| 1. |
- McGinn, Steven, et al.
(author)
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New Technologies for DNA analysis-A review of the READNA Project.
- 2016
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In: New Biotechnology. - : Elsevier BV. - 1876-4347 .- 1871-6784.
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Research review (peer-reviewed)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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| 2. |
- Alizadehheidari, Mohammadreza, 1987, et al.
(author)
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Nanoconfined Circular and Linear DNA: Equilibrium Conformations and Unfolding Kinetics
- 2015
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In: Macromolecules. - : American Chemical Society (ACS). - 0024-9297 .- 1520-5835. ; 48:3, s. 871-878
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Journal article (peer-reviewed)abstract
- Studies of circular DNA confined to nanofluidic channels are relevant both from a fundamental polymer-physics perspective and due to the importance of circular DNA molecules in vivo. We here observe the unfolding of confined DNA from the circular to linear configuration as a light-induced double-strand break occurs, characterize the dynamics, and compare the equilibrium conformational statistics of linear and circular configurations. This is important because it allows us to determine to what extent existing statistical theories describe the extension of confined circular DNA. We find that the ratio of the extensions of confined linear and circular DNA configurations increases as the buffer concentration decreases. The experimental results fall between theoretical predictions for the extended de Gennes regime at weaker confinement and the Odijk regime at stronger confinement. We show that it is possible to directly distinguish between circular and linear DNA molecules by measuring the emission intensity from the DNA. Finally, we determine the rate of unfolding and show that this rate is larger for more confined DNA, possibly reflecting the corresponding larger difference in entropy between the circular and linear configurations.
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| 3. |
- Alizadehheidari, Mohammadreza, 1987, et al.
(author)
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Unfolding of nanoconfined circular DNA
- 2015
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In: BIOPHYSICAL JOURNAL. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 108:2 Supplement 1
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Journal article (other academic/artistic)
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| 4. |
- Barrett, Michael P., et al.
(author)
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Microfluidics-based approaches to the isolation of African trypanosomes
- 2017
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In: Pathogens. - : MDPI AG. - 2076-0817. ; 6:4
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Research review (peer-reviewed)abstract
- African trypanosomes are responsible for significant levels of disease in both humans and animals. The protozoan parasites are free-living flagellates, usually transmitted by arthropod vectors, including the tsetse fly. In the mammalian host they live in the bloodstream and, in the case of human-infectious species, later invade the central nervous system. Diagnosis of the disease requires the positive identification of parasites in the bloodstream. This can be particularly challenging where parasite numbers are low, as is often the case in peripheral blood. Enriching parasites from body fluids is an important part of the diagnostic pathway. As more is learned about the physicochemical properties of trypanosomes, this information can be exploited through use of different microfluidic-based approaches to isolate the parasites from blood or other fluids. Here, we discuss recent advances in the use of microfluidics to separate trypanosomes from blood and to isolate single trypanosomes for analyses including drug screening.
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| 5. |
- Beech, Jason P., et al.
(author)
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Active Posts in Deterministic Lateral Displacement Devices
- 2019
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In: Advanced Materials Technologies. - : Wiley. - 2365-709X. ; 4:9
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Journal article (peer-reviewed)abstract
- Using electrically connected metal-coated posts in a deterministic lateral displacement (DLD) device and applying electric fields, electrokinetics is used to tune separations, significantly decrease the critical size for separation, and increase the dynamic range with switching times on the order of seconds. The strength of DLD stems from its binary behavior. To first approximation, particles move in one out of two trajectories based on their effective size. For particles that are close to the threshold size, a small external force is sufficient to nudge the particles from one trajectory to another. The devices consist of arrays of cylindrical metal-coated SU-8 posts connected by an underlying metal layer. This allows the application of voltages at the post surfaces and the generation of electric field gradients between neighboring posts, causing polarizable particles to experience a dielectrophoretic (DEP) force. This force, which depends on the volume and polarizability of the particle, can be made sufficient to push particles from one trajectory into another. In this way, the critical size in a device, normally fixed by the geometry, can be tuned. What's more, adding DEP in this way allows for the simultaneous creation of multiple size fractions.
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| 6. |
- Beech, Jason P., et al.
(author)
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Separation of pathogenic bacteria by chain length
- 2018
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In: Analytica Chimica Acta. - : Elsevier BV. - 0003-2670 .- 1873-4324. ; 1000, s. 223-231
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Journal article (peer-reviewed)abstract
- Using Deterministic Lateral Displacement devices optimized for sensitivity to particle length, we separate subpopulations of bacteria depending on known properties that affect their capability to cause disease (virulence). For the human bacterial pathogen Streptococcus pneumoniae, bacterial chain length and the presence of a capsule are known virulence factors contributing to its ability to cause severe disease. Separation of cultured pneumococci into subpopulations based on morphological type (single cocci, diplococci and chains) will enable more detailed studies of the role they play in virulence. Moreover, we present separation of mixed populations of almost genetically identical encapsulated and non-encapsulated pneumococcal strains in our device.
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| 7. |
- Beech, Jason P., et al.
(author)
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Sorting bacteria by chain length - A factor of virulence?
- 2016
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In: 20th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2016. - 9780979806490 ; , s. 250-251
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Conference paper (peer-reviewed)abstract
- Using Deterministic Lateral Displacement (DLD), we are able to separate bacteria by their size and their chain length. This separation enables the study of these properties as factors of virulence.
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| 8. |
- Beech, Jason P., et al.
(author)
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The separation and identification of parasite eggs from horse feces
- 2019
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In: 23rd International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2019. - 9781733419000 ; , s. 602-603
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Conference paper (peer-reviewed)abstract
- Freely grazing horses are at risk of infection by parasites such as Parascaris equorum (roundworm), Strongylus spp. (large bloodworms), Cyathostomes (small bloodworms), and Anoplochephala perfoliata (tapeworms). Mixed infections are common and diagnosis is based on demonstrations of eggs in feces followed by identification of larvae after fecal culture. Drug resistance is a growing problem, not least because treatments tend to be cheaper than diagnosis and “just in case” treatments common. There is a need for improved methods that are easy to use, rapid and cheap. Furthermore, a successful approach may find use with other livestock such as ruminants and pigs.
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| 9. |
- Beech, Jason P., et al.
(author)
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The separation of nano-sized particles in micro-scaled post arrays
- 2019
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In: 23rd International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2019. - 9781733419000 ; , s. 10-11
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Conference paper (peer-reviewed)abstract
- The precise separation of nanoscale particles has proven challenging due to diffusion and the need to use nanoscale devices. We show the separation of particles in the 100 nm size range in Deterministic Lateral Displacement (DLD) devices with feature sizes in the 10 µm size range. We achieve this using Dielectrophoretic (DEP) forces, generated between the metal coated posts that act as active electrodes. This opens up for the separation of submicron particles based not only on size but also on electric and dielectric properties.
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| 10. |
- Beech, Jason P., et al.
(author)
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Tunable separation and DNA manipulation in metal coated pillar arrays
- 2018
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In: 22nd International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2018. - 9781510897571 ; 4, s. 2090-2093
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Conference paper (peer-reviewed)abstract
- Implementing electrically connected metal-coated posts in a Deterministic Lateral Displacement (DLD) device and applying electric fields, we use electrokinetics to achieve tunable particle separations and to trap and manipulate DNA. The strength of DLD stems from its typically binary behavior. Particles move in one out of two trajectories based on their effective size. For particles that are close to the threshold size, a minute external force is sufficient to nudge the particles from one trajectory to another. Dielectrophoresis (DEP) provides such a force and also gives specificity based on the dielectric properties of the particles.
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| 11. |
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| 12. |
- Bogas, Diana, et al.
(author)
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Applications of optical DNA mapping in microbiology
- 2017
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In: BioTechniques. - : Future Science Ltd. - 0736-6205 .- 1940-9818. ; 62:6, s. 255-267
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Research review (peer-reviewed)abstract
- Optical mapping (OM) has been used in microbiology for the past 20 years, initially as a technique to facilitate DNA sequence-based studies; however, with decreases in DNA sequencing costs and increases in sequence output from automated sequencing platforms, OM has grown into an important auxiliary tool for genome assembly and comparison. Currently, there are a number of new and exciting applications for OM in the field of microbiology, including investigation of disease outbreaks, identification of specific genes of clinical and/or epidemiological relevance, and the possibility of single-cell analysis when combined with cell-sorting approaches. In addition, designing lab-on-a-chip systems based on OM is now feasible and will allow the integrated and automated microbiological analysis of biological fluids. Here, we review the basic technology of OM, detail the current state of the art of the field, and look ahead to possible future developments in OM technology for microbiological applications.
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| 13. |
- Faridi, Muhammad Asim
(author)
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Bioparticle Manipulation using Acoustophoresis and Inertial Microfluidics
- 2017
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Doctoral thesis (other academic/artistic)abstract
- Despite the many promising advances made in microfluidics, sample preparation remains the single largest challenge and bottleneck in the field of miniaturised diagnostics. This thesis is focused on the development of sample preparation methods using active and passive particle manipulation techniques for point of care diagnostic applications. The active technique is based on acoustophoresis (acoustic manipulation) while the passive method is based on inertial microfluidics (hydrodynamic manipulation). In paper I, acoustic capillary-based cavity resonator was used to study aggregation of silica and polystyrene particles. We found that silica particles show faster aggregation time (5.5 times) and larger average area of aggregates (3.4 times) in comparison to polystyrene particles under the same actuation procedure. The silica particles were then used for acoustic based bacteria up-concentration. In paper II, a microfluidic-based microbubbles activated acoustic cell sorting technique was developed for affinity based cell separation. As a proof of principle, separation of cancer cell line in a suspension with better than 75% efficiency is demonstrated. For the passive sample preparation, inertial and elasto-inertial microfluidic approach that uses geometry-induced hydrodynamic forces for continuous size-based sorting of particles in a flow-through fashion were studied and applied for blood processing (paper III-V). In paper III, a simple ushaped curved channel was used for inertial microfluidics based enrichment of white blood cells from diluted whole blood. A filtration efficiency of 78% was achieved at a flow rate of 2.2 ml/min. In paper IV, elasto-inertial microfluidics where viscoelastic flow enables size-based migration of cells into a non- Newtonian solution, was used to continuously separate bacteria from unprocessed whole blood for sepsis diagnostics. Bacteria were continuously separated at an efficiency of 76% from undiluted whole blood sample. Finally, in paper V, the inertial and elasto-inertial techniques were combined with a detection platform to demonstrate an integrated miniaturized flow cytometer. The all-optical-fiber technology based system allows for simultaneous measurements of fluorescent and scattering data at 2500 particles/s. The use of inertial and acoustic techniques for sample preparation and development of an integrated detection platform may allow for further development and realization of point of care testing (POCT) systems.
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| 14. |
- Freitag, C., et al.
(author)
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Visualizing the entire DNA from a chromosome in a single frame
- 2015
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In: Biomicrofluidics. - : AIP Publishing. - 1932-1058. ; 9:4
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Journal article (peer-reviewed)abstract
- The contiguity and phase of sequence information are intrinsic to obtain complete understanding of the genome and its relationship to phenotype. We report the fabrication and application of a novel nanochannel design that folds megabase lengths of genomic DNA into a systematic back-and-forth meandering path. Such meandering nanochannels enabled us to visualize the complete 5.7 Mbp (1mm) stained DNA length of a Schizosaccharomyces pombe chromosome in a single frame of a CCD. We were able to hold the DNA in situ while implementing partial denaturation to obtain a barcode pattern that we could match to a reference map using the Poland-Scheraga model for DNA melting. The facility to compose such long linear lengths of genomic DNA in one field of view enabled us to directly visualize a repeat motif, count the repeat unit number, and chart its location in the genome by reference to unique barcode motifs found at measurable distances from the repeat. Meandering nanochannel dimensions can easily be tailored to human chromosome scales, which would enable the whole genome to be visualized in seconds. (C) 2015 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution 3.0 Unported License.
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| 15. |
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| 16. |
- Henry, Ewan, et al.
(author)
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Sorting cells by their dynamical properties
- 2016
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In: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 6
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Journal article (peer-reviewed)abstract
- Recent advances in cell sorting aim at the development of novel methods that are sensitive to various mechanical properties of cells. Microfluidic technologies have a great potential for cell sorting; however, the design of many micro-devices is based on theories developed for rigid spherical particles with size as a separation parameter. Clearly, most bioparticles are non-spherical and deformable and therefore exhibit a much more intricate behavior in fluid flow than rigid spheres. Here, we demonstrate the use of cells' mechanical and dynamical properties as biomarkers for separation by employing a combination of mesoscale hydrodynamic simulations and microfluidic experiments. The dynamic behavior of red blood cells (RBCs) within deterministic lateral displacement (DLD) devices is investigated for different device geometries and viscosity contrasts between the intra-cellular fluid and suspending medium. We find that the viscosity contrast and associated cell dynamics clearly determine the RBC trajectory through a DLD device. Simulation results compare well to experiments and provide new insights into the physical mechanisms which govern the sorting of non-spherical and deformable cells in DLD devices. Finally, we discuss the implications of cell dynamics for sorting schemes based on properties other than cell size, such as mechanics and morphology.
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| 17. |
- Ho, Bao D., et al.
(author)
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Combining electrokinetics with deterministic lateral displacement
- 2016
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In: 20th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2016. - 9780979806490 ; , s. 1559-1560
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Conference paper (peer-reviewed)abstract
- We make Deterministic Lateral Displacement (DLD) flexible by combining it with AC electrokinetics.
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| 18. |
- Ho, Bao D., et al.
(author)
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Viable/non-viable cell assay using electrokinetic deterministic lateral displacement
- 2019
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In: 23rd International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2019. - 9781733419000 ; , s. 596-597
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Conference paper (peer-reviewed)abstract
- To evaluate the effect of antibiotic drugs, it is important that the cells that are killed can be sorted from the cells that are resistant to the antibiotics. Here we report a simple label-free method for the continuous separation of viable/non-viable cells using Electrokinetic Deterministic Lateral Displacement. In general, this opens up for continuous sorting of a population of cells that is homogeneous in size but heterogeneous in surface charge or dielectric properties, properties that change as a function of viability.
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| 19. |
- Holm, Stefan H., et al.
(author)
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Microfluidic Particle Sorting in Concentrated Erythrocyte Suspensions
- 2019
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In: Physical Review Applied. - 2331-7019. ; 12:1
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Journal article (peer-reviewed)abstract
- An important step in diagnostics is the isolation of specific cells and microorganisms of interest from blood. Since such bioparticles are often present at very low concentrations, throughput needs to be as high as possible. In addition, to ensure simplicity, a minimum of sample preparation is important. Therefore, sorting schemes that function for whole blood are highly desirable. Deterministic lateral displacement (DLD) devices have proven to be very precise and versatile in terms of a wide range of sorting parameters. To better understand how DLD devices perform for blood as the hematocrit increases, we carry out measurements and simulations for spherical particles in the micrometer range which move through DLD arrays for different flow velocities and hematocrits ranging from pure buffer to concentrated erythrocyte suspensions mimicking whole blood. We find that the separation function of the DLD array is sustained even though the blood cells introduce a shift in the trajectories and a significant dispersion for particles whose diameters are close to the critical size in the device. Simulations qualitatively replicate our experimental observations and help us identify fundamental mechanisms for the effect of hematocrit on the performance of the DLD device.
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| 20. |
- Iarko, V., et al.
(author)
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Extension of nanoconfined DNA: Quantitative comparison between experiment and theory
- 2015
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In: Physical Review E (Statistical, Nonlinear, and Soft Matter Physics). - 1539-3755 .- 1550-2376. ; 92:6, s. Art. Nr. 062701-
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Journal article (peer-reviewed)abstract
- The extension of DNA confined to nanochannels has been studied intensively and in detail. However, quantitative comparisons between experiments and model calculations are difficult because most theoretical predictions involve undetermined prefactors, and because the model parameters (contour length, Kuhn length, effective width) are difficult to compute reliably, leading to substantial uncertainties. Here we use a recent asymptotically exact theory for the DNA extension in the "extended de Gennes regime" that allows us to compare experimental results with theory. For this purpose, we performed experiments measuring the mean DNA extension and its standard deviation while varying the channel geometry, dye intercalation ratio, and ionic strength of the buffer. The experimental results agree very well with theory at high ionic strengths, indicating that the model parameters are reliable. At low ionic strengths, the agreement is less good. We discuss possible reasons. In principle, our approach allows us to measure the Kuhn length and the effective width of a single DNA molecule and more generally of semiflexible polymers in solution.
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| 21. |
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| 22. |
- Kim, Rebekah, et al.
(author)
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DNA sample cleanup using deterministic lateral displacement
- 2016
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In: 20th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2016. - 9780979806490 ; , s. 1527-1528
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Conference paper (peer-reviewed)abstract
- Optical mapping relies on the preparation of fluorescent DNA. DNA must be imaged with good signal to noise and therefore the background of unwanted DNA fragments, fluorescent dyes and other reagents need to be removed. We use deterministic lateral displacement to separate 48.5 kbp DNA from < 10 kbp DNA. We also show the removal of 48.5 kbp DNA fragments from a background of fluorescent ATTO647N molecules and the recovery of >50 kbp molecules from a background of shorter digested fragments. In both cases improving signal to noise during imaging.
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| 23. |
- Krog, Jens, et al.
(author)
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Stochastic unfolding of nanoconfined DNA: Experiments, model and Bayesian analysis
- 2018
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In: Journal of Chemical Physics. - : AIP Publishing. - 0021-9606 .- 1089-7690. ; 149:21
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Journal article (peer-reviewed)abstract
- Nanochannels provide a means for detailed experiments on the effect of confinement on biomacro-molecules, such as DNA. Here we introduce a model for the complete unfolding of DNA from the circular to linear configuration. Two main ingredients are the entropic unfolding force and the friction coefficient for the unfolding process, and we describe the associated dynamics by a non-linear Langevin equation. By analyzing experimental data where DNA molecules are photo-cut and unfolded inside a nanochannel, our model allows us to extract values for the unfolding force as well as the friction coefficient for the first time. In order to extract numerical values for these physical quantities, we employ a recently introduced Bayesian inference framework. We find that the determined unfolding force is in agreement with estimates from a simple Flory-type argument. The estimated friction coefficient is in agreement with theoretical estimates for motion of a cylinder in a channel. We further validate the estimated friction constant by extracting this parameter from DNA's center-of -mass motion before and after unfolding, yielding decent agreement. We provide publically available software for performing the required image and Bayesian analysis. Published by AIP Publishing.
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| 24. |
- Månsson, Linda K., et al.
(author)
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Preparation of colloidal molecules with temperature-tunable interactions from oppositely charged microgel spheres
- 2019
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In: Soft Matter. - : Royal Society of Chemistry (RSC). - 1744-683X .- 1744-6848. ; 15:42, s. 8512-8524
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Journal article (peer-reviewed)abstract
- The self-assembly of small colloidal clusters, so-called colloidal molecules, into crystalline materials has proven extremely challenging, the outcome often being glassy, amorphous states where positions and orientations are locked. In this paper, a new type of colloidal molecule is therefore prepared, assembled from poly(N-isopropylacrylamide) (PNIPAM)-based microgels that due to their well documented softness and temperature-response allow for greater defect tolerance compared to hard spheres and for convenient in situ tuning of size, volume fraction and inter-particle interactions with temperature. The microgels (B) are assembled by electrostatic adsorption onto oppositely charged, smaller-sized microgels (A), where the relative size of the two determines the valency (n) of the resulting core-satellite ABn-type colloidal molecules. Following assembly, a microfluidic deterministic lateral displacement (DLD) device is used to effectively isolate AB4-type colloidal molecules of tetrahedral geometry that possess a repulsive-to-attractive transition on crossing the microgels' volume phase transition temperature (VPTT). These soft, temperature-responsive colloidal molecules constitute highly promising building blocks for the preparation of new materials with emergent properties, and their optical wavelength-size makes them especially interesting for optical applications.
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| 25. |
- Noble, Charleston, et al.
(author)
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A fast and scalable kymograph alignment algorithm for nanochannel-based optical DNA mappings.
- 2015
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In: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 10:4
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Journal article (peer-reviewed)abstract
- Optical mapping by direct visualization of individual DNA molecules, stretched in nanochannels with sequence-specific fluorescent labeling, represents a promising tool for disease diagnostics and genomics. An important challenge for this technique is thermal motion of the DNA as it undergoes imaging; this blurs fluorescent patterns along the DNA and results in information loss. Correcting for this effect (a process referred to as kymograph alignment) is a common preprocessing step in nanochannel-based optical mapping workflows, and we present here a highly efficient algorithm to accomplish this via pattern recognition. We compare our method with the one previous approach, and we find that our method is orders of magnitude faster while producing data of similar quality. We demonstrate proof of principle of our approach on experimental data consisting of melt mapped bacteriophage DNA.
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