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- Jarvius, Jonas, et al.
(author)
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Digital Quantification using Amplified Single-Molecule Detection
- 2006
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In: Nature Methods. - 1548-7091 .- 1548-7105. ; 3:9, s. 725-727
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Journal article (peer-reviewed)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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| 5. |
- Melin, Jonas, et al.
(author)
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Homogeneous amplified single-molecule detection : Characterization of key parameters
- 2007
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In: Analytical Biochemistry. - : Elsevier BV. - 0003-2697 .- 1096-0309. ; 368:2, s. 230-238
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Journal article (peer-reviewed)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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| 7. |
- Melin, Jonas, et al.
(author)
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Ligation-based molecular tools for lab-on-a-chip devices
- 2008
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In: New Biotechnology. - : Elsevier BV. - 1871-6784. ; 25:1, s. 42-8
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Journal article (peer-reviewed)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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| 9. |
- Melin, Jonas, et al.
(author)
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Thermoplastic microfluidic platform for single-molecule detection, cell culture and actuation
- 2005
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In: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 77:22, s. 7122-30
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Journal article (peer-reviewed)abstract
- We have developed a multipurpose microfluidic platform that allows for sensitive fluorescence detection on inexpensive disposable chips. The fabrication scheme involves rapid injection molding of thermoplastics, followed by silica deposition and covalent attachment of an unstructured flexible lid. This combines the virtues of elastomer technology with high-throughput compact disk injection molding. Using this technique, the time to produce 100 chips using a single master can be lowered from more than 1 week by standard PDMS technologies to only a couple hours. The optical properties of the fabricated chips were evaluated by studying individual fluorescence-labeled DNA molecules in a microchannel. Concatemeric DNA molecules were generated through rolling circle replication of circular DNA molecules, which were labeled by hybridization of fluorescence-tagged oligonucleotides. Rolling circle products (RCPs) were detected after as little as 5 min of DNA polymerization, and the RCPs in solution showed no tendency for aggregation. To illustrate the versatility of the platform, we demonstrate two additional applications: The flexible property of the lid was used to create a peristaltic pump generating a flow rate of 9 nL/s. Biocompatibility of the platform was illustrated by culturing Chinese hamster ovary cells for 7 days in the microfluidic channels.
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| 10. |
- Pinidiyaarachchi, Amalka, et al.
(author)
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Digital image processing for multiplexing of single molecule detection
- 2005
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In: Medicinteknikdagarna.
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Conference paper (other academic/artistic)abstract
- Using padlock and proximity probing techniques, individual molecular identification events are converted to long DNA molecules, carrying repeated sequence motifs used for identification of the detected molecules. We show that identification events can be amplified using rolling circle replication, and randomly attached to a surface for repeated access by identification probes. Repeated hybridization with detection probes carrying fluorescing nano-crystals (quantum dots) of varying spectral properties opens the possibility to search for large numbers of different identification events simultaneously. Methods for digital image processing of the resulting multi-spectral data include spatial as well as spectral data clustering. Spatial data processing includes registration of images from repeated hybridization events as well as delineation of clustered reporter events. Spectral data processing and analysis includes classification of spectral data into groups of either pre-defined or unknown patterns representing different molecular identification events.
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