We present an electrical sensor that uses rolling circle amplification (RCA) of DNA to stretch across the gap between two electrodes, interact with metal nanoparticle seeds to generate an electrically conductive nanowire, and produce electrical signals upon detection of specific target DNA sequences. RCA is a highly specific molecular detection mechanism based on DNA probe circularization. With this technique, long single-stranded DNA with simple repetitive sequences are produced. Here we show that stretched RCA products can be metalized using silver or gold solutions to form metal wires. Upon metallization, the resistance drops from TΩ to kΩ for silver and to Ω for gold. Metallization is seeded by gold nanoparticles aligned along the single-stranded DNA product through hybridization of functionalized oligonucleotides. We show that combining RCA with electrical DNA detection produces results in readout with very high signal-to-noise ratio, an essential feature for sensitive and specific detection assays. Finally, we demonstrate detection of 10 ng of Escherichia coli genomic DNA using the sensor concept.

Detection and identification of pathogens in environmental samples for biosecurity applications are challenging due to the strict requirements on specificity, sensitivity and time. We have developed a concept for quick, specific and sensitive pathogen identification in environmental samples. Target identification is realized by padlock- and proximity probing, and reacted probes are amplified by RCA (rolling-circle amplification). The individual RCA products are labeled by fluorescence and enumerated by an instrument, developed for sensitive and rapid digital analysis. The concept is demonstrated by identification of simili biowarfare agents for bacteria (Escherichia coli and Pantoea agglomerans) and spores (Bacillus atrophaeus) released in field.

The sensitive detection and quantification of DNA targets in the food industry and in environmental and clinical settings are issues of utmost importance in ensuring contamination-free food, monitoring the environment, and battling disease. Selective probes coupled with powerful amplification techniques are therefore of major interest. In this study, we set out to create an integrated microchemical chip that benefits from microfluidic chip technology in terms of sensitivity and a strong detection methodology provided jointly by padlock probes and rolling circle amplification (RCA). Here, we have integrated padlock probes and RCA into a microchip. The chip uses solid phase capture in a microchannel to enable washing cycles and decrease analytical area, and employs on-bead RCA for single-molecule amplification and detection. We investigated the effects of reagent concentration and amount of padlock probes, and demonstrated the feasibility of detecting Salmonella.

The cost of developing new drugs is a major obstacle for pharmaceutical companies and academia with many drugs identified in the drug discovery process failing approval for clinical use due to lack of intended effect or because of severe side effects. Since the early 1990s, high throughput screening of drug compounds has increased enormously in capacity but has not resulted in a higher success rate of the identified drugs. Thus, there is a need for methods that can identify biologically relevant compounds and more accurately predict in vivo effects early in the drug discovery process. To address this, we developed a proximity ligation-based assay for high content screening of drug effects on signaling pathways. As a proof of concept, we used the assay to screen through a library of previously identified kinase inhibitors, including six clinically used tyrosine kinase inhibitors, to identify compounds that inhibited the platelet-derived growth factor (PDGF) receptor beta signaling pathway in stimulated primary human fibroblasts. Thirteen of the 80 compounds were identified as hits, and the dose responses of these compounds were measured. The assay exhibited a very high Z' factor (0.71) and signal to noise ratio (11.7), demonstrating excellent ability to identify compounds interfering with the specific signaling event. A comparison with regular immunofluorescence detection of phosphorylated PDGF receptor demonstrated a far superior ability by the in situ proximity ligation assay to reveal inhibition of receptor phosphorylation. In addition, inhibitor-induced perturbation of protein-protein interactions of the PDGF signaling pathway could be quantified, further demonstrating the usefulness of the assay in drug discovery. Molecular & Cellular Proteomics 9: 178-183, 2010.

Using an amplified single molecule detection platform based on padlock probe and proximity ligation, detection of very low amounts of nucleic acid or protein molecules is achieved. Protein and nucleic acid analyses are performed in a similar fashion using circularizing DNA probes, rolling circle amplification and fluorescence-based counting of single reporter molecules. This allows a single system to be used for detection and identification of different types of biological threats, such as bacteria, bacterial spores, viruses, and toxins.

Gradients of secreted signaling proteins guide growing blood vessels during both normal and pathological angiogenesis. However, the mechanisms by which endothelial cells integrate and respond to graded distributions of chemotactic factors are still poorly understood. We have in this study investigated endothelial cell migration in response to hill-shaped gradients of vascular endothelial growth factor A (VEGFA) and fibroblast growth factor 2 (FGF2) using a novel microfluidic chemotaxis chamber (MCC). Cell migration was scored at the level of individual cells using time-lapse microscopy. A stable gradient of VEGFA165 ranging from 0 to 50 ng/ml over a distance of 400 μm was shown to strongly induce chemotaxis of endothelial cells of different vascular origin. VEGFA121, unable to bind proteoglycan and neuropilin coreceptors, was also shown to induce chemotaxis in this setup. Furthermore, a gradient of FGF2 was able to attract venular but not arterial endothelial cells, albeit less efficiently than VEGFA165. Notably, constant levels of VEGFA165, but not of FGF2, were shown to efficiently reduce chemokinesis. Systematic exploration of different gradient shapes led to the identification of a minimal gradient steepness required for efficient cell guidance. Finally, analysis of cell migration in different regions of the applied gradients showed that chemotaxis is reduced when cells reach the high end of the gradient. Our findings suggest that chemotactic growth factor gradients may instruct endothelial cells to shift toward a nonmigratory phenotype when approaching the growth factor source.

Molecular diagnostics can offer early detection of disease, improved diagnostic accuracy, and qualified follow-up. Moreover, the use of microfluidic devices can in principle render these analyses quickly and user-friendly, placing them within the reach of the general practitioner and maybe even in households. However, the progress launching such devices has been limited so far. We propose that an important limiting factor has been the difficulty of establishing molecular assays suitable for microfabricated formats. The assays should be capable of monitoring a wide range of molecules, including genomic DNA, RNA and proteins with secondary modifications and interaction partners, and they must exhibit excellent sensitivity and specificity. We discuss these problems and describe a series of molecular tools that may present new opportunities for lab-on-a-chip devices at the point-of-care.

DNA microarrays serve to monitor a wide range of molecular events, but emerging applications like measurements of weakly expressed genes or of proteins and their interaction patterns will require enhanced performance to improve specificity of detection and dynamic range. To further extend the utility of DNA microarray-based approaches we present a high-performance tag microarray procedure that enables probe-based analysis of as little as 100 target cDNA molecules, and with a linear dynamic range close to 10(5). Furthermore, the protocol radically decreases the risk of cross-hybridization on microarrays compared to current approaches, and it also allows for quantification by single-molecule analysis and real-time on-chip monitoring of rolling-circle amplification. We provide proof of concept for microarray-based measurement of both mRNA molecules and of proteins, converted to tag DNA sequences by padlock and proximity probe ligation, respectively.

We present a new random array format together with a decoding scheme for targeted multiplex digital molecular analyses. DNA samples are analyzed using multiplex sets of padlock or selector probes that create circular DNA molecules upon target recognition. The circularized DNA molecules are amplified through rolling-circle amplification (RCA) to generate amplified single molecules (ASMs). A random array is generated by immobilizing all ASMs on a microscopy glass slide. The ASMs are identified and counted through serial hybridizations of small sets of tag probes, according to a combinatorial decoding scheme. We show that random array format permits at least 10 iterations of hybridization, imaging and dehybridization, a process required for the combinatorial decoding scheme. We further investigated the quantitative dynamic range and precision of the random array format. Finally, as a demonstration, the decoding scheme was applied for multiplex quantitative analysis of genomic loci in samples having verified copy-number variations. Of 31 analyzed loci, all but one were correctly identified and responded according to the known copy-number variations. The decoding strategy is generic in that the target can be any biomolecule which has been encoded into a DNA circle via a molecular probing reaction.

We recently presented a method that enables single-molecule enumeration by transforming specific molecular recognition events at nanometer dimensions to micrometer-sized DNA macromolecules. This transformation process is mediated by target-specific padlock probe ligation, followed by rolling circle amplification (RCA), resulting in the creation of one rolling circle product (RCP) for each recognized target. The transformation makes optical detection and quantification possible using standard fluorescence microscopy by counting the number of generated RCPs in a sample pumped through a microfluidic channel. In this study, we demonstrate that confocal volume definition is crucial to achieve high-precision measurements in the microfluidic quantification (coefficient of variance typically 3%). We further demonstrate that complementary sequence motifs between RCPs is only a weak inducer of aggregates and that all detection sites of the RCPs are occupied at detection oligonucleotide concentrations greater than 5nM if hybridized in the proper buffer conditions. Therefore, the signal/noise ratio is limited by the number of detection sites. By increasing the density of detection sites in the RCP by a factor of 1.9, we show that the optical signal/noise level can be increased from 42 to 75.

Knowledge about the total human genome sequence now provides opportunities to study its myriad gene products. However, the presence of alternative splicing, post-translational modifications, and innumerable protein-protein interactions among proteins occurring at widely different concentrations, all combine to place extreme demands on the specificity and sensitivity of assays. The choice of method also depends on matters such as whether proteins will be analyzed in body fluids and lysates, or localized inside single cells. In this review we discuss commonly used detection methods and compare these to the recently-developed proximity ligation technique.

We describe a scheme for biomolecule enumeration by converting nanometer-scale specific molecular recognition events mediated by rolling-circle amplification to fluorescent micrometer-sized DNA molecules amenable to discrete optical detection. Our amplified single-molecule detection (SMD) approach preserves the discrete nature of the molecular population, allowing multiplex detection and highly precise quantification of molecules over a dynamic range of seven orders of magnitude. We apply the method for sensitive detection and quantification of the bacterial pathogen Vibrio cholerae .

Cellular processes can only be understood as the dynamic interplay of molecules. There is a need for techniques to monitor interactions of endogenous proteins directly in individual cells and tissues to reveal the cellular and molecular architecture and its responses to perturbations. Here we report our adaptation of the recently developed proximity ligation method to examine the subcellular localization of protein-protein interactions at single-molecule resolution. Proximity probes—oligonucleotides attached to antibodies against the two target proteins—guided the formation of circular DNA strands when bound in close proximity. The DNA circles in turn served as templates for localized rolling-circle amplification (RCA), allowing individual interacting pairs of protein molecules to be visualized and counted in human cell lines and clinical specimens. We used this method to show specific regulation of protein-protein interactions between endogenous Myc and Max oncogenic transcription factors in response to interferon-γ (IFN-γ) signaling and low-molecular-weight inhibitors.