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- Jarvius, Jonas, et al.
(författare)
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Digital Quantification using Amplified Single-Molecule Detection
- 2006
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Ingår i: Nature Methods. - 1548-7091 .- 1548-7105. ; 3:9, s. 725-727
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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- Leuchowius, Karl-Johan, et al.
(författare)
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High content screening for inhibitors of protein interactions and post-translational modifications in primary cells by proximity ligation
- 2010
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Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 9:1, s. 178-183
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Tidskriftsartikel (refereegranskat)abstract
- 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 1990 s, 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.
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- Melin, Jonas, et al.
(författare)
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Homogeneous amplified single-molecule detection : Characterization of key parameters
- 2007
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 368:2, s. 230-238
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Tidskriftsartikel (refereegranskat)abstract
- 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 5 nM 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.
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- Melin, Jonas, et al.
(författare)
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Ligation-based molecular tools for lab-on-a-chip devices
- 2008
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Ingår i: New Biotechnology. - : Elsevier BV. - 1871-6784. ; 25:1, s. 42-8
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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