Single-bead assay

Obvious exceptions to this are compound libraries generated on beads by the mix-split technique, which are subsequently tested in single-bead assays. Here, the amount of compound obtained after cleavage from a single bead is sufficient to perform the biological assay. This is the classical one bead - one compound situation, whereby resin-bound compounds are synthesized in mixtures and sequestered before cleavage. In cases where... 

The compound library is divided into mixtures of up to several hundred compounds (Fig. 1.11.). In the first step, about one-third of each respective compound bound on a macrobead (about 600-800 pmol compound per bead) is split off into solution for biological evaluation. Experiments performed indicate that an optimal mixture complexity is about 20 compounds per mixture [135]. This can be accomplished, e.g., by the use of a threearmed linker that selectively releases compounds at different pH optima [132,134] or of a photochemically cleavable linker which releases compounds into solution by controlled irradiation [126, 136]. In a second step, the collection of beads corresponding to the greatest biological activity is then redistributed in smaller mixtures. A further aliquot of the respective compound is then released and assayed. Ideally, the beads are rearrayed separately for direct identification of the compound responsible for biological activity ( single-bead assay ). The structure of the bioactive compound can then be determined from the remaining resin-bound compound (often in only femtomole quantity) by the analytical methods described in Section 1.4.1. 

The identification of positives from an SP pool library usually requires cleavage from the beads, a screening campaign, and finally the crucial step of linking the observed activity with the structure of one, or more, of the library components. A powerful approach, generally named bead based, consists of submitting the libraries to assays that allow detection of the activities of single beads, and thus evenmally to determine the structure of the individual that was loaded on the specific, active bead. Some formats for bead-based stmcture determination will be examined in detail in Sections 7.4 and 7.5, while here we will present the so-called on-bead approach. 

A broad variety of particle sizes are available, from microspheres with less than 5-pm particle size up to resins of 700-pm particle size. The large beads can be used as polymeric microreactors.P l As the separation between resin and liquid is still performed by filtration, different glass filters have to be used the porosity and nominal pore size of these filters are listed in Table 5. In those applications where single beads are used (split/mix, single-bead analysis, bead-based assays) it is essential to know the capacity of the single beads. For practical purposes a correlation between bead size and capacity per bead is reported in Table 6. 

FIGURE 7 Illumina BeadArrays. The Sentrix Array Matrix contains 96 1.4-mm fiber-optic bundles (bottom left). Each bundle is an individual array consisting of 50,000 5-pm fiber-optic strands, each of which is chemically etched to create a microwell for a single bead (top left). The Sentrix BeadChips can assay 1-16 samples at a time on a silicon slide (bottom right) that has been processed to provide microwells for individual beads (top right). Both BeadArray platforms rely on 3-pm silica beads that randomly self-assemble (center and top). 

FIGURE 8.12 The decoding process from the biological assay results that reveal the active wells from the solution plates. The beads that correspond to these wells are identified in the bead plates. The five beads in each active well are distributed as single beads and the code on each bead is acid-chemically cleaved and analyzed by mass spectrometry. Each code is determined and then identified with the appropriate compound structure. 

FIGURE 8.13 A representation of the melanophore lawn assay. A lawn of receptors is grown on a petri dish. Library beads are spread across the surface of the assay plate. The compounds interact with receptors on the cell lawn after photocieavage. Active compounds generate a color change on the cell lawn. The active beads are identified, isolated as single beads, and the code on each bead is chemically cleaved and analyzed by mass spectrometry. Each code is determined and associated with the compound s structure. 

Another technique of single-bead, direct deconvolution is based on on-bead screening. When the biological assay can be run in this format, each library component is tested as resin bound in an assay medium where a soluble receptor is present and the receptor/ligand, or inhibitor, interaction takes place at the surface of the bead. After the biological assay a few positive beads are identified, usually through colorimetric, fluorescent, or radiolabeled detection. They are removed from the assay medium and characterized analytically after washing off the receptor. This technique was first described by Lam et al. [78], has since been... 

---