| 1. |
|
|
| 2. |
- Fornander, Louise, 1984, et al.
(författare)
-
Using nanofluidic channels to probe dynamics of RAD51-Filaments
- 2015
-
Ingår i: 18th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2014. - 9780979806476 ; , s. 1826-1828
-
Konferensbidrag (refereegranskat)abstract
- Using nanochannels, passivated with a lipid bilayer to avoid sticking of proteins, we study Rad51 filaments bound to single- and double stranded DNA. We demonstrate how we can discern different properties of the filaments by studying them at different degrees of confinement. Unlike the bacterial homologue RecA, that forms homogeneous filaments along DNA, Rad51 forms heterogeneous filaments containing both rigid kinks as well as flexible regions. Varying the counterion, the DNA substrate as well as the initial protein concentration, we try to understand the factors governing the structure of the filaments.
|
|
| 3. |
|
|
| 4. |
- Fornander, Louise, 1984, et al.
(författare)
-
Visualizing the Nonhomogeneous Structure of RAD51 Filaments Using Nanofluidic Channels
- 2016
-
Ingår i: Langmuir. - : American Chemical Society (ACS). - 0743-7463 .- 1520-5827. ; 32:33, s. 8403-8412
-
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.
|
|
| 5. |
- Fritzsche, Joachim, 1977, et al.
(författare)
-
Single Particle Nanoplasmonic Sensing in Individual Nanofluidic Channels
- 2016
-
Ingår i: Nano Letters. - : American Chemical Society (ACS). - 1530-6992 .- 1530-6984. ; 16:12, s. 7857-7864
-
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.
|
|
| 6. |
- Fritzsche, Michael, et al.
(författare)
-
A Highly UV-transparent Fused Silica Biochip for Sensitive Hepatotoxicity Testing by Autofluorescence
- 2014
-
Ingår i: Biochip Journal. - : Springer Science and Business Media LLC. - 2092-7843 .- 1976-0280. ; 8:2, s. 115-121
-
Tidskriftsartikel (refereegranskat)abstract
- Fabrication and application of a non-fluorescent and UV-transparent microfluidic biochip in fused silica that allows sensitive autofluorescence detection are described. The biochip is particularly useful in cell-based assays where the most informative autofluorescence signals from the cells reside in the ultraviolet spectral range and where plastic labware materials commonly used in cell culture work severely disturb such measurements. In this study the fused silica biochip was used for measuring intrinsic autofluorescence from liver cells in order to assess hepatotoxic effects of drugs. The assessment assay was carried out with the human liver cell line HepG2 under perfusion conditions in the microfluidics of the biochip. The autofluorescence from the.liver cells exposed to quinidine was readily recorded without background disturbance and correlated well with reference toxicity data.
|
|
| 7. |
- Frykholm, Karolin, 1977, et al.
(författare)
-
Fast size-determination of intact bacterial plasmids using nanofluidic channels
- 2015
-
Ingår i: Lab on a Chip. - : Royal Society of Chemistry (RSC). - 1473-0189 .- 1473-0197. ; 15:13, s. 2739-2743
-
Tidskriftsartikel (refereegranskat)abstract
- We demonstrate how nanofluidic channels can be used as a tool to rapidly determine the number and sizes of plasmids in bacterial isolates. Each step can be automated at low cost, opening up opportunities for general use in microbiology labs.
|
|
| 8. |
- Frykholm, Karolin, 1977, et al.
(författare)
-
Probing Physical Properties of a DNA- Protein Complex Using Nanofluidic Channels
- 2014
-
Ingår i: Small. - : Wiley. - 1613-6810 .- 1613-6829. ; 10:5, s. 884-887
-
Tidskriftsartikel (refereegranskat)abstract
- A method to investigate physical properties of a DNA-protein complex in solution is demonstrated. By using tapered nanochannels and lipid passivation the persistence length of a RecA filament formed on double-stranded DNA is determined to 1.15 μm, in agreement with the literature, without attaching protein or DNA to any handles or surfaces.
|
|
| 9. |
- Frykholm, Karolin, 1977, et al.
(författare)
-
Probing physical properties of DNA-protein complexes using nanofluidic channels
- 2013
-
Ingår i: 17th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2013; Freiburg; Germany; 27 October 2013 through 31 October 2013. - 9781632666246 ; 2, s. 1311-1313
-
Konferensbidrag (refereegranskat)abstract
- We present the use of nanofluidic channels as a tool for determining physical properties of single DNA-protein complexes. By coating the nanochannels with a lipid bilayer we avoid sticking of proteins to the channel walls. RecA is a prokaryotic protein involved in recombination and DNA repair. We study filaments of RecA, bound to both double stranded (ds) and single stranded (ss) DNA. We determine the persistence length of RecA filaments on both dsDNA and ssDNA and obtain values in agreement with the literature. Neither the DNA nor the protein has to be attached to handles or surfaces, and the technique is directly transferable to Lab-on-a-Chip technologies for high throughput measurements in solution.
|
|
| 10. |
- Müller, Vilhelm, 1990, et al.
(författare)
-
Direct identification of antibiotic resistance genes on single plasmid molecules using CRISPR/Cas9 in combination with optical DNA mapping.
- 2016
-
Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322 .- 2045-2322. ; 6, s. 37938-
-
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.
|
|
