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Long-read genomic applications, such as genome mapping in nanochannels, require long DNA that is free of small-DNA impurities. We have developed a chip-based system based on entropic trapping that can simultaneously concentrate and purify a long DNA sample under the alternating application of an applied pressure (for sample injection) and an electric field (for filtration and concentration). In contrast, short DNA tends to pass through the filter owing to its comparatively weak entropic penalty for entering the nanoslit. The single-stage prototype developed here, which operates in a continuous pulsatile manner, achieves selectivities of up to 3.5 for λ-phage DNA (48.5 kilobase pairs) compared to a 2 kilobase pair standard based on experimental data for the fraction filtered using pure samples of each species. The device is fabricated in fused silica using standard clean-room methods, making it compatible for integration with long-read genomics technologies.

Bioincorporation of the methionine analogue S-(2-fluoroethyl)-l-homocysteine (l-MFE) into bacteriophage lysozyme overproduced in Escherichia coli results not only in the expected l-MFE incorporation but surprisingly substantial l-vinthionine incorporation into the labeled lysozymes. Synthetic l-vinthionine itself however is not activated by purified Escherichia coli methionyl-tRNA synthetase. The indirect preparation of vinthionine-containing proteins has the potential to be an alternate strategy to prepare vinyl thioether moieties for click chemistry applications on proteins.

Interactions of proteins with nucleic acids arise at all levels of cellular function, from chromosomal packing to biological regulation. These interactions can be analyzed in a high-throughput fashion by immobilizing the DNA sequences of interest, possibly numbering in the thousands, at discrete locations on a solid support and identifying those sequences that a protein analyte binds. Ideally, such surface assays would use unlabeled analyte to simplify protocols and avoid the possibility of perturbing the protein/DNA interaction. The present study compares three electrochemical modalities for simultaneously detecting binding of unlabeled transcription factor proteins to immobilized DNA duplexes based on (i) changes in the duplex diffusive motions, (ii) variations in the surface potential, and (iii) variations in the interfacial charging impedance, all of which can be conveniently derived from AC voltammetry traces. Cro protein from bacteriophage lambda is used as a model transcription factor. Specific binding of protein was successfully detected through modalities (i) and (ii), but not (iii). The effectiveness of these techniques is compared as a function of sampling frequency and protein concentration. Binding of 15 kDa Cro slowed down rotational diffusion of immobilized duplexes approximately 3-fold, and induced up to 5 mV changes in the surface potential. Moreover, by assessing Cro binding to bacteriophage operators of variable affinity, the study illustrates how contrast between specific and nonspecific interactions impacts detection.

Herein we report the specific labelling of the epigenetic modification 5-hydroxymethyl-cytosine along genomic DNA molecules with a fluorescent reporter molecule. Enzymatic glucosylation followed by a click chemistry reaction enables single molecule detection as well as global quantification of 5hmC in genomic DNA.

Transportation of magnetic beads between different reagents plays a crucial role in many biological assays e.g. for purification of biomolecules or cells where the beads act as a mobile solid support. Therefore, usually a complex set-up either for fluidic processing or for manipulation of magnetic beads is required. To circumvent these drawbacks, we present a facile and automated method for the transportation of magnetic beads between multiple microfluidic chambers on a centrifugal microfluidic cartridge "LabDisk". The method excels by requiring only one stack of stationary permanent magnets, a specific microfluidic layout without actively controlled valves and a predefined frequency protocol for rotation of the LabDisk. Magnetic beads were transported through three fluidically separated chambers with a yield of 82.6% ± 3.6%. Bead based DNA purification from a dilution series of a Listeria innocua lysate and from a lambda phage DNA standard was demonstrated where the three chambers were used for binding, washing and elution of DNA. Recovery of L. innocua DNA was up to 68% ± 24% and for lambda phage DNA 43% ± 10% compared to manual reference purification in test tubes. Complete purification was conducted automatically within 12.5 min. Since all reagents can be preloaded onto the LabDisk prior to purification, no further hands-on steps are required during processing. Due to its modular and generic character, the presented method could also be adapted to other magnetic bead based assays e.g. to immunoassays or protein affinity purification, solely requiring the adjustment of number and volumes of the fluidic chambers.

A complex entropy gradient for confined DNA molecules was engineered for the first time. Following the second law of thermodynamics, this enabled the directed self-transport and self-concentration of DNA molecules. This new nanofluidic method is termed entropophoresis. As implemented in experiments, long DNA molecules were dyed with cyanine dimers, dispersed in a high ionic strength buffer, and confined by a nanofluidic channel with a depth profile approximated by a staircase function. The staircase step depths spanned the transition from strong to moderate confinement. The diffusion of DNA molecules across slitlike steps was ratcheted by entropic forces applied at step edges, so that DNA molecules descended and collected at the bottom of the staircase, as observed by fluorescence microscopy. Different DNA morphologies, lengths, and stoichiometric base pair to dye molecule ratios were tested and determined to influence the rate of transport by entropophoresis. A model of ratcheted diffusion was used to interpret a shifting balance of forces applied to linear DNA molecules of standard length in a complex free energy landscape. Related metrics for the overall and optimum performance of entropophoresis were developed. The device and method reported here transcend current limitations in nanofluidics and present new possibilities in polymer physics, biophysics, separation science, and lab-on-a-chip technology.

With the advent of next-generation sequencing (NGS) systems and the associated high throughput they afford, the input to these machines requires manageable lengths of fragments (~1000 bp) produced from chromosomal DNAs. Therefore, it is critical to develop devices that can shear DNA in a controlled fashion. We report a polymer-based microfluidic device that establishes an efficient and inexpensive platform with performance comparable to a commercially available bench-top system.

Fabrication of nanochannels is drawing considerable interest due to its broad applications in nanobiotechnology (e.g. biomolecular sensing and single DNA manipulation). Nanochannels offer distinct advantages in allowing a slower translocation and multiple sensing spots along the channel, both of which improve the read-out resolution. However, implementing electrodes inside the nanochannel has rarely been demonstrated to our knowledge. The device described in this work is a Si-Glass anodically bonded Lab-on-a-Chip (LOC) device of a few millimetres in size capable of performing DNA manipulation. The LOC device structure is based on two mainstream microchannels interconnected by nanochannels. DNA, once trapped within the nanochannel, has been tracked throughout the length of the channel and the data have been recorded and analysed., (This journal is © The Royal Society of Chemistry 2011)

A Focused Ion Beam (FIB)-patterned silicon mould is used to fabricate elastomeric nanostructures, whose cross-section can be dynamically and reversibly tuned by applying a controlled mechanical stress. Direct-write, based on FIB milling, allows the fabrication of nanostructures with a variety of different geometries, aspect ratio, spacing and distribution offering a higher flexibility compared to other nanopatterning approaches. Moreover, a simple double replication process based on poly(dimethylsiloxane) permits a strong reduction of the fabrication costs that makes this approach well-suited for the production of low cost nanofluidic devices. DNA stretching and single molecule manipulation capabilities of these platforms have been successfully demonstrated., (This journal is © The Royal Society of Chemistry 2011)

We demonstrate that fabrication of well-defined nanofluidic systems can be greatly simplified by injection molding of thermoplastic polymers. Chips featuring nanochannel arrays, microchannels and integrated interconnects are produced in a single processing step by injection molding. The resulting open channel structures are subsequently sealed by facile plasma-enhanced thermal bonding of a polymer film. This fast, inexpensive and industry-compatible method thus provides a single-use all-polymer platform for nanofluidic lab-on-a-chip applications. Its applicability for nanofluidics is demonstrated by DNA stretching experiments performed on individual double-stranded DNA molecules confined in the injection molded nanochannels. The obtained results are consistent with measurements performed in costly state-of-the-art silica nanochannels, for both straight and tapered channel geometries.

Bacteriophage lambda-DNA molecules are frequently used as a scaffold to characterize the action of single proteins unwinding, translocating, digesting or repairing DNA. However, scaling up such single-DNA-molecule experiments under identical conditions to attain statistically relevant sample sizes remains challenging. Additionally the movies obtained are frequently noisy and difficult to analyse with any precision. We address these two problems here using, firstly, a novel variable-angle total internal reflection fluorescence (VA-TIRF) reflector composed of a minimal set of optical reflective elements, and secondly, using single value decomposition (SVD) to improve the signal-to-noise ratio prior to analysing time-lapse image stacks. As an example, we visualize under identical optical conditions hundreds of surface-tethered single lambda-DNA molecules, stained with the intercalating dye YOYO-1 iodide, and stretched out in a microcapillary flow. Another novelty of our approach is that we arrange on a mechanically driven stage several capillaries containing saline, calibration buffer and lambda-DNA, respectively, thus extending the approach to high-content, high-throughput screening of single molecules. Our length measurements of individual DNA molecules from noise-reduced kymograph images using SVD display a 6-fold enhanced precision compared to raw-data analysis, reaching approximately 1 kbp resolution. Combining these two methods, our approach provides a straightforward yet powerful way of collecting statistically relevant amounts of data in a semi-automated manner. We believe that our conceptually simple technique should be of interest for a broader range of single-molecule studies, well beyond the specific example of lambda-DNA shown here.

A fluorescence-based integrated optics microfluidic device is presented, capable of detecting single DNA molecules in a high throughput and reproducible manner. The device integrates microfluidics for DNA stretching with two optical elements for single molecule detection (SMD): a plano-aspheric refractive lens for fluorescence excitation (illuminator) and a solid parabolic reflective mirror for fluorescence collection (collector). Although miniaturized in size, both optical components were produced and assembled onto the microfluidic device by readily manufacturable fabrication techniques. The optical resolution of the device is determined by the small and relatively low numerical aperture (NA) illuminator lens (0.10 effective NA, 4.0 mm diameter) that delivers excitation light to a diffraction limited 2.0 microm diameter spot at full width half maximum within the microfluidic channel. The collector (0.82 annular NA, 15 mm diameter) reflects the fluorescence over a large collection angle, representing 71% of a hemisphere, toward a single photon counting module in an infinity-corrected scheme. As a proof-of-principle experiment for this simple integrated device, individual intercalated lambda-phage DNA molecules (48.5 kb) were stretched in a mixed elongational-shear microflow, detected, and sized with a fluorescence signal to noise ratio of 9.9 +/-1.0. We have demonstrated that SMD does not require traditional high numerical aperture objective lenses and sub-micron positioning systems conventionally used in many applications. Rather, standard manufacturing processes can be combined in a novel way that promises greater accessibility and affordability for microfluidic-based single molecule applications.

A laser-induced fluorescence imaging method was developed to simultaneously measure flow velocities in the middle and near wall of a channel with particles or single molecules, by selectively switching from the wide field excitation mode to the evanescent wave excitation mode. Fluorescent microbeads with a diameter of 175 nm were used to calibrate the system, and the collisions of microbeads with channel walls were directly observed. The 175 nm microbeads velocities in the main flow and at 275 nm from the bottom of the channel were measured. The measured velocities of particles or single molecules in two positions in a microchannel were consistent with the calculated value based on Poiseuille flow theory when the diameter of a microbead was considered. The errors caused by Brownian diffusion in our measurement were negligible compared to the flow velocity. Single lambda DNA molecules were then used as a flowing tracer to measure the velocities. The velocity can be obtained at a distance of 309.0 +/- 82.6 nm away from bottom surface of the channel. The technique may be potentially useful for studying molecular transportation both in the center and at the bottom of the channel, and interactions between molecules and microchannel surfaces. It is especially important that the technique can be permitted to measure both velocities in the same experiment to eliminate possible experimental inconsistencies.