Bronwyn J. Battersby

Novel miniaturized systems in high-throughput screening.

High-throughput screening (HTS) using high-density microplates is the primary method for the discovery of novel lead candidate molecules. However, new strategies that eschew 2D microplate technology, including technologies that enable mass screening of targets against large combinatorial libraries, have the potential to greatly increase throughput and decrease unit cost. This review presents an overview of state-of-the-art microplate-based HTS technology and includes a discussion of emerging miniaturized systems for HTS. We focus on new methods of encoding combinatorial libraries that promise throughputs of as many as 100,000 compounds per second.

Optical barcoding of colloidal suspensions: applications in genomics, proteomics and drug discovery

The enormous amount of information generated through sequencing of the human genome has increased demands for more economical and flexible alternatives in genomics, proteomics and drug discovery. Many companies and institutions have recognised the potential of increasing the size and complexity of chemical libraries by producing large chemical libraries on colloidal support beads. Since colloid-based compounds in a suspension are randomly located, an encoding system such as optical barcoding is required to permit rapid elucidation of the compound structures. We describe in this article innovative methods for optical barcoding of colloids for use as support beads in both combinatorial and non-combinatorial libraries. We focus in particular on the difficult problem of barcoding extremely large libraries, which if solved, will transform the manner in which genomics, proteomics and drug discovery research is currently performed.

Optical encoding of microbeads for gene screening: alternatives to microarrays

Abstract Rapid access to genetic information is central to the revolution presently occurring in the pharmaceutical industry, particularly in relation to novel drug target identification and drug development. Genetic variation, gene expression, gene function and gene structure are just some of the important research areas requiring efficient methods of DNA screening. Here, we highlight state-of-the-art techniques and devices for gene screening that promise cheaper and higher-throughput yields than currently achieved with DNA microarrays. We include an overview of existing and proposed bead-based strategies designed to dramatically increase the number of probes that can be interrogated in one assay. We focus, in particular, on the issue of encoding and/or decoding (bar-coding) large bead-based libraries for HTS.

Synthesis and Application of FRET Nanoparticles in the Profiling of a Protease

A novel colloidal bar coding method utilizing nanoscale fluorescence resonance energy transfer (FRET) reporters decoded by confocal laser scanning microscopy combined with spectral unmixing, was reported. Succinimidyl ester dyes Atto-488, Atto-550, and Atto-590 were covalently linked to a silane coupling agent (3-aminopropyl)-trimethoxysilane, forming an APS-dye conjugate. The three dyes were incorporated into the reporters at five molar ratios. The reporters were loaded on 10μm amine functionalized polystyrene microspheres in eleven different combinations of between three and seven codes to evaluate the utility of the reporters to form a colloidal barcode. Following characterization, the reporters were used to encode a combinatorial peptide library that was applied to profiling the affinity of the West Nile flavivirus protease (WNV-pro). Varying the ratios of energy transfer dyes served as an innovative route to produce unique spectral signatures.

A structural study of hybrid organosilica materials for colloid-based DNA biosensors

Organosilane hybrid materials are of interest in the development of diagnostic devices and drug-delivery applications. Here we report a spectroscopic study involving the chemical and structural modification of thiol-functionalised organosilica particles with aminosilane to produce a bifunctional silica hybrid. The aminosilane was revealed to be distributed throughout the microsphere as opposed to being surface-localised as is commonly reported for modifications of pure silica. Spectroscopic methods including NMR, XPS, Ninhydrin and gravimetric measurements were employed to investigate the surface and internal elemental composition of the particles independently. A multiplexed model bioassay is presented to demonstrate the advantage of organosilane bifunctionality, enabling separate covalent attachment strategies for both homogeneous incorporation of fluorescent dyes and surface-specific biomolecule attachment. This study represents an advance in the understanding of organosilane chemistry resulting in versatile materials with a range of functionalities for covalent attachment.

A dual-purpose synthetic colloidal platform for protease mapping: substrate profiling for Dengue and West Nile virus proteases.

In a proof of concept study, we created a small focused fluorescent hexapeptide library onto 14 multiplexed barcoded sets of silica particles to probe the substrate recognition specificity of West Nile and Dengue virus proteases. A flow cytometric analysis demonstrated that the optical signature of each bead population remained distinguishable throughout the solid-phase peptide synthesis and proteolytic assay. As expected, both proteases displayed a narrow specificity for lysine and arginine residues in the P(1) and P(2) substrate positions. This open-ended platform enables the fast and simultaneous identification of peptide substrates and is applicable to other proteases.

Optically encoded particles and their applications in multiplexed biomedical assays

In the future, the rapid discovery of new cures, vaccines, and diagnostics for common diseases will depend on the ability of biomedical researchers to investigate complex mixtures of proteins or DNA. The need to measure the abundance of these entities, together with their level of interaction, has driven the development of new research tools that enable simultaneous analysis of multiple analytes (multiplexing). Optically encoded particles are emerging as the multiplexing tools of choice, especially for clinical research. In this Review, an overview of various new optical encoding methods will be presented, together with important biomedical applications in which particle-based assays are currently being used.

Dimyristoyl phosphatidylcholine: equilibrium spreading behaviour

Abstract The equilibrium spreading behaviour of dimyristoyl phophatidylcholine has been studied at the air—water interface. The lipid was examined both as a dispersion (prepared by vortexing) and as the bulk solid (dropped onto the surface of water). The physical properties of the lipid in the aqueous media were examined by optical microscopy and cryo transmission electron microscopy. It was found that the nature of the sample was a key factor in the formation of a monolayer, spreading being dependent on the hydration of the bulk lipid and the formation of bilayers in the subphase. A model of bilayer formation is used to relate discrepancies in previously reported values of the equilibrium spreading pressure to a shift in the chain melting transition temperature Tm.

Particle-by-particle quantification of protein adsorption onto poly(ethylene glycol) grafted surfaces

The use and advantage of flow cytometry as a particle-by-particle, low sampling volume, high-throughput screening technique for quantitatively examining the non-specific adsorption of proteins onto surfaces is presented. The adsorption of three proteins: bovine serum albumin (BSA), immunoglobulin gamma (IgG) and protein G, incubated at room temperature for 2 h onto organosilica particles modified with poly(ethylene glycol) (PEG) of increasing MW (2000, 3400, 6000, 10,000 and 20,000 g mol−1) and grafted amounts (0.14–1.4 mg m−2) was investigated as a model system. Each protein exhibited Langmuir-like, high affinity monolayer limited adsorption on unmodified particles with the proteins reaching surface saturation at 1.8, 4.0 and 2.5 mg m−2 for BSA, IgG and protein G, respectively. Protein adsorption on PEG-modified surfaces was found to decrease with increasing amounts of grafted polymer. PEG grafting amounts >0.6 mg m−2 effectively prevented the adsorption of the larger two proteins (BSA and IgG) while a PEG grafting amount >1.3 mg m−2 was required to prevent the adsorption of the smaller protein G.

Vesicle formation in aqueous dimyristoyl phosphatidylcholine suspensions: a cryo-electron microscopy study

The swelling of phospholipid in water above the chain-melting transition temperature produces lamellar structures in various shapes and sizes. These structures are potentially useful as drug, gene or cosmetic delivery systems. Cryo-electron microscopy has been used to demonstrate that the morphology of the structures in aqueous dimyristoyl phosphatidylcholine suspensions is affected by the degree of agitation. Long, undulating bilayer tubes are created, as hydrating phospholipid crystals flow through water. The proportion of unilamellar bilayer tubes to multilamellar tubes can be manipulated by varying the degree of agitation. Evidence is presented to show that agitation can transform multilamellar bilayer structures into unilamellar structures. A mechanism for the formation of unilamellar and multilamellar vesicles from these structures is discussed