The production and verification of pristine semi-fluorinated thiol monolayers on gold.

The presence of adventitious contamination of self-assembled monolayers (SAMs) is a well-known phenomenon that is often overlooked or underestimated in the literature. Herein, we demonstrate that it is possible to produce pristine self-assembled monolayers (SAMs) on gold surfaces that are devoid of adventitious species. The chemical purity or the pristine quality of the SAM was verified by the experimental relative atomic ratios measured by X-ray photoelectron spectroscopy (XPS) of all elements including carbon and corresponded to within 5% of the stoichiometric ratios.

'On-the-fly' optical encoding of combinatorial peptide libraries for profiling of protease specificity.

Large solid-phase combinatorial libraries currently play an important role in areas such as infectious disease biomarker discovery, profiling of protease specificity and anticancer drug discovery. Because compounds on solid support beads are not positionally-encoded as they are in microarrays, innovative methods of encoding are required. There are many advantages associated with optical encoding and several strategies have been described in the literature to combine fluorescence encoding methods with solid-phase library synthesis.

Antifouling surface layers for improved signal-to-noise of particle-based immunoassays.

A 10-fold improvement in the signal-to-noise (S/N) ratio of an optically encoded silica particle-based immunoassay was achieved through incorporating a protein resistant poly(ethylene glycol) (PEG) surface layer and optimizing antibody immobilization conditions. PEG was activated using 2,2,2-trifluoroethanesulfonyl chloride (tresyl) and required a minimum reaction time of 1.5 h.

Modification and optimization of organosilica microspheres for peptide synthesis and microsphere-based immunoassays.

A new generation of optically encoded organosilica microspheres, suitable for both solid phase synthesis and multiplexed microsphere-based assays, has recently been described. One of the challenges of producing this type of dual-purpose solid support is that the particles must maintain their morphology as well as their encoding during exposure to the solvents used for solid phase synthesis. In this article, organosilica microspheres are subjected to ammonia treatment methods for enhancing the condensation of the silica matrix and their subsequent resilience toward organic solvents and peptide synthesis reagents is described.

Development of a multiplexed bead-based assay for detection of DNA methylation in cancer-related genes.

Herein we report a method for the detection of methylated CpG dinucleotides located within CpG islands in genomic DNA using multiplexed bead-based assays and standard flow cytometry instrumentation. Four CpG "clusters" were identified in the TFPI2 and SPARC CpG islands whose methylation status was highly correlated with the incidence of invasive cervical cancer in our previous studies. Eight probes in total were designed for both the methylated and unmethylated forms of each cluster and attached to different fluorescently-encoded organosilica bead sets.

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.

Flow cytometric detection of proteolysis in peptide libraries synthesised on optically encoded supports.

The concept of optically encoding particles for solid phase organic synthesis has existed in the literature for several years. However, there remains a significant challenge to producing particles that are capable of withstanding harsh solvents and reagents whilst maintaining the integrity and range of the optical encoding. In this study, a new generation of fluorescently encoded support particles was used for both solid phase peptide synthesis and on-particle analysis of proteolysis in a multiplexed, flow cytometric assay.

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.

Multiplexed microsphere diagnostic tools in gene expression applications: factors and futures.

Microarrays have received significant attention in recent years as scientists have firstly identified factors that can produce reduced confidence in gene expression data obtained on these platforms, and secondly sought to establish laboratory practices and a set of standards by which data are reported with integrity. Microsphere-based assays represent a new generation of diagnostics in this field capable of providing substantial quantitative and qualitative information from gene expression profiling. However, for gene expression profiling, this type of platform is still in the demonstration phase, with issues arising from comparative studies in the literature not yet identified.

Tailoring surface properties to build colloidal diagnostic devices: controlling interparticle associations.

The ability to control the surface properties and subsequent colloidal stability of dispersed particles has widespread applicability in many fields. Sub-micrometer fluorescent silica particles (reporters) can be used to actively encode the combinatorial synthesis of peptide libraries through interparticle association. To achieve these associations, the surface chemistry of the small fluorescent silica reporters is tailored to encourage robust adhesion to large silica microparticles onto which the peptides are synthesized.

Porous functionalised silica particles: a potential platform for biomolecular screening.

Novel, porous, functionalised silica particles have been developed with controlled morphology, which promote covalent attachment of fluorescent dyes which can act as an optical barcode.

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.