Improving the dielectric properties of ethylene-glycol alkanethiol self-assembled monolayers.

Self-assembled monolayers (SAMs) can be formed at the interface between solids and fluids, and are often used to modify the surface properties of the solid. One of the most widely employed SAM systems is exploiting thiol-gold chemistry, which, together with alkane-chain-based molecules, provides a reliable way of SAM formation to modify the surface properties of electrodes. Oligo ethylene-glycol (OEG) terminated alkanethiol monolayers have shown excellent antifouling properties and have been used extensively for the coating of biosensor electrodes to minimize nonspecific binding. Here, we report the investigation of the dielectric properties of COOH-capped OEG monolayers and demonstrate a strategy to improve the dielectric properties significantly by mixing the OEG SAM with small concentrations of 11-mercaptoundecanol (MUD). The monolayer properties and composition were characterized by means of impedance spectroscopy, water contact angle, ellipsometry and X-ray photoelectron spectroscopy. An equivalent circuit model is proposed to interpret the EIS data and to determine the conductivity of the monolayer. We find that for increasing MUD concentrations up to about 5% the resistivity of the SAM steadily increases, which together with a considerable decrease of the phase of the impedance, demonstrates significantly improved dielectric properties of the monolayer. Such monolayers will find widespread use in applications which depend critically on good dielectric properties such as capacitive biosensor.

Highly specific label-free protein detection from lysed cells using internally referenced microcantilever sensors.

We report the investigation of label-free protein detection directly from lysed cells using microcantilever sensors. The integration of an internally referenced microcantilever sensor combined with peptide aptamer technology enables scalable and label-free detection of proteins from a complex biological environment (e.g. cell lysate). The internally referenced microcantilever sensor was found to be effective in minimizing both the effects of thermal drift and non-specific binding interactions with the backside of the cantilever, thereby allowing protein detection in a complex biological background. Highly specific peptide aptamers are used to modify the cantilever surface to specifically detect less than 80 nM CDK2 protein from yeast cell lysate. This binding of CDK2 on the microcantilever generates a tensile surface stress of average magnitude equal to 70+/-22 mN/m. Similar experiments conducted with quartz crystal microbalance (QCM) technology are consistent with the response observed using microcantilever sensors.

Electrical protein detection in cell lysates using high-density peptide-aptamer microarrays.

BACKGROUND: The dissection of biological pathways and of the molecular basis of disease requires devices to analyze simultaneously a staggering number of protein isoforms in a given cell under given conditions. Such devices face significant challenges, including the identification of probe molecules specific for each protein isoform, protein immobilization techniques with micrometer or submicrometer resolution, and the development of a sensing mechanism capable of very high-density, highly multiplexed detection. RESULTS: We present a novel strategy that offers practical solutions to these challenges, featuring peptide aptamers as artificial protein detectors arrayed on gold electrodes with feature sizes one order of magnitude smaller than existing formats. We describe a method to immobilize specific peptide aptamers on individual electrodes at the micrometer scale, together with a robust and label-free electronic sensing system. As a proving proof of principle experiment, we demonstrate the specific recognition of cyclin-dependent protein kinases in whole-cell lysates using arrays of ten electrodes functionalized with individual peptide aptamers, with no measurable cross-talk between electrodes. The sensitivity is within the clinically relevant range and can detect proteins against the high, whole-cell lysate background. CONCLUSION: The use of peptide aptamers selected in vivo to recognize specific protein isoforms, the ability to functionalize each microelectrode individually, the electronic nature and scalability of the label-free detection and the scalability of the array fabrication combine to yield the potential for highly multiplexed devices with increasingly small detection areas and higher sensitivities that may ultimately allow the simultaneous monitoring of tens or hundreds of thousands of protein isoforms.

Surface-immobilized peptide aptamers as probe molecules for protein detection.

We demonstrate the use of surface-immobilized, oriented peptide aptamers for the detection of specific target proteins from complex biological solutions. These peptide aptamers are target-specific peptides expressed within a protein scaffold engineered from the human protease inhibitor stefin A. The scaffold provides stability to the inserted peptides and increases their binding affinity owing to the resulting three-dimensional constraints. A unique cysteine residue was introduced into the protein scaffold to allow orientation-specific surface immobilization of the peptide aptamer and to ensure exposure of the binding site to the target solution. Using dual-polarization interferometry, we demonstrate a strong relationship between binding affinity and aptamer orientation and determine the affinity constant KD for the interaction between an oriented peptide aptamer ST(cys+)_(pep9) and the target protein CDK2. Further, we demonstrate the high selectivity of the peptide aptamer STM_(pep9) by exposing surface-immobilized ST(cys+)_(pep9) to a complex biological solution containing small concentrations of the target protein CDK2.

Peptide aptamers in label-free protein detection: 1. Characterization of the immobilized scaffold.

Protein microarray development is absolutely dependent upon the ability to construct interfaces capable of specific, stable, sensitive, and designable recognition of specific proteins. Peptide aptamers, being peptide recognition moieties presented and constrained by a robust scaffold protein, offer one possible solution. The relative uniformity of a scaffold protein across potentially many thousands of arrayed peptide aptamers is predicted to simplify the production of microarrays. This paper describes the generation and assaying characteristics of a scaffold protein adlayer. Orientational control of the scaffold protein STM, a triply mutated form of the stable intracellular protein inhibitor stefin A is achieved with a surface cysteine residue, which leads to the presentation of the scaffold recognition surface to solution. Operational stability of the system is excellent, with only a minor decrease in detection sensitivity over time (less than 1% h-1). We use this system to establish a surface plasmon resonance assay offering a limit of detection of 1 nM (150 ng mL-1) and determine the affinity constant of interaction of STM for a cognate antibody to be KD = 1.47 +/- 0.23 nM. Thus, we have established a solid foundation for the future creation of highly multiplexed peptide aptamer microarrays that will be compatible with a broad range of label-free detection technologies.

Whole organism approaches to chemical genomics: the promising role of zebrafish (Danio rerio).

Chemical genomics is a new and rapidly developing field. It refers to the use of cell-permeable small molecules, which are highly specific for their protein targets, in order to dissect biological pathways and to discover new drug leads. Small-molecule screening is usually limited to high-throughput approaches that use defined cell lines; however, whole organism screening is gaining increasing attention. This review addresses the latter concept and highlights the advances in whole organism-based screening, with an emphasis on the use of the zebrafish (Danio rerio).

Design and validation of a neutral protein scaffold for the presentation of peptide aptamers.

Peptide aptamers are peptides constrained and presented by a scaffold protein that are used to study protein function in cells. They are able to disrupt protein-protein interactions and to constitute recognition modules that allow the creation of a molecular toolkit for the intracellular analysis of protein function. The success of peptide aptamer technology is critically dependent on the performance of the scaffold. Here, we describe a rational approach to the design of a new peptide aptamer scaffold. We outline the qualities that an ideal scaffold would need to possess to be broadly useful for in vitro and in vivo studies and apply these criteria to the design of a new scaffold, called STM. Starting from the small, stable intracellular protease inhibitor stefin A, we have engineered a biologically neutral scaffold that retains the stable conformation of the parent protein. We show that STM is able to present peptides that bind to targets of interest, both in the context of known interactors and in library screens. Molecular tools based on our scaffold are likely to be used in a wide range of studies of biological pathways, and in the validation of drug targets.

Chemical discovery and global gene expression analysis in zebrafish.

The zebrafish (Danio rerio) provides an excellent model for studying vertebrate development and human disease because of its ex utero, optically transparent embryogenesis and amenability to in vivo manipulation. The rapid embryonic developmental cycle, large clutch sizes and ease of maintenance at large numbers also add to the appeal of this species. Considerable genomic data has recently become publicly available that is aiding the construction of zebrafish microarrays, thus permitting global gene expression analysis. The zebrafish is also suitable for chemical genomics, in part as a result of the permeability of its embryos to small molecules and consequent avoidance of external confounding maternal effects. Finally, there is increasing characterization and analysis of zebrafish models of human disease. Thus, the zebrafish offers a high-quality, high-throughput bioassay tool for determining the biological effect of small molecules as well as for dissecting biological pathways.