| 1. |
- Fritzsche, Joachim, 1977, et al.
(författare)
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Single Particle Nanoplasmonic Sensing in Individual Nanofluidic Channels
- 2016
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Ingår i: Nano Letters. - : American Chemical Society (ACS). - 1530-6992 .- 1530-6984. ; 16:12, s. 7857-7864
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Tidskriftsartikel (refereegranskat)abstract
- Nanoplasmonics allows label-free optical sensing and spectroscopy at the single nanoparticle level by exploiting plasmonic excitations in metal nanoparticles. Nanofluidics offers exclusive possibilities for applying and controlling fluid flow and mass transport at the nanoscale and toward nanosized objects. Here, we combine these two concepts in a single device, by integrating single particle nanoplasmonic sensing with nanofluidics using advanced nanofabrication. The developed devices enable on-chip referenced parallel single particle nanoplasmonic sensing inside multiple individual nanofluidic channels with dimensions down to the 100 nm range. Beyond detailed discussion of the nanofabrication, general device characterization, and parallelized single particle plasmonic readout concepts, we demonstrate device function on two examples: (i) in situ measurements of local buffer concentrations inside a nanofluidic channel; (ii) real time binding kinetics of alkanethiol molecules to a single plasmonic nanonatenna sensor in a single nanochannel. Our concept thus provides a powerful solution for controlling mass transport to and from individual (plasmonic) nanoparticles, which in a long-term perspective offers unique opportunities for label-free detection of analyte molecules at low concentrations and for fundamental studies of fluids in extreme confinement.
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| 2. |
- Fornander, Louise, 1984, et al.
(författare)
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Visualizing the Nonhomogeneous Structure of RAD51 Filaments Using Nanofluidic Channels
- 2016
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Ingår i: Langmuir. - : American Chemical Society (ACS). - 0743-7463 .- 1520-5827. ; 32:33, s. 8403-8412
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Tidskriftsartikel (refereegranskat)abstract
- RAD51 is the key component of the homologous recombination pathway in eukaryotic cells and performs its task by forming filaments on DNA. In this study we investigate the physical properties of RAD51 filaments formed on DNA using nanofluidic channels and fluorescence microscopy. Contrary to the bacterial ortholog RecA, RAD51 forms inhomogeneous filaments on long DNA in vitro, consisting of several protein patches. We demonstrate that a permanent "kink" in the filament is formed where two patches meet if the stretch of naked DNA between the patches is short. The kinks are readily seen in the present microscopy approach but would be hard to identify using conventional single DNA molecule techniques where the DNA is more stretched. We also demonstrate that protein patches separated by longer stretches of bare DNA roll up on each other and this is visualized as transiently overlapping filaments. RAD51 filaments can be formed at several different conditions, varying the cation (Mg2+ or Ca2+), the DNA substrate (single-stranded or double-stranded), and the RAD51 concentration during filament nucleation, and we compare the properties of the different filaments formed. The results provide important information regarding the physical properties of RAD51 filaments but also demonstrate that nanofluidic channels are perfectly suited to study protein-DNA complexes.
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| 3. |
- McGinn, Steven, et al.
(författare)
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New Technologies for DNA analysis-A review of the READNA Project.
- 2016
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Ingår i: New Biotechnology. - : Elsevier BV. - 1876-4347 .- 1871-6784.
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Forskningsöversikt (refereegranskat)abstract
- The REvolutionary Approaches and Devices for Nucleic Acid analysis (READNA) project received funding from the European Commission for 4 1/2 years. The objectives of the project revolved around technological developments in nucleic acid analysis. The project partners have discovered, created and developed a huge body of insights into nucleic acid analysis, ranging from improvements and implementation of current technologies to the most promising sequencing technologies that constitute a 3(rd) and 4(th) generation of sequencing methods with nanopores and in situ sequencing, respectively.
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| 4. |
- Müller, Vilhelm, 1990, et al.
(författare)
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Direct identification of antibiotic resistance genes on single plasmid molecules using CRISPR/Cas9 in combination with optical DNA mapping.
- 2016
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322 .- 2045-2322. ; 6, s. 37938-
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Tidskriftsartikel (refereegranskat)abstract
- Bacterial plasmids are extensively involved in the rapid global spread of antibiotic resistance. We here present an assay, based on optical DNA mapping of single plasmids in nanofluidic channels, which provides detailed information about the plasmids present in a bacterial isolate. In a single experiment, we obtain the number of different plasmids in the sample, the size of each plasmid, an optical barcode that can be used to identify and trace the plasmid of interest and information about which plasmid that carries a specific resistance gene. Gene identification is done using CRISPR/Cas9 loaded with a guide-RNA (gRNA) complementary to the gene of interest that linearizes the circular plasmids at a specific location that is identified using the optical DNA maps. We demonstrate the principle on clinically relevant extended spectrum beta-lactamase (ESBL) producing isolates. We discuss how the gRNA sequence can be varied to obtain the desired information. The gRNA can either be very specific to identify a homogeneous group of genes or general to detect several groups of genes at the same time. Finally, we demonstrate an example where we use a combination of two gRNA sequences to identify carbapenemase-encoding genes in two previously not characterized clinical bacterial samples.
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| 5. |
- Müller, Vilhelm, 1990, et al.
(författare)
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Rapid Tracing of Resistance Plasmids in a Nosocomial Outbreak Using Optical DNA Mapping
- 2016
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Ingår i: Acs Infectious Diseases. - : American Chemical Society (ACS). - 2373-8227. ; 2:5, s. 322-328
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Tidskriftsartikel (refereegranskat)abstract
- Resistance to life-saving antibiotics increases rapidly worldwide, and multiresistant bacteria have become a global threat to human health. Presently, the most serious threat is the increasing spread of Enterobacteriaceae carrying genes coding for extended spectrum beta-lactamases (ESBL) and carbapenemases on highly mobile plasmids. We here demonstrate how optical DNA maps of single plasmids can be used as fingerprints to trace plasmids, for example, during resistance outbreaks. We use the assay to demonstrate a potential transmission route of an ESBL-carrying plasmid between bacterial strains/species and between patients, during a polyclonal outbreak at a neonatal ward at Sahlgrenska University Hospital (Gothenburg, Sweden). Our results demonstrate that optical DNA mapping is an easy and rapid method for detecting the spread of plasmids mediating resistance. With the increasing prevalence of multiresistant bacteria, diagnostic tools that can aid in solving ongoing routes of transmission, in particular in hospital settings, will be of paramount importance.
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| 6. |
- Nyberg, Lena, 1979, et al.
(författare)
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Rapid identification of intact bacterial resistance plasmids via optical mapping of single DNA molecules
- 2016
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322 .- 2045-2322. ; 6
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Tidskriftsartikel (refereegranskat)abstract
- The rapid spread of antibiotic resistance - currently one of the greatest threats to human health according to WHO - is to a large extent enabled by plasmid-mediated horizontal transfer of resistance genes. Rapid identification and characterization of plasmids is thus important both for individual clinical outcomes and for epidemiological monitoring of antibiotic resistance. Toward this aim, we have developed an optical DNA mapping procedure where individual intact plasmids are elongated within nanofluidic channels and visualized through fluorescence microscopy, yielding barcodes that reflect the underlying sequence. The assay rapidly identifies plasmids through statistical comparisons with barcodes based on publicly available sequence repositories and also enables detection of structural variations. Since the assay yields holistic sequence information for individual intact plasmids, it is an ideal complement to next generation sequencing efforts which involve reassembly of sequence reads from fragmented DNA molecules. The assay should be applicable in microbiology labs around the world in applications ranging from fundamental plasmid biology to clinical epidemiology and diagnostics.
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