Fluorescent vesicles for signal amplification in reverse phase protein microarray assays.

Developments in microarray technology promise to lead to great advancements in the biomedical and biological field. However, implementation of these analytical tools often relies on signal amplification strategies that are essential to reach the sensitivity levels required for a variety of biological applications. This is true especially for reverse phase arrays where a complex biological sample is directly immobilized on the chip. We present a simple and generic method for signal amplification based on the use of antibody-tagged fluorescent vesicles as labels for signal generation. To assess the gain in assay sensitivity, we performed a model assay for the detection of rabbit immunoglobulin G (IgG) and compared the limit of detection (LOD) of the vesicle assay with the LOD of a conventional assay performed with fluorescent reporter molecules. We evaluated the improvements for two fluorescence-based transduction setups: a high-sensitivity microarray reader (ZeptoREADER) and a conventional confocal scanner. In all cases, our strategy led to an increase in sensitivity. However, gain in sensitivity widely depended on the type of illumination; whereas an approximately 2-fold increase in sensitivity was observed for readout based on evanescent field illumination, the contribution was as high as more than 200-fold for confocal scanning.

Protein microarrays based on polymer brushes prepared via surface-initiated atom transfer radical polymerization.

Polymer brushes represent an interesting platform for the development of high-capacity protein binding surfaces. Whereas the protein binding properties of polymer brushes have been investigated before, this manuscript evaluates the feasibility of poly(glycidyl methacrylate) (PGMA) and PGMA-co-poly(2-(diethylamino)ethyl methacrylate) (PGMA-co-PDEAEMA) (co)polymer brushes grown via surface-initiated atom transfer radical polymerization (SI-ATRP) as protein reactive substrates in a commercially available microarray system using tantalum-pentoxide-coated optical waveguide-based chips. The performance of the polymer-brush-based protein microarray chips is assessed using commercially available dodecylphosphate (DDP)-modified chips as the benchmark. In contrast to the 2D planar, DDP-coated chips, the polymer-brush-covered chips represent a 3D sampling volume. This was reflected in the results of protein immobilization studies, which indicated that the polymer-brush-based coatings had a higher protein binding capacity as compared to the reference substrates. The protein binding capacity of the polymer-brush-based coatings was found to increase with increasing brush thickness and could also be enhanced by copolymerization of 2-(diethylamino)ethyl methacrylate (DEAEMA), which catalyzes epoxide ring-opening of the glycidyl methacrylate (GMA) units. The performance of the polymer-brush-based microarray chips was evaluated in two proof-of-concept microarray experiments, which involved the detection of biotin-streptavidin binding as well as a model TNFα reverse assay. These experiments revealed that the use of polymer-brush-modified microarray chips resulted not only in the highest absolute fluorescence readouts, reflecting the 3D nature and enhanced sampling volume provided by the brush coating, but also in significantly enhanced signal-to-noise ratios. These characteristics make the proposed polymer brushes an attractive alternative to commercially available, 2D microarray surface coatings.

Reverse protein arrays as novel approach for protein quantification in muscular dystrophies.

The definite molecular diagnosis in patients with muscular dystrophies often requires the assessment of muscular expression of multiple proteins in small amounts of muscle tissue. The sample material obtained in muscle biopsies is limited and the measurement of multiple proteins is often restricted to conventional, non-quantitative assays, i.e. immunohistochemistry and immunoblotting. Here, we demonstrate that reverse protein arrays are a novel and excellent material-saving method for the measurement and quantification of changes in protein expression between healthy and diseased muscle tissue as well as cultured primary myotubes. We evaluated a set of antibodies and found reproducible differences between Duchenne muscular dystrophy/limb-girdle muscular dystrophy patients and control samples for dystrophin, the sarcoglycans and the dystroglycans. As little as 10 mg of tissue is sufficient for the analysis of all diagnostically relevant proteins. The average coefficient of variation calculated for the sample signals confirmed that the method is highly reproducible. Thus, our experiments provide strong evidence that quantitative protein detection from very small amounts of muscle tissue is possible using reverse protein arrays. This technology may not only be of interest for diagnostic purposes, but also for protein quantification of multiple, follow-up biopsies during clinical trials when protein expression in muscle is considered an important outcome measure or biomarker.

Antibody-based proteomics: analysis of signaling networks using reverse protein arrays.

Protein kinases drive the cellular signal transduction networks that underlie the regulation of growth, survival and differentiation. To repair the deregulations of signaling cascades that are associated with numerous disease states, therapeutic strategies, based on controlling aberrant protein kinase activity, are emerging. To develop such therapies it is crucial to have knowledge of the full complexity of signaling networks at a molecular level in order to understand the information flow through signaling cascades and their cell and tissue specificity. Antibody-based proteomic approaches (such as reverse-phase protein microarrays) are a powerful tool for using to obtain those signaling maps, through the study of phosphorylation states of pathway components using antibodies that specifically recognize the phosphorylated form of kinase substrates.

Tracing pathway activities with kinase inhibitors and reverse phase protein arrays.

Controlling aberrant protein kinase activity is a promising strategy for a variety of diseases, particularly cancer. Hence, the development of kinase inhibitors is currently a focal point for pharmaceutical research. In this study we utilize a chip-based reverse phase protein array (RPA) platform for profiling of kinase inhibitors in cell-based assays. In combination with the planar wave-guide technology the assay system has an absolute LOD down to the low zeptomole range. A431 cell lysates were analyzed for the activation state of key effectors in the epidermal growth factor (EGF) and insulin signaling pathways to validate this model for compound screening. A microtiter-plate format for growing, treating, and lysing cells was shown to be suitable for this approach, establishing the value of the technology as a screening tool for characterization of large numbers of kinase inhibitors against a wide variety of cellular signaling pathways. Moreover, the reverse array format allows rapid development of site-specific phosphorylation assays, since in contrast to ELISA type systems only a single antigen-specific antibody is required.

Rapid bacterial identification using evanescent-waveguide oligonucleotide microarray classification.

Bacterial identification relies primarily on culture-based methodologies and requires 48-72 h to deliver results. We developed and used i) a bioinformatics strategy to select oligonucleotide signature probes, ii) a rapid procedure for RNA labelling and hybridization, iii) an evanescent-waveguide oligoarray with exquisite signal/noise performance, and iv) informatics methods for microarray data analysis. Unique 19-mer signature oligonucleotides were selected in the 5'-end of 16s rDNA genes of human pathogenic bacteria. Oligonucleotides spotted onto a Ta(2)O(5)-coated microarray surface were incubated with chemically labelled total bacterial RNA. Rapid hybridization and stringent washings were performed before scanning and analyzing the slide. In the present paper, the eight most abundant bacterial pathogens representing >54% of positive blood cultures were selected. Hierarchical clustering analysis of hybridization data revealed characteristic patterns, even for closely related species. We then evaluated artificial intelligence-based approaches that outperformed conventional threshold-based identification schemes on cognate probes. At this stage, the complete procedure applied to spiked blood cultures was completed in less than 6 h. In conclusion, when coupled to optimal signal detection strategy, microarrays provide bacterial identification within a few hours post-sampling, allowing targeted antimicrobial prescription.

Tuned graft copolymers as controlled coatings for DNA microarrays.

DNA microarrays have become a powerful tool for expression profiling and other genomics applications. A critical factor for their sensitivity is the interfacial coating between the chip substrate and the bound DNA. Such a coating has to embrace the divergent requirements of tightly binding the capture probe DNA during the spotting process and of minimizing the nonspecific binding of target DNA during the hybridization assay. To fulfill these conditions, most coatings require a passivation step. Here we demonstrate how the chain density of a graft copolymer with a polycationic backbone, poly(l-lysine)-graft-poly(ethylene glycol), can be tuned such that the binding capacity during capture probe deposition is maximized while the nonspecific binding during hybridization assays is kept to a minimum, thus alleviating the requirement for a separate passivation procedure. Evidence for the superior performance of such coatings in terms of signal-to-noise ratio and spot quality is presented using an evanescent field-based fluorescent sensing technique (the ZeptoREADER). The surface architecture is further characterized using optical waveguide lightmode spectroscopy and time-of-flight secondary ion mass spectrometry. Finally, in a model assay, we demonstrate that expression changes can be detected from 1 microg of total mRNA sample material with a limit of detectable differential expression of +/-1.5.

Zeptosens' protein microarrays: a novel high performance microarray platform for low abundance protein analysis.

Protein microarrays are considered an enabling technology, which will significantly expand the scope of current protein expression and protein interaction analysis. Current technologies, such as two-dimensional gel electrophoresis (2-DE) in combination with mass spectrometry, allowing the identification of biologically relevant proteins, have a high resolving power, but also considerable limitations. As was demonstrated by Gygi et al. (Proc. Nat. Acad. Sci. USA 2000,97, 9390-9395), most spots in 2-DE, observed from whole cell extracts, are from high abundance proteins, whereas low abundance proteins, such as signaling molecules or kinases, are only poorly represented. Protein microarrays are expected to significantly expedite the discovery of new markers and targets of pharmaceutical interest, and to have the potential for high-throughput applications. Key factors to reach this goal are: high read-out sensitivity for quantification also of low abundance proteins, functional analysis of proteins, short assay analysis times, ease of handling and the ability to integrate a variety of different targets and new assays. Zeptosens has developed a revolutionary new bioanalytical system based on the proprietary planar waveguide technology which allows us to perform multiplexed, quantitative biomolecular interaction analysis with highest sensitivity in a microarray format upon utilizing the specific advantages of the evanescent field fluorescence detection. The analytical system, comprising an ultrasensitive fluorescence reader and microarray chips with integrated microfluidics, enables the user to generate a multitude of high fidelity data in applications such as protein expression profiling or investigating protein-protein interactions. In this paper, the important factors for developing high performance protein microarray systems, especially for targeting low abundant messengers of relevant biological information, will be discussed and the performance of the system will be demonstrated in experimental examples.

Immunoaffinity screening with capillary electrochromatography.

Highly efficient capillary electrochromatographic separations of cardiac glycosides and other steroids are presented. Employing butyl-derivatized silica particles as stationary phase resulted in a nearly three times faster electroosmotic flow (EOF) compared to capillary electrochromatography (CEC) with octadecyl silica particles. On-column focusing with a preconcentration factor of 180 was performed and separation efficiencies of up to 240,000 plates per meter were obtained. Using label-free standard UV absorbance, detection limits of 10-80 nM were reached for all steroids tested. For screening of cardiac glycosides, e.g., digoxin and digitoxin in mixtures of steroids, CEC was combined with immunoaffinity extraction using immobilized polyclonal anti-digoxigenin antibodies and F(ab) fragments. Simply adding small amounts of antibody carrying particles to the samples and comparing chromatograms before and after antibody addition allowed screening for high affinity antigens in mixtures with moderate numbers of compounds. Under conditions of competing antigens, affinity fingerprints of immobilized anti-digoxigenin and anti-digitoxin antibodies were obtained, reflecting the cross-reactivity of eleven steroids. The method provides high selectivity due to the combination of bioaffinity interaction with highly efficient CEC separation and UV detection at several wavelengths in parallel. This selectivity was exploited for the detection of four cardiac glycosides in submicromolar concentrations in an untreated urine sample.