Review
Gene to ScreenOptical biosensors: where next and how soon?
Gene to Screen
Introduction
From the earliest days of screening and molecular profiling in drug discovery, assay development has exploited a variety of labelled assays to report an interaction of a drug candidate with a receptor or cell. These include ELISAs, radiolabelled pull-down assays, scintillation proximity assays (SPA) and an ever-expanding suite of intensity and time-resolved fluorescence assays [-intensity, -lifetime, -polarization, -fluorescence resonance energy transfer (FRET) and so on]. Such assays are used extensively in most stages of preclinical drug discovery and form the basis for dedicated high-throughput screening (HTS) instrumentation developed by the major technology suppliers to the pharmaceutical industry. Formats cover pathway-based screens (e.g. cellular Ca2+ flux), activity-based screens (e.g. phosphorylation) and interaction-based screens (e.g. AlphaScreen™, enhanced chemiluminescence, fluorometric microvolume assay technology (FMAT™), LeadSeeker™, SPA). The development of a label-based assay requires additional time and cost allocation, a component of which is not required for true label-free assays.
Following a decade of major investment in compound generation, storage and characterization, and the industrialization of assay development, implementation and data handling, major Pharma companies can now run several major screening campaigns each quarter, with each encompassing over a million drug candidates. Unfortunately, most HTS platforms still give high hit rates and do not always discriminate causal perturbation of a biological pathway from non-specific or concomitant activation of non-relevant cellular processes. More importantly, the label can, in some cases, interfere with the molecular interaction by occluding a binding site, leading to false negatives. For many fluorescent and chemiluminescent reporter compounds, background binding can be a significant problem, leading to false positives. There are also a large number of other artefacts inherent with label-based assays that originate from the screened compounds themselves – in particular, autofluorescence. These artefacts can be offset by the use of multicomponent fitting (e.g. fluorescence lifetime and fluorescence intensity) and other proprietary software algorithms; however, these approaches all add to the complexity of the screening assay. These effects and their impact on the data quality originating from HTS have been reviewed in detail elsewhere [1]. For applications of emergent technologies for multiplexed optical arrays and higher information content application of dual polarization interferometry, see Refs 2, 3, 4, 5.
Section snippets
The label-free technology landscape
Although labelled assays require significant initial effort to develop the assay platform, it is important to note that the end user is only exposed to these additional R&D requirements through higher instrumentation pricing. The technology supplier usually carries out the development work that provides the foundation for most assay platforms. In other words, many tools companies put a lot of effort into developing robust assays tailored specifically to a particular assay class, a signalling
Biacore
For more than 15 years, Biacore systems have been used by scientists to profile the specificity, affinity and kinetics of protein interactions. Over the past 3–5 years, Biacore instruments, such as S51 and 3000, and more recently Biacore A100 and T100, have demonstrated the sensitivity required for typical drug candidates (Figure 2) and lower molecular weight (∼150 Da) drug fragments or ‘needles’. The response levels for drug ‘needles’ are lower than those for larger molecular weight drugs
SRU Biosystems
The SRU BIND™ system is comprised of SBS-standard 96, 384, and 1536-well microplates and a selection of two types of detection instruments 17, 18. Photonic crystal optical biosensors are incorporated into the bottom surface of the microplate wells, and are designed to reflect only a very narrow band of wavelengths when illuminated with a broad band of incident wavelengths. The photonic crystal tightly confines resonantly coupled light to the device surface, resulting in a shift of the reflected
Corning
Corning has also developed a label-free detection platform that utilizes resonant waveguide grating (RWG) sensors [19]. The Corning® Epic™ system consists of a Society for Biomolecular Sciences (SBS; http://www.sbsonline.org) standard 384-well microplate with RWG sensors and attachment surface chemistry within each well (Figure 3) and an HTS-compatible microplate reader capable of reading up to 40 000 wells in an eight-hour period. The optical reader head inside the Epic™ instrument reads 16
ForteBio
In 2006, ForteBio (http://www.fortebio.com) released the Octet system, based on a proprietary technique called BioLayer Interferometry (BLI) [22]. The Octet system uses disposable sensors with an optical coating layer at the tip of each sensor (Figure 5). This optical surface is coated with a biocompatible matrix that can interact with molecules from a surrounding solution. A minimum sample volume of 80 μL should be used in low-volume microplate wells to make accurate measurements because
Conclusion
The past five years have witnessed the emergence of an increasing number of commercially available technology platforms that are driving the development of novel label-free assays. Several new platform technologies have been developed and were launched during 2006. These have come from both small companies and from more established players in the drug discovery tools market. In theory, a label-free screening system imparts additional flexibility and efficiency to the process of assay design,
Acknowledgements
I thank my numerous colleagues in pharma and biosensor companies who agreed to contribute material and quotations to this article, and also John Comley (HTStec; http://www.htstec.com), who agreed to release data from his ‘Label Free Detection Trends 2004’ market report for publication. The author is a Founder of Akubio and a former consultant to Biacore. This article represents the sole opinion of the author; not that of Akubio, nor Biacore, nor any employee, shareholders, consultants,
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