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. Perfluoro-octyl-thiolate (F8) was used as a model compound in this study, where monolayers were assembled from solutions of an acetylated F8 precursor. Quantitative elemental characterization of the acetylated F8 precursor by cold-stage XPS provided valuable reference data for the analysis of the subsequent SAMs. Comprehensive analysis of high-resolution XPS C 1s spectra proved to be essential for establishing the purity of the SAMs, since the peaks of the adventitious species were easily distinguished from those of the F8. Analyses of deliberately contaminated F8 SAMs showed that the adventitious species persisted during the process of self-assembly and therefore co-existed with the SAM in the interfacial region. The work also established that even a lengthy deposition time of 18 h was incapable of displacing the adventitious species present at the interface.

'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. We have previously introduced an alternative fluorescence-based encoding method ("colloidal barcoding"), which involves encoding 10-20 mum support beads during a split-and-mix synthesis with smaller 0.6-0.8 mum silica colloids that contain specific and identifiable combinations of fluorescent dye. The power of this 'on-the-fly' encoding approach lies in the efficient use of a small number of fluorescent dyes to encode millions of compounds. Described herein, for the first time, is the use of a colloid-barcoded library in a biological assay (i.e., protease profiling) combined with the use of confocal microscopy to decode the colloidal barcode. In this proof-of-concept demonstration, a small focussed peptide library was optically-encoded during a combinatorial synthesis, incubated with a protease (trypsin), analysed by flow cytometry and decoded via confocal microscopy. During assay development, a range of parameters were investigated and optimised, including substrate (or probe) loading, barcode stability, characteristics of the peptide-tagging fluorophore, and spacer group configuration. Through successful decoding of the colloidal barcodes, it was confirmed that specific peptide sequences presenting one or two cleavage sites were recognised by trypsin while peptide sequences not cleavable by trypsin remained intact.

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. The success of the assay was demonstrated through the use of a model protease, trypsin. Our results show that the use of solid supports with high peptide yield, high swellability in water and high penetration of the enzyme into the interior of the particle is not absolutely necessary for proteolysis assays.

Functional molecular wires.

The properties of self-assembled molecules may be tuned by sequentially coupling components on a gold surface, the molecular electronics toolbox of chemically reactive building blocks yielding molecular wires with diode-like current-voltage (I-V) characteristics. The bias for rectification in each case is dependent upon the sequence of electron-donating and electron-accepting moieties and similar behaviour has been achieved for four different contacting techniques.

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. It is desirable to identify potential parameters that are established as important in controlling the information derived from microsphere-based hybridizations to quantify gene expression. As these evolve, a standard set of parameters will be established that are required to be provided when data are submitted for publication. Here we initiate this process by identifying a number of parameters we have found to be important in microsphere-based assays designed for the quantification of low abundant genes which are variable between studies.