillation, luminescence and fluorescence for HTS. 7.
Allen M, Reeves J, Mellor G: High throughput fluorescence polarization: a homogeneous alternative to radioligand binding for cell surface receptors. J Biomol Screen 2000, 5:63-69.
8.
Parker GJ, Law TL, Lenoch FJ, Bolger RE: Development of high throughput screening assays using fluorescence polarization: nuclear receptor-ligand-binding and kinase/phosphatase assays. J Biomol Screen 2000, 5:77-88.
9. •
Li Z, Mehdi S, Patel I, Kaooya J, Judkins M, Zhang W, Diener K, Lzada A, Dunnington D: An ultra-high throughput screening approach for an adenine transferase using fluorescence polarization. J Biomol Screen 2000, 5:31-38. HTS application paper using fluorescence anisotropy including 1536-well plate work. 10. LJL Biosciences homepage on World Wide Web URL: http://www.ljlbio.com/ 11. Packard Instrument Corporation homepage on World Wide Web URL: http://www.packardinst.com/ 12. Mathis G: HTRF technology. J Biomol Screen 1999, 4:309-313. 13. PerkinElmer Life Sciences homepage on World Wide Web URL: http://lifesciences.perkinelmer.com/ 14. Hemmila I: LANCETM: homogeneous assay platform for HTS. J Biomol Screen 1999, 4:303-307.
15. Zhou G, Cummings R, Li Y, Mitra S, Wilkinson HA, Elbriecht A, Hermes J, Schaeffer JM, Smith RG, Moller DE: Nuclear receptors have distinct affinities for coactivators: characterization by fluorescence resonance energy transfer. Mol Endocrinol 1999, 12:1594-1604. 16. Earnshaw DL, Moore KJ, Greenwood CJ, Djaballah H, Jurewicz AJ, Murray KJ, Pope AJ: Time-resolved energy transfer DNA helicase assays for high throughput screening. J Biomol Screen 1999, 4:239-248. 17.
Moore KJ, Turconi S, Miles-Williams A, Djaballah H, Hurskainen P, Harrop J, Murray KJ, Pope AJ: A homogeneous 384-well high throughput screen for novel tumor necrosis factor receptor: ligand interactions using time-resolved energy transfer. J Biomol Screen 1999, 4:205-214.
18. Biosignal homepage on World Wide Web URL: http://www.biosignal.com/ 19. Xu Y, Piston DW, Johnson CH: A bioluminescence resonance energy transfer (BRET) system: applications to interacting circadian clock proteins. Proc Natl Acad Sci USA 1999, 96:151-156. 20. French T, Owicki JC, Modlin DN, Desphande SS, Mineyev I, Crwaford K, Burton W: Fluorescence-lifetime technologies for high-throughput screening. Proc Soc Optical Engineering 1998, 3259:209-218.
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21. French T, Bailey B, Stumbo DP, Modlin DN: A time resolved • fluorimeter for high-throughput screening. Proc Soc Optical Engineering 1999, 3603:272-280. Description of the fluorescence lifetime assay repertoire (FLARe) system for phase-modulation detection of lifetime and lifetime-discriminated fluorescence in HTS. 22. Lakowicz JP, Grynczynski I, Gryzinski Z: High throughput screening with multiphoton excitation. J Biomol Screen 1999, 4:355-361. 23. Helms MK, French T: Time resolved fluorescence measurements • of actin phalloidin interactions. Proc Soc Optical Engineering 2000, 3926:158-165. Application of FLARe in measurement of actin-phalloidin interactions and showing power of lifetime discrimination to eliminate compound library interferences. 24. Molecular Probes Inc. homepage on World Wide Web URL: • http://www.probes.com/ Excellent website for comprehensive information on fluorescent dyes, reagents and applications from Molecular Probes as well as background literature. 25. Amersham Pharmacia Biotech homepage on World Wide Web URL: http://www.apbiotech.com/ 26
Bruchez M Jr, Moronne M, Gin P, Weiss S, Alivisatos AP: Semiconductor nanocrystals as fluorescent biological labels. Science 1998, 281:2013-2016.
27.
Chan WCW, Nie S: Quantum dot bioconjugates for ultrasensitive nonisotopic detection. Science 1998, 281:2016-2020.
28. Auer M, Moore KJ, Meyer-Almes FJ, Guenther R, Pope AJ, Stoekli KA: Fluorescence correlation spectroscopy: lead discovery by miniaturized HTS. Drug Discov Today 1998, 3:457-465. 29. Moore KJ, Turconi S, Ashman S, Ruediger M, Haupts U, Emerick V, • Pope AJ: Single molecule detection technologies in high throughput screening: fluorescence correlation spectroscopy. J Biomol Screen 1999, 4:335-353. Applications of FCS to a wide range of therapeutic target types. 30. Evotec Biosystems homepage on World Wide Web URL: http://www.evotec.com/ 31. Ullman D, Busch M, Mander T: Fluorescence correlation • spectroscopy-based screening technology. J Pharm Technol 1999, 99:30-40. This paper describes new applications and instrumentation for confocal fluctuation fluorescence-based HTS. The authors also describe new two-dimensional applications of this methodology in which molecular brightness analysis (FIDA) is combined with molecular anisotropy measurements. 32. Kask P, Palo K, Ullmann D, Gall K: Fluorescence-intensity •• distribution analysis and its application in biomolecular detection technology. Proc Natl Acad Sci USA 1999, 96:13756-13761. Description of a rapid confocal fluctuation method for determination of molecular brightness, FIDA. This paper also describes application of this technique to resolve different labelled oligonucleotides, with a brightness resolution of two-fold. 33. Chen Y, Muller J, So PTC, Gratton E: The photon counting histogram in fluorescence fluctuation spectroscopy. Biophys J 1999, 77:553-567. 34. Muller JD, Chen Y, Gratton E: Resolving heterogeneity on the single molecular level with the photon counting histogram. Biophys J 2000, 78:474-486. 35. Fries J, Brand L, Eggleling C, Köllner M, Seidel CA: Quantitative identification of different single molecules be selective timeresolved confocal fluorescence spectroscopy. J Phys Chem 1998, 102:6601-6613. 36. Kolterman A, Kettling U, Bieschke J, Winkler T, Eigen M: Rapid assay processing by integration of dual-color fluorescence correlation spectroscopy: high throughput screening for enzyme activity. Proc Natl Acad Sci USA 1998, 95:1421-1426. 37.
Kettling U, Kolterman A, Schwille P, Eigen M: Real-time enzyme kinetics monitored by dual-color fluorescence cross-correlation spectroscopy. Proc Natl Acad Sci USA 1998, 95:1416-1420.
38. Winkler T, Kettling U, Koltermann, Eigen M: Confocal fluorescence coincidence analysis: an approach to ultra high-throughput screening. Proc Natl Acad Sci USA 1999, 96:1375-1378. 39. Fox SJ: High Throughput Screening: Trends in Assay Design. Moraga, USA: High Tech Business Decisions; 1999.
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Next generation therapeutics
40. Cook ND: Scintillation proximity assays – a versatile high throughput screening technology. Drug Discov Today 1996, 1:287-294. 41. NEN Life Science Products homepage on World Wide Web URL: http://www.nen.com/ 42. McDonald OB, Chen WJ, Ellis B, Hoffman C, Overton L, Rink M, •• Smith A, Marshall CJ, Wood ER: A scintillation proximity-assay for the Raf/MEK/ERK kinase cascade: high-throughput screening and identification of selective enzyme inhibitors. Anal Biochem 1999, 268:318-329. The authors describe an assay that reproduces a kinase signal transduction pathway and can detect inhibitors of cRaf1, MEK1 or ERK2. cRaf-1 phosphorylates and activates MEK1, MEK1 phosphorylates and activates ERK2, and ERK2 phosphorylates a biotinylated peptide. The resultant [P-33]labeled peptide is bound to streptavidin-coated SPA beads and detected. 43. Park YW, Cummings RT, Wu L, Zheng S, Cameron PM, Woods A, •• Zaller DM, Marcy AI, Hermes JD: Homogeneous proximity tyrosine kinase assays: scintillation proximity assay versus homogeneous time-resolved fluorescence. Anal Biochem 1999, 269:94-104. The authors provide data comparing the relative merits of two important screening technologies. Scintillation proximity assay offers the advantages of fewer secondary reagents, easier optimization and the potential for fewer false positives, while homogeneous time-resolved fluorescence offers the advantages of safety, faster read times, increased sensitivity and reagent savings. 44. Macarrón R, Mensah L, Cid C, Carranza C, Benson N, Pope A, • Diez E: A homogeneous method to measure aminoacyl-tRNA synthetase activities using scintillation proximity assay technology. Anal Biochem 2000, in press. This homogeneous assay detects incorporation of radiolabeled amino acids into cognate tRNA catalyzed by specific aminoacyl-tRNA synthetases. The authors take advantage of the ability of yttrium silicate SPA beads to bind tRNA aggregates under acidic conditions. 45. Nare B, Allocco JJ, Kuningas R, Galuska S, Myers RW, Bednarek MA, Schmatz DM: Development of a scintillation proximity assay for histone deacetylase using a biotinylated peptide derived from histone H4. Anal Biochem 1999, 267:390-396. 46. Kahl SD, Hubbard FR, Sittampalam GS, Zock JM: Validation of a high throughput scintillation proximity assay for 5-hydroxytryptamine(1E) receptor binding activity. J Biomol Screen 1997, 2:33-40. 47.
Graziani F, Aldegheri L, Terstappen GC: High throughput scintillation proximity assay for the identification of FKBP-12 ligands. J Biomol Screen 1999, 4:3-7.
48. Tian YE, Wu LH, Mueller WT, Chung FZ: A screening strategy based • on differential binding of ligand to receptor and to binding proteins: screening for compounds interacting with corticotrophinreleasing factor-binding protein. J Biomol Screen 1999, 4:319-326. The authors describe an SPA-based screening strategy that can identify compounds that interact with multiple components. 49. Kariv I, Stevens ME, Behrens DL, Oldenburg KR: High throughput quantitation of cAMP production mediated by activation of seven transmembrane domain receptors. J Biomol Screen 1999, 4:27-32. 50. Bosse R, Garlick R, Brown B, Menard L: Development of nonseparation binding and functional assays for G protein-coupled receptors for high throughput screening: pharmacological characterization of the immobilized CCR5 receptor on FlashPlate™. J Biomol Screen 1998, 3:285-292. 51. Molecular Devices homepage on World Wide Web URL: http://www.molecular-devices.com/ 52. Schroeder KS, Neagle BD: FLIPR: A new instrument for accurate, high throughput optical screening. J Biomol Screen 1996, 1:75-80. 53. Gonzalez JE, Oades K, Leychkis Y, Harootunian A, Negulescu PA: • Cell-based assays and instrumentation for screening ion-channel targets. Drug Discov Today 1999, 4:431-439. The authors describe voltage-sensitive sensor dyes based on fluorescence resonance energy transfer for measuring ion channel function. This method is sensitive, generic and compatible with HTS. 54. Chambers J, Ames RS, Bergsma D, Muir A, Fitzgerald LR, Hervieu G, Dytko GM, Foley JJ, Martin J, Liu WS et al.: Melanin-concentrating hormone is the cognate ligand for the orphan G-protein-coupled receptor SLC-1. Nature 1999, 400:261-265. 55. Smart D, Gunthorpe MJ, Jerman JC, Nasir S, Gray J, Muir AI, Chambers JK, Randall AD, Davis JB: The endogenous lipid anandamide is a full agonist at the human vanilloid receptor (hVR1). Brit J Pharmacol 2000, 129:227-230.
56. Simpson PB, Woollacott AJ, Hill RG, Seabrook GR: Functional characterization of bradykinin analogues on recombinant human bradykinin B-1 and B-2 receptors. Eur J Pharmacol 2000, 392:1-9. 57. •
Jurewicz AJ, Foley JJ, Stewart R, Francis T, Vaidya K, Tempeton L, Sarau HM: Fluorometric imaging plate reader: using the orexin receptor to screen 145,000 compounds. Genet Eng News 1999, 19:44-46. The authors describe a successful cell-based high-throughput screen using FLIPR leading to the discovery of selective antagonists of the orexin receptor. 58. Velicelebi G, Stauderman KA, Varney MA, Akong M, Hess SD, Johnson EC: Fluorescence techniques for measuring ion channel activity. Meth Enzymol 1999, 294:20-47. 59. Aurora Biosciences homepage on World Wide Web URL: http://www.aurorabiosciences.com/ 60. Maffia AM, Kariv I, Oldenburg KR: Miniaturization of a mammalian cell-based assay: luciferase reporter gene readout in a 3 microliter 1536-well plate. J Biomol Screen 1999, 4:137-142. 61. Comley JC, Reeves T, Robinson P: A 1536 colorimetric SPAP reporter assay: comparison with 96- and 384-well formats. J Biomol Screen 1998, 3:217-225. 62. Zlokarnik G, Negulescu PA, Knapp TE, Mere L, Burres N, Feng LX, Whitney M, Roemer K, Tsien RY: Quantitation of transcription and clonal selection of single living cells with beta-lactamase as reporter. Science 1998, 279:84-88. 63. Mattheakis LC, Olivan SE, Dias JM, Northrop JP: Expression of Cre • recombinase as a reporter of signal transduction in mammalian cells. Chem Biol 1999, 6:835-844. This reporter system converts transient activation of a signal transduction pathway to DNA recombination, offering the advantage of high sensitivity and a long timeframe for signal detection. 64. Klein C, Paul JI, Sauve K, Schmidt MM, Arcangeli L, Ransom J, Trueheart J, Manfredi JP, Broach JR, Murphy AJ: Identification of surrogate agonists for the human FPRL-1 receptor by autocrine selection in yeast. Nat Biotechnol 1998, 16:1334-1337. 65. Berg M, Undisz K, Thiericke R, Moore T, Posten C: Miniaturization of a functional transcription assay in yeast (human progesterone receptor) in the 384- and 1536-well plate format. J Biomol Screen 2000, 5:71-76. 66. Carrithers MD, Marotti LA, Yoshimura A, Lerner MR: A melanophore based screening assay for erythropoietin receptors. J Biomol Screen 1999, 4:9-14. 67. •
Zaccolo M, De Giorgi F, Cho CY, Feng LX, Knapp T, Negulescu PA, Taylor SS, Tsien RY, Pozzan T: A genetically encoded fluorescent indicator for cyclic AMP in living cells. Nat Cell Biol 2000, 2:25-29. Traditional cAMP measurements are typically carried out in cell lysates. This paper describes a new method that involves tagging protein kinase A with green fluorescent protein mutants and is capable of measuring cAMP in living cells. 68. Conway BR, Minor LK, Xu JZ, Gunnet JW, DeBiasio R, D’Andrea MR, Rubin R, Giuliano K, Zhou LB, Demarest KT: Quantification of G-protein coupled receptor internalization using G-protein coupled receptor-green fluorescent protein conjugates with the ArrayScan™ high-content screening system. J Biomol Screen 1999, 4:75-86. 69. Cellomics homepage on World Wide Web URL: http://www.cellomics.com/ 70. Giuliano KA, DeBiaso RL, Dunlay T, Dunlay RT, Gough A, Volosky JM, Zock J, Pavlakis GN, Taylor L: High-content screening: a new approach to easing key bottlenecks in the drug discovery process. J Biomol Screen 1997, 2:249-259. 71. Zuck P, Lao ZG, Skwish S, Glickman JF, Yang K, Burbaum J, Inglese J: Ligand-receptor binding measured by laser-scanning imaging. Proc Natl Acad Sci USA 1999, 96:11122-11127. 72. Mellentin-Michelotti J, Evangelista LT, Swartzman EE, Miraglia SJ, Werner WE, Yuan PM: Determination of ligand binding affinities for endogenous seven-transmembrane receptors using fluorometric microvolume assay technology. Anal Biochem 1999, 272:182-190. 73. Hanley QS, Verrbeer PJ, Jovin TM: Optical sectioning fluorescence spectroscopy in a programmable array microscope. Appl Spectroscopy 1998, 52:783-789. 74. Mere L, Bennett T, Coassin P, England P, Hamman B, Rink T, Zimmerman S, Negulescu P: Miniaturized FRET assays and microfluidics: key components for ultra-high-throughput screening. Drug Discov Today 1999, 4:363-369.
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75. Robbins Scientific homepage on World Wide Web URL: http://www.robsci.com/
81. ACLARA Biosciences homepage on World Wide Web URL: http://www.aclara.com/
76. Rose D: Microdispensing technologies in drug discovery. Drug Discov Today 1999, 4:411-419.
82. Gibbons I: Microfluidic assays for high-throughput submicroliter • assays using capillary electrophoresis. Drug Discov Today 2000, 1(suppl):33-37. Applications of microchannel-based separation and detection for ultra low volume HTS enzyme assays.
77.
Burbaum JJ: The evolution of miniaturized well plates. J Biomol Screen 2000, 5:5-8.
78. Greiner Labortechnik homepage on World Wide Web URL: http://www.greiner-lab.com/ 79. Knapp MR, Sundberg S, Parce JW: Test tube’s end. J Biomol Screen 2000, 5:9-12. 80. Caliper homepage on World Wide Web URL: http://www.calipertech.com/
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Unger MA, Chou H, Thorsen T, Scherer A, Quake SR: Monolithic microfabricated valves and pumps by multilayer soft lithography. Science 2000, 288:113-116. Description of a new method for fabrication of micochannel systems incorporating valves and pumps using soft lithography. Such devices could have exciting applications in screening and diagnostics.
High-throughput screening: new technology for the 21st century Robert P Hertzberg* and Andrew J Pope† New technologies in high-throughput screening have significantly increased throughput and reduced assay volumes. Key advances over the past few years include new fluorescence methods, detection platforms and liquid-handling technologies. Screening 100,000 samples per day in miniaturized assay volumes will soon become routine. Furthermore, new technologies are now being applied to information-rich cellbased assays, and this is beginning to remove one of the key bottlenecks downstream from primary screening.
What were (and are) the major stumbling blocks that had to (and have to) be overcome to achieve these goals? We can sum it up in three major categories:
Addresses *Molecular Screening Technologies, SmithKline Beecham Pharmaceuticals, 709 Swedeland Road, King of Prussia, PA 19406, USA; e-mail: [email protected] † Molecular Interactions & New Assay Technologies, SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW, UK; e-mail: [email protected]
In this review, we discuss several successful strategies in fluorescence and radiometric detection that are compatible with miniaturized uHTS, and new methods of liquid handling that are being developed to meet these challenges. While developing appropriate bioware and hardware is difficult, building reliable software to control processes and extract knowledge from millions of data points is perhaps the most challenging aspect of uHTS — and this problem remains to be solved.
Current Opinion in Chemical Biology 2000, 4:445–451 1367-5931/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved. Abbreviations CCD charge-coupled device FA fluorescence anisotropy FCS fluorescence correlation spectroscopy FIDA fluorescence intensity distribution analysis GPCR G-protein-coupled receptor HTS high-throughput screening SPA scintillation proximity assay TRET time-resolved energy transfer uHTS ultraHTS
Introduction Advances in molecular biology, human genetics and functional genomics continue to produce increasing numbers of molecular targets available for therapeutic intervention. This, coupled with major increases in compound collections produced by combinatorial technologies, has fueled an important need for improvements in high-throughput screening (HTS) capabilities. As a result, HTS technologies have undergone a revolution in the latter half of the 1990s. Today, most pharmaceutical companies use HTS as the primary engine driving lead discovery. The increased reliance on HTS labs meant that screening had to change. Assay systems and robotics that were capable of screening thousands of compounds per day in the mid-1990s had to evolve into ultraHTS (uHTS) methods capable of 100,000 assays per day, or more. This explosion in assay throughput meant that reagent production had to be scaled to meet the challenge — or else the HTS labs would have to miniaturize assays to meet the reagent suppliers halfway. Fortunately, many of the technological advances required to reach the goals of miniaturized uHTS have been established and are on their way toward routine use.
1. Assay methods and detection (bioware). 2. Liquid handling and robotics (hardware). 3. Process flow and information management (software).
Homogeneous fluorescence methods It is now clear that fluorescence-based techniques are likely to be amongst the most important detection approaches used for HTS in the future, given the industry-wide drive to simplify, miniaturize and speed up assays. Fluorescence techniques are well suited to uHTS because they give very high sensitivity, which allows fairly straightforward miniaturization. This is illustrated by the fact that simple fluorescence intensity measurements have been successfully applied in an ultra-miniaturized format [1]. However, HTS assays based upon fluorescence intensity measurements are mainly restricted to fluorogenic enzyme substrates (for example, see [2]). A more powerful aspect of other, more complex, fluorescence readouts is their ability to yield information on fluorophore environment, which allows predictive design for a wide range of target types [3••,4]. Two factors have made fluorescence readouts more widely used in HTS/uHTS: the development of new microtiter plate readers [5,6•]; and, to a lesser extent, the development of new dyes and molecular reagents. The latest instrumentation enables routine measurement of a range of fluorescence readouts in 1536-well plate formats [4,5,6•]. One such technique is fluorescence anisotropy (FA; also known as polarization), which yields information on molecular rotation, which is related to mass. Anisotropy can be used to measure bimolecular association events within a specific range, as determined by the fluorophore lifetime [3••]. Many examples of HTS applications of FA have now been reported, including ligand−receptor binding [7,8] and enzyme assays [8,9•]. A number of groups have also demonstrated that FA measurements work well in
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1536-well plates when using high-sensitivity plate readers (for example, see [5,9•,10]). Another important fluorescence readout is time-resolved energy transfer (TRET) [3••]. This is a dual labeling approach that is based upon long-range energy transfer between fluorescent Ln3+ complexes and a suitable resonance energy acceptor. Packard/CysBio (Meriden, CT) [11,12] and PerkinElmer/Wallac Oy (Turku, Finland) [13,14] market reagents and instrumentation for this technique. These approaches have an advantage in that time-gating of the long-lived donor and acceptor signals gives high sensitivity by reducing background. A large number of HTS assays have now been configured using TRET (for example, see [15–17]), including the successful miniaturization of the technique to 1536-well plates [16,17]. Given the large distance for effective energy transfer (~90 Å [3••]), TRET is highly suited to measurements of protein−protein interactions (for example, see [15]). Recently, two-alternative approaches with similar capabilities have been developed by Packard/Biosignal in AlphaScreen [18] and bioluminescence energy transfer [19]. In addition to the readouts described above, there have also been recent advances in the development of microplate instrumentation for other, previously inaccessible, fluorescence methods such as lifetime (and lifetime discriminated) measurements (LJL Biosystems FLARe) [10,20,21•]; and two-photon excitation [22]. The FLARe system from LJL (Sunnyvale, CA) uses phase modulation approaches to measure changes in probe lifetimes that may occur via environmental effects or energy transfer. FLARe can also use knowledge of the probe lifetime to discriminate signal from background, which has been shown to be highly useful in eliminating interference from test compounds [23•]. One source of frustration to those involved in the development of fluorescence approaches is the lack of identification of fundamentally new chemical classes of dyes. However, successful modifications (to improve brightness, photostability, physical properties, etc.) to existing dye chemical families have been achieved, most notably by Molecular Probes (e.g. Alexa dyes [24•]) and Amerhsam (Cy dyes [25]). The use of semi-conductor nano-crystals as fluorescent tags [26,27] is intriguing, particularly because these systems seem to be of highly tunable wavelength, allowing the prospect of multiplexed or very high efficiency energy transfer applications. However, these probes have not yet been used in HTS applications so it is too early to tell how their performance is likely to compare with more conventional fluorophores. One area of fluorescence measurements that has developed very strongly in terms of fundamental approaches is that of single molecule fluctuation-based measurements. One attraction of these methods for HTS/uHTS is their
high information content and intrinsic sensitivity to miniaturization [3••,4]. All are performed using confocal optics in which the observation volume is extremely small (~1 fl) and very few molecules are present in the confocal volume optics at any one time, so the output fluorescence signal fluctuates with time as molecules enter and exit. The classical form of confocal fluctuation spectroscopy, known as fluorescence correlation spectroscopy (FCS) has now been demonstrated to be a viable approach to HTS for a wide range of therapeutic targets (for example, see [28,29•]). FCS can be used to detect binding interactions via changes in translational diffusion rates or, where there is a significant change in brightness during a binding or catalytic reaction, can be configured in a way that is not dependent upon changes in diffusion rates (i.e. mass) [29•]. It is only during the past few years that advances in optics, electronics and computation tools have made FCS a viable proposition for use in routine HTS applications. These same advances have led to a renaissance in the whole area of single molecule fluctuation methods, driven in particular by a number of academic labs and by Evotec Biosystems (Hamburg, Germany) [30], who are developing an integrated miniaturized screening system based upon this detection technology [31•]. Several new methods for analyzing fluctuation data have now been reported, including methods to determine molecular brightness (fluorescence intensity distribution analysis; FIDA [32••] or related methods [33–35]). These methods can be used to configure assays in a variety of ways; molecular brightness can change either by environment or energy transfer, or via changes in the stoichiometry of fluorophore molecules present on a single particle. There has been significant progress, using a number of approaches, to extend these methods by simultaneously analyzing data from more than one output channel. For example, Evotec Biosystems recently showed that combining FIDA with molecular anisotropy (so-called 2D-FIDA) resulted in more robust measurements of kinase activity than when either technique was used individually [31•]. In addition, a number of approaches have been developed that measure the coincidence of two discrete fluors within the same particle [36–38].
Miniaturized radiometric readouts While fluorescence assay technologies are growing in importance, the predicted demise of radiometric assays as an important facet of HTS labs has not yet occurred. Current estimates from various surveys of HTS laboratories indicate that radiometric assays presently constitute between 20% and 50% of all screens performed [39]. Although we expect the fraction of radiometric assays to decrease over the coming years, this technology is unlikely to disappear completely. In the early 1990s, several advances in radiometric assay technology were introduced including scintillation proximity assay (SPA) (Amersham Pharmacia Biotech) and
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FlashPlates™ (NEN Life Science Products, Boston, MA). With these approaches, the target of interest is immobilized onto a solid support (e.g. SPA beads or FlashPlate™ surface) that contains a scintillant. When a radiolabeled molecule binds to the target molecule, the radioisotope is brought in close proximity to the solid support, and energy transfer between the emitted beta particle and the scintillant results in the emission of light. Radioisotope remaining free in solution is too distant from the scintillant and the beta particle dissipates energy into the aqueous environment. Thus, scintillation proximity technologies facilitate a homogeneous approach to radiometric assays [25,40,41]. SPA has been used in a wide variety of applications and it is a standard technique in HTS labs. The technology has been applied to kinases [42••,43••], nucleic-acid-processing enzymes [44•], other enzymes [45] and is widely used for ligand−receptor interactions [46,47,48•]. FlashPlate™ technology is similar to SPA but the solid surface is a microtiter plate rather than a bead. Recent FlashPlate™ applications include the detection of cAMP levels [49] and ligand−receptor interactions [50]. Of course, radiometric assays have several disadvantages including safety, limited reagent stability, relatively long read-times and little intrinsic information on the isotope environment. However, new technologies are now emerging to address the issue of read-time and assay miniaturization. The most important of these are the emergence of imaging platereaders such as Leadseeker™ (Amersham Pharmacia) [6•,25] and CLIPR™ (Molecular Devices, Sunnyvale CA) [51]. These instruments consist of high sensitivity CCD (charge-coupled device) cameras and special lenses capable of rapidly flat-field imaging an entire microtiter plate. These systems are capable of reading 1536-well plates (or plates of even higher density) and can rapidly quantify the light emitted by each microtiter well. In addition to sensitivity and speed, imaging platereaders demonstrate further advantages such as low backgrounds and low variability. While the Leadseeker™ and CLIPR™ can read SPA, the former can also read fluorescence and both have the capability of imaging luminescence with high sensitivity. Additional fluorescence imaging platereaders are available such as the ViewLux™ (manufactured by PerkinElmer/Wallac Oy) [13]. Imaging platereaders are likely to become increasingly important relative to point-detection devices in the coming years, given the desire to achieve higher measurement speeds and therefore throughput. In addition to the emergence of imaging platereaders, new SPA beads have recently been introduced by Amersham Pharmacia Biotech [25]. These beads emit light at 615 nm, in contrast to traditional SPA beads, which emit around 420 nm. This longer wavelength is well-suited to the response of the CCD camera in the Leadseeker™ imager. Furthermore, light emission at 615 nm may result in fewer
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problems with color quench by test compounds, which tend to be yellow and red, although this has yet to be definitively shown.
Cell-based assay technologies Advances in technology and instrumentation for cell-based assays have occurred over the past few years. Among these is the emergence of HTS-compatible technology to measure G-protein-coupled receptor (GPCR) [52] and ion channel function [53•], confocal imaging platforms for rapid cellular and sub-cellular imaging, and the continued development of reporter gene technology. The FLIPR™ (Molecular Devices) is a fluorescence imaging platereader with integrated liquid handling that facilitates the simultaneous fluorescence imaging of 384 samples to measure intracellular calcium mobilization in real time [52]. Historically, these measurements have only been possible on single cuvette fluorimeters and by microscopic imaging. FLIPR™ has been used to identify cognate ligands for orphan GPCRs [54,55], to characterize GPCR pharmacology [56] and to screen compound libraries [57•]. The instrument is also capable of measuring ion channel function by coupling the activity of a target channel to a voltage-gated calcium channel [58]. An alternative and promising HTS technology for ion channels is based on voltage-sensitive fluorescence resonance energy transfer (VIPR™; Aurora Biosciences, La Jolla, CA) [53•,59]. Although kinetic platereaders facilitate cell-based functional screens, they are currently limited to 96/384-well plates and are somewhat labor-intensive. In contrast, cellbased reporter gene screens require fewer cells, are easier to automate and can be performed in 1536-well plates. Recent descriptions of miniaturized reporter gene readouts include luciferase [60] and secreted alkaline phosphate [61]. A novel and sensitive beta-lactamase reporter system has been described that allows the clonal selection of living cells and is amenable to miniaturized HTS [62]. Finally, a Cre recombinase reporter system links signal transduction to DNA recombination and results in a permanent readout of gene expression [63•]. Human GPCRs can be screened in yeast to find agonists and antagonists by taking advantage of the pheromone signaling pathway [64]. Although this technology can offer the advantage of a null background for the expression of human receptors, there is only a moderate correlation between ligand activation in yeast and mammalian cells (R Hertzberg, unpublished data). A yeast-based transcription assay for the human progesterone receptor has recently been performed in 1536-well plates [65]. In addition, receptor assays based on cell darkening can be performed in frog melanophores [66]. Cyclic AMP measurements in cell extracts can be performed with SPA, FlashPlate™ [49], fluorescence
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polarization [10,41] or AlphaScreen™ technologies [18]. A novel fluorescent indicator for cyclic AMP in living cells involves tagging protein kinase A with green fluorescent protein mutants [67•]. Recently, laser scanning imaging systems have been developed to measure cellular and sub-cellular quantitation of fluorescence in whole cells. These systems have the capability of bringing low throughput biological studies with high information content into the world of HTS. One of the most advanced systems is the ArrayScan™ (Cellomics, Pittsburgh, PA) which has been used to measure GPCR internalization [68,69] as well as a range of other applications [70]. Other imaging systems have been used to measure ligand-receptor binding in whole cells [71,72]. Current instrumentation does not combine sufficient resolution and speed to allow uHTS-level throughput for true sub-cellular imaging. However, a number of approaches are being developed for high speed cellular imaging (for example, see [73]), which look likely to enable the use of this approach for first-line uHTS in the future.
Technology for assay miniaturization Several advances in liquid handling and microplates have occurred in the past few years, and true miniaturized uHTS is getting closer to being a regular feature of the modern HTS laboratory. The 1536-well plate is now a reality, and although only a few full screening campaigns have been performed in this format, enough proof-of-concept work on assay readouts (e.g. [2,4,5,6•,9•,12,16,17,28, 29•,31•,60,61,65,74]) has been done to show that miniaturization is here to stay. However, an equally important aspect of assay miniaturization is liquid handling. In fact, it is the development of robust technology in this area, particularly for high speed low volume reformatting of test compounds, that is now largely limiting uHTS implementation. Conventional syringe pump pipetters are now capable of delivering volumes as low as 100 nl with touch-off dispensing [75]. Syringe-solenoid technology, a non-contact method that can dispense droplets in the low nanoliter to low microliter range, is well-suited for ‘bulk’ dispensing biological reagents [76]. Piezoelectric dispensers can create drops in the picoliter range and dispense thousands of drops per second [30,59,74]. These devices are well-suited to compound reformatting and will be important tools for miniaturized uHTS, although problems such as clogging and gas bubbles can occur. Assay vessels are also undergoing a revolution. Openwell formats still predominate, but 96- and 384-well plates have now evolved into 1536- [31•,77,78] and 3456well formats [74]. Such carriers probably represent the ultimate development of the equivalent of test tubes. Beyond this are closed ‘lab-chip’ microfluidic systems such as those being developed by Caliper
(Mountainview, CA) [79,80] and ACLARA Biosciences (Mountainview, CA) [81]. These systems offer potential advantages for ultra miniaturization, because they are not prone to evaporation problems and are compatible with sub-microliter volumes. These systems also have some unique properties, as illustrated by the development of rapid separation-based assays in microfluidic systems [82•]. The development of microfluidic systems and microsystems-technology in general is a very rapidly moving area in which important advances will impact HTS in the future. For example, the use of new materials has recently enabled the construction of devices containing monolithic pumps and valves [83•]. However, there are a number of issues with these types of systems that will take time to resolve, such as how they interface with very large chemical libraries. Although these systems are very promising, many in the HTS industry believe that the 1536-well plate will continue as the standard uHTS medium at least until the middle of this decade.
Conclusions HTS technologies have undergone a revolution and the field shows no signs of slowing down. Fluorescence methods such as intensity, anisotropy and time-resolved energy transfer are now routine in HTS labs. Newer techniques such as fluorescence lifetime measurements, FCS and FIDA are showing promise and will be important screening formats in the next few years. Major advances in HTS hardware have also been made. State-of-the-art detection platforms demonstrate significant improvements in sensitivity and throughput. In particular, imaging platereaders show great promise in reducing assay read-times and facilitating miniaturization. Methods are available to perform information-rich cell-based assays with throughputs approaching those of uHTS. New liquid-handling methods are finally coming on line that allow the dispensement of compounds and biological reagents in volumes consistent with miniaturized assay formats. Lab-chip microfluidic systems are on the horizon; these have the promise to drive assay volumes down even further and provide unique separation capabilities. Now that most of the technologies have been developed, the main hurdles to solve are problems of implementation. Applying these exciting new technologies to routine HTS and keeping these systems running on a day-to-day basis will continue to be a challenge. Nevertheless, in a short period of time, the promise of uHTS capabilities will be fully realized and we can look forward to producing higher quality leads to fuel drug discovery.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest •• of outstanding interest 1.
Oldenburg KR, Zhang JH, Chen T, Maffia A, Blom KF, Combs AP, Chung TDY: Assay miniaturization for ultra-high throughput
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screening of combinatorial and discrete compound libraries: a 9600-well (0.2 microliter) assay system. J Biomol Screen 1998, 3:55-62. 2.
Abriola L, Chin M, Fuerst P, Schweitzer R, Sills MA: Digital imaging as a detection method for a fluorescent protease assay in 96-well and miniaturized assay plate formats. J Biomol Screen 1999, 4:121-128.
3. ••
Pope AJ, Haupts U, Moore KJ: Homogeneous fluorescence readouts for miniaturized high-throughput screening; theory and practice. Drug Discov Today 1999, 4:350-362. A review of the theoretical background to homogeneous fluorescence-based methods currently used in HTS. Includes relevant theory and simulations plus practical examples. 4.
Haupts U, Ruediger M, Pope AJ: Macroscopic versus microscopic fluorescence techniques in (ultra)-high-throughput screening. Drug Discov Today 2000, 1(suppl):3-9.
5.
Sportsman JR, Leytes L: Miniaturization of homogeneous assays using fluorescence polarization. Drug Discov Today 2000, 1(suppl):27-32.
6. Ramm P: Imaging systems in assay screening. Drug Discov Today • 1999, 4:401-410. Review of CCD-based systems for rapid measurement of scintillation, luminescence and fluorescence for HTS. 7.
Allen M, Reeves J, Mellor G: High throughput fluorescence polarization: a homogeneous alternative to radioligand binding for cell surface receptors. J Biomol Screen 2000, 5:63-69.
8.
Parker GJ, Law TL, Lenoch FJ, Bolger RE: Development of high throughput screening assays using fluorescence polarization: nuclear receptor-ligand-binding and kinase/phosphatase assays. J Biomol Screen 2000, 5:77-88.
9. •
Li Z, Mehdi S, Patel I, Kaooya J, Judkins M, Zhang W, Diener K, Lzada A, Dunnington D: An ultra-high throughput screening approach for an adenine transferase using fluorescence polarization. J Biomol Screen 2000, 5:31-38. HTS application paper using fluorescence anisotropy including 1536-well plate work. 10. LJL Biosciences homepage on World Wide Web URL: http://www.ljlbio.com/ 11. Packard Instrument Corporation homepage on World Wide Web URL: http://www.packardinst.com/ 12. Mathis G: HTRF technology. J Biomol Screen 1999, 4:309-313. 13. PerkinElmer Life Sciences homepage on World Wide Web URL: http://lifesciences.perkinelmer.com/ 14. Hemmila I: LANCETM: homogeneous assay platform for HTS. J Biomol Screen 1999, 4:303-307.
15. Zhou G, Cummings R, Li Y, Mitra S, Wilkinson HA, Elbriecht A, Hermes J, Schaeffer JM, Smith RG, Moller DE: Nuclear receptors have distinct affinities for coactivators: characterization by fluorescence resonance energy transfer. Mol Endocrinol 1999, 12:1594-1604. 16. Earnshaw DL, Moore KJ, Greenwood CJ, Djaballah H, Jurewicz AJ, Murray KJ, Pope AJ: Time-resolved energy transfer DNA helicase assays for high throughput screening. J Biomol Screen 1999, 4:239-248. 17.
Moore KJ, Turconi S, Miles-Williams A, Djaballah H, Hurskainen P, Harrop J, Murray KJ, Pope AJ: A homogeneous 384-well high throughput screen for novel tumor necrosis factor receptor: ligand interactions using time-resolved energy transfer. J Biomol Screen 1999, 4:205-214.
18. Biosignal homepage on World Wide Web URL: http://www.biosignal.com/ 19. Xu Y, Piston DW, Johnson CH: A bioluminescence resonance energy transfer (BRET) system: applications to interacting circadian clock proteins. Proc Natl Acad Sci USA 1999, 96:151-156. 20. French T, Owicki JC, Modlin DN, Desphande SS, Mineyev I, Crwaford K, Burton W: Fluorescence-lifetime technologies for high-throughput screening. Proc Soc Optical Engineering 1998, 3259:209-218.
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21. French T, Bailey B, Stumbo DP, Modlin DN: A time resolved • fluorimeter for high-throughput screening. Proc Soc Optical Engineering 1999, 3603:272-280. Description of the fluorescence lifetime assay repertoire (FLARe) system for phase-modulation detection of lifetime and lifetime-discriminated fluorescence in HTS. 22. Lakowicz JP, Grynczynski I, Gryzinski Z: High throughput screening with multiphoton excitation. J Biomol Screen 1999, 4:355-361. 23. Helms MK, French T: Time resolved fluorescence measurements • of actin phalloidin interactions. Proc Soc Optical Engineering 2000, 3926:158-165. Application of FLARe in measurement of actin-phalloidin interactions and showing power of lifetime discrimination to eliminate compound library interferences. 24. Molecular Probes Inc. homepage on World Wide Web URL: • http://www.probes.com/ Excellent website for comprehensive information on fluorescent dyes, reagents and applications from Molecular Probes as well as background literature. 25. Amersham Pharmacia Biotech homepage on World Wide Web URL: http://www.apbiotech.com/ 26
Bruchez M Jr, Moronne M, Gin P, Weiss S, Alivisatos AP: Semiconductor nanocrystals as fluorescent biological labels. Science 1998, 281:2013-2016.
27.
Chan WCW, Nie S: Quantum dot bioconjugates for ultrasensitive nonisotopic detection. Science 1998, 281:2016-2020.
28. Auer M, Moore KJ, Meyer-Almes FJ, Guenther R, Pope AJ, Stoekli KA: Fluorescence correlation spectroscopy: lead discovery by miniaturized HTS. Drug Discov Today 1998, 3:457-465. 29. Moore KJ, Turconi S, Ashman S, Ruediger M, Haupts U, Emerick V, • Pope AJ: Single molecule detection technologies in high throughput screening: fluorescence correlation spectroscopy. J Biomol Screen 1999, 4:335-353. Applications of FCS to a wide range of therapeutic target types. 30. Evotec Biosystems homepage on World Wide Web URL: http://www.evotec.com/ 31. Ullman D, Busch M, Mander T: Fluorescence correlation • spectroscopy-based screening technology. J Pharm Technol 1999, 99:30-40. This paper describes new applications and instrumentation for confocal fluctuation fluorescence-based HTS. The authors also describe new two-dimensional applications of this methodology in which molecular brightness analysis (FIDA) is combined with molecular anisotropy measurements. 32. Kask P, Palo K, Ullmann D, Gall K: Fluorescence-intensity •• distribution analysis and its application in biomolecular detection technology. Proc Natl Acad Sci USA 1999, 96:13756-13761. Description of a rapid confocal fluctuation method for determination of molecular brightness, FIDA. This paper also describes application of this technique to resolve different labelled oligonucleotides, with a brightness resolution of two-fold. 33. Chen Y, Muller J, So PTC, Gratton E: The photon counting histogram in fluorescence fluctuation spectroscopy. Biophys J 1999, 77:553-567. 34. Muller JD, Chen Y, Gratton E: Resolving heterogeneity on the single molecular level with the photon counting histogram. Biophys J 2000, 78:474-486. 35. Fries J, Brand L, Eggleling C, Köllner M, Seidel CA: Quantitative identification of different single molecules be selective timeresolved confocal fluorescence spectroscopy. J Phys Chem 1998, 102:6601-6613. 36. Kolterman A, Kettling U, Bieschke J, Winkler T, Eigen M: Rapid assay processing by integration of dual-color fluorescence correlation spectroscopy: high throughput screening for enzyme activity. Proc Natl Acad Sci USA 1998, 95:1421-1426. 37.
Kettling U, Kolterman A, Schwille P, Eigen M: Real-time enzyme kinetics monitored by dual-color fluorescence cross-correlation spectroscopy. Proc Natl Acad Sci USA 1998, 95:1416-1420.
38. Winkler T, Kettling U, Koltermann, Eigen M: Confocal fluorescence coincidence analysis: an approach to ultra high-throughput screening. Proc Natl Acad Sci USA 1999, 96:1375-1378. 39. Fox SJ: High Throughput Screening: Trends in Assay Design. Moraga, USA: High Tech Business Decisions; 1999.
450
Next generation therapeutics
40. Cook ND: Scintillation proximity assays – a versatile high throughput screening technology. Drug Discov Today 1996, 1:287-294. 41. NEN Life Science Products homepage on World Wide Web URL: http://www.nen.com/ 42. McDonald OB, Chen WJ, Ellis B, Hoffman C, Overton L, Rink M, •• Smith A, Marshall CJ, Wood ER: A scintillation proximity-assay for the Raf/MEK/ERK kinase cascade: high-throughput screening and identification of selective enzyme inhibitors. Anal Biochem 1999, 268:318-329. The authors describe an assay that reproduces a kinase signal transduction pathway and can detect inhibitors of cRaf1, MEK1 or ERK2. cRaf-1 phosphorylates and activates MEK1, MEK1 phosphorylates and activates ERK2, and ERK2 phosphorylates a biotinylated peptide. The resultant [P-33]labeled peptide is bound to streptavidin-coated SPA beads and detected. 43. Park YW, Cummings RT, Wu L, Zheng S, Cameron PM, Woods A, •• Zaller DM, Marcy AI, Hermes JD: Homogeneous proximity tyrosine kinase assays: scintillation proximity assay versus homogeneous time-resolved fluorescence. Anal Biochem 1999, 269:94-104. The authors provide data comparing the relative merits of two important screening technologies. Scintillation proximity assay offers the advantages of fewer secondary reagents, easier optimization and the potential for fewer false positives, while homogeneous time-resolved fluorescence offers the advantages of safety, faster read times, increased sensitivity and reagent savings. 44. Macarrón R, Mensah L, Cid C, Carranza C, Benson N, Pope A, • Diez E: A homogeneous method to measure aminoacyl-tRNA synthetase activities using scintillation proximity assay technology. Anal Biochem 2000, in press. This homogeneous assay detects incorporation of radiolabeled amino acids into cognate tRNA catalyzed by specific aminoacyl-tRNA synthetases. The authors take advantage of the ability of yttrium silicate SPA beads to bind tRNA aggregates under acidic conditions. 45. Nare B, Allocco JJ, Kuningas R, Galuska S, Myers RW, Bednarek MA, Schmatz DM: Development of a scintillation proximity assay for histone deacetylase using a biotinylated peptide derived from histone H4. Anal Biochem 1999, 267:390-396. 46. Kahl SD, Hubbard FR, Sittampalam GS, Zock JM: Validation of a high throughput scintillation proximity assay for 5-hydroxytryptamine(1E) receptor binding activity. J Biomol Screen 1997, 2:33-40. 47.
Graziani F, Aldegheri L, Terstappen GC: High throughput scintillation proximity assay for the identification of FKBP-12 ligands. J Biomol Screen 1999, 4:3-7.
48. Tian YE, Wu LH, Mueller WT, Chung FZ: A screening strategy based • on differential binding of ligand to receptor and to binding proteins: screening for compounds interacting with corticotrophinreleasing factor-binding protein. J Biomol Screen 1999, 4:319-326. The authors describe an SPA-based screening strategy that can identify compounds that interact with multiple components. 49. Kariv I, Stevens ME, Behrens DL, Oldenburg KR: High throughput quantitation of cAMP production mediated by activation of seven transmembrane domain receptors. J Biomol Screen 1999, 4:27-32. 50. Bosse R, Garlick R, Brown B, Menard L: Development of nonseparation binding and functional assays for G protein-coupled receptors for high throughput screening: pharmacological characterization of the immobilized CCR5 receptor on FlashPlate™. J Biomol Screen 1998, 3:285-292. 51. Molecular Devices homepage on World Wide Web URL: http://www.molecular-devices.com/ 52. Schroeder KS, Neagle BD: FLIPR: A new instrument for accurate, high throughput optical screening. J Biomol Screen 1996, 1:75-80. 53. Gonzalez JE, Oades K, Leychkis Y, Harootunian A, Negulescu PA: • Cell-based assays and instrumentation for screening ion-channel targets. Drug Discov Today 1999, 4:431-439. The authors describe voltage-sensitive sensor dyes based on fluorescence resonance energy transfer for measuring ion channel function. This method is sensitive, generic and compatible with HTS. 54. Chambers J, Ames RS, Bergsma D, Muir A, Fitzgerald LR, Hervieu G, Dytko GM, Foley JJ, Martin J, Liu WS et al.: Melanin-concentrating hormone is the cognate ligand for the orphan G-protein-coupled receptor SLC-1. Nature 1999, 400:261-265. 55. Smart D, Gunthorpe MJ, Jerman JC, Nasir S, Gray J, Muir AI, Chambers JK, Randall AD, Davis JB: The endogenous lipid anandamide is a full agonist at the human vanilloid receptor (hVR1). Brit J Pharmacol 2000, 129:227-230.
56. Simpson PB, Woollacott AJ, Hill RG, Seabrook GR: Functional characterization of bradykinin analogues on recombinant human bradykinin B-1 and B-2 receptors. Eur J Pharmacol 2000, 392:1-9. 57. •
Jurewicz AJ, Foley JJ, Stewart R, Francis T, Vaidya K, Tempeton L, Sarau HM: Fluorometric imaging plate reader: using the orexin receptor to screen 145,000 compounds. Genet Eng News 1999, 19:44-46. The authors describe a successful cell-based high-throughput screen using FLIPR leading to the discovery of selective antagonists of the orexin receptor. 58. Velicelebi G, Stauderman KA, Varney MA, Akong M, Hess SD, Johnson EC: Fluorescence techniques for measuring ion channel activity. Meth Enzymol 1999, 294:20-47. 59. Aurora Biosciences homepage on World Wide Web URL: http://www.aurorabiosciences.com/ 60. Maffia AM, Kariv I, Oldenburg KR: Miniaturization of a mammalian cell-based assay: luciferase reporter gene readout in a 3 microliter 1536-well plate. J Biomol Screen 1999, 4:137-142. 61. Comley JC, Reeves T, Robinson P: A 1536 colorimetric SPAP reporter assay: comparison with 96- and 384-well formats. J Biomol Screen 1998, 3:217-225. 62. Zlokarnik G, Negulescu PA, Knapp TE, Mere L, Burres N, Feng LX, Whitney M, Roemer K, Tsien RY: Quantitation of transcription and clonal selection of single living cells with beta-lactamase as reporter. Science 1998, 279:84-88. 63. Mattheakis LC, Olivan SE, Dias JM, Northrop JP: Expression of Cre • recombinase as a reporter of signal transduction in mammalian cells. Chem Biol 1999, 6:835-844. This reporter system converts transient activation of a signal transduction pathway to DNA recombination, offering the advantage of high sensitivity and a long timeframe for signal detection. 64. Klein C, Paul JI, Sauve K, Schmidt MM, Arcangeli L, Ransom J, Trueheart J, Manfredi JP, Broach JR, Murphy AJ: Identification of surrogate agonists for the human FPRL-1 receptor by autocrine selection in yeast. Nat Biotechnol 1998, 16:1334-1337. 65. Berg M, Undisz K, Thiericke R, Moore T, Posten C: Miniaturization of a functional transcription assay in yeast (human progesterone receptor) in the 384- and 1536-well plate format. J Biomol Screen 2000, 5:71-76. 66. Carrithers MD, Marotti LA, Yoshimura A, Lerner MR: A melanophore based screening assay for erythropoietin receptors. J Biomol Screen 1999, 4:9-14. 67. •
Zaccolo M, De Giorgi F, Cho CY, Feng LX, Knapp T, Negulescu PA, Taylor SS, Tsien RY, Pozzan T: A genetically encoded fluorescent indicator for cyclic AMP in living cells. Nat Cell Biol 2000, 2:25-29. Traditional cAMP measurements are typically carried out in cell lysates. This paper describes a new method that involves tagging protein kinase A with green fluorescent protein mutants and is capable of measuring cAMP in living cells. 68. Conway BR, Minor LK, Xu JZ, Gunnet JW, DeBiasio R, D’Andrea MR, Rubin R, Giuliano K, Zhou LB, Demarest KT: Quantification of G-protein coupled receptor internalization using G-protein coupled receptor-green fluorescent protein conjugates with the ArrayScan™ high-content screening system. J Biomol Screen 1999, 4:75-86. 69. Cellomics homepage on World Wide Web URL: http://www.cellomics.com/ 70. Giuliano KA, DeBiaso RL, Dunlay T, Dunlay RT, Gough A, Volosky JM, Zock J, Pavlakis GN, Taylor L: High-content screening: a new approach to easing key bottlenecks in the drug discovery process. J Biomol Screen 1997, 2:249-259. 71. Zuck P, Lao ZG, Skwish S, Glickman JF, Yang K, Burbaum J, Inglese J: Ligand-receptor binding measured by laser-scanning imaging. Proc Natl Acad Sci USA 1999, 96:11122-11127. 72. Mellentin-Michelotti J, Evangelista LT, Swartzman EE, Miraglia SJ, Werner WE, Yuan PM: Determination of ligand binding affinities for endogenous seven-transmembrane receptors using fluorometric microvolume assay technology. Anal Biochem 1999, 272:182-190. 73. Hanley QS, Verrbeer PJ, Jovin TM: Optical sectioning fluorescence spectroscopy in a programmable array microscope. Appl Spectroscopy 1998, 52:783-789. 74. Mere L, Bennett T, Coassin P, England P, Hamman B, Rink T, Zimmerman S, Negulescu P: Miniaturized FRET assays and microfluidics: key components for ultra-high-throughput screening. Drug Discov Today 1999, 4:363-369.
High-throughput screening Hertzberg and Pope
451
75. Robbins Scientific homepage on World Wide Web URL: http://www.robsci.com/
81. ACLARA Biosciences homepage on World Wide Web URL: http://www.aclara.com/
76. Rose D: Microdispensing technologies in drug discovery. Drug Discov Today 1999, 4:411-419.
82. Gibbons I: Microfluidic assays for high-throughput submicroliter • assays using capillary electrophoresis. Drug Discov Today 2000, 1(suppl):33-37. Applications of microchannel-based separation and detection for ultra low volume HTS enzyme assays.
77.
Burbaum JJ: The evolution of miniaturized well plates. J Biomol Screen 2000, 5:5-8.
78. Greiner Labortechnik homepage on World Wide Web URL: http://www.greiner-lab.com/ 79. Knapp MR, Sundberg S, Parce JW: Test tube’s end. J Biomol Screen 2000, 5:9-12. 80. Caliper homepage on World Wide Web URL: http://www.calipertech.com/
83 •
Unger MA, Chou H, Thorsen T, Scherer A, Quake SR: Monolithic microfabricated valves and pumps by multilayer soft lithography. Science 2000, 288:113-116. Description of a new method for fabrication of micochannel systems incorporating valves and pumps using soft lithography. Such devices could have exciting applications in screening and diagnostics.