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The transmission of light through sub-wavelength apertures (zero-mode waveguides, ZMW) in metal films is well-explored. It introduces both an amplitude modulation as well as a phase shift to the oscillating electromagnetic field. We propose a nanophotonic interferometer by bringing two ZMW (∼100 nm diameter) in proximity and monitoring the distribution of transmitted light in the back-focal plane of collecting microscope objective (1.3 N.A.). We demonstrate that both an asymmetry induced by the binding of a quantum dot in one of the two ZMW, as well as an asymmetry in ZMW diameter yield qualitatively similar transmission patterns. We find that the complex pattern can be quantified through a scalar measure of asymmetry along the symmetry axis of the aperture pair. In a combined experimental and computational exploration of detectors with differing ZMW diameters, we find that the scalar asymmetry is a monotonous function of the diameter difference of the two apertures, and that the scalar asymmetry measure is higher if the sample is slightly displaced from the focal plane of the collecting microscope objective. An optimization of the detector geometry determined that the maximum response is achieved at an aperture separation that is comparable to the wavelength on the exit side of the sensor. For small separations of apertures, on the order of a quarter of the wavelength and less, the signal is strongly polarization dependent, while for larger separations, on the order of the wavelength or larger, the signal becomes essentially polarization-independent. [ABSTRACT FROM AUTHOR]

Background: MeCP2 and MBD2 are members of a family of proteins that possess a domain that selectively binds 5-methylcytosine in a CpG context. Members of the family interact with other proteins to modulate DNA packing. Stretching of DNA–protein complexes in nanofluidic channels with a cross-section of a few persistence lengths allows us to probe the degree of compaction by proteins. Results: We demonstrate DNA compaction by MeCP2 while MBD2 does not affect DNA configuration. By using atomic force microscopy (AFM), we determined that the mechanism for compaction by MeCP2 is the formation of bridges between distant DNA stretches and the formation of loops. Conclusions: Despite sharing a similar specific DNA-binding domain, the impact of full-length 5-methylcytosine-binding proteins can vary drastically between strong compaction of DNA and no discernable large-scale impact of protein binding. We demonstrate that ATTO 565-labeled MBD2 is a good candidate as a staining agent for epigenetic mapping. [ABSTRACT FROM AUTHOR]

We report an experimental investigation of the magnitude of length and density fluctuations in DNA that has been stretched in nanofluidic channels. We find that the experimental data can be described using a one-dimensional overdamped oscillator chain with nonzero equilibrium spring length and that a chain of discrete oscillators yields a better description than a continuous chain. We speculate that the scale of these discrete oscillators coincides with the scale at which the finite extensibility of the polymer manifests itself. We discuss how the measurement process influences the apparent measured dynamic properties, and outline requirements for the recovery of true physical quantities. [ABSTRACT FROM AUTHOR]

Separation of blood cells by native susceptibility and by the selective attachment of magnetic beads has recently been demonstrated on microfluidic devices. We discuss the basic principles of how forces are generated via the magnetic susceptibility of an object and how microfluidics can be combined with micron-scale magnetic field gradients to greatly enhance in principle the fractionating power of magnetic fields. We discuss our efforts and those of others to build practical microfluidic devices for the magnetic separation of blood cells. We also discuss our attempts to integrate magnetic separation with other microfluidic features for developing handheld medical diagnostic tools. [ABSTRACT FROM AUTHOR]

Intercalating fluorescent probes are widely used to visualize DNA in studies on DNA-protein interactions. Some require the presence of adenosine triphosphate (ATP). We have investigated the mechanical properties of DNA stained with the fluorescent intercalating dyes YOYO-1 and YOYO-3 as a function of ATP concentrations (up to 2 mM) by stretching single molecules in nanofluidic channels with a channel cross-section as small as roughly 100 × 100 nm. The presence of ATP reduces the length of the DNA by up to 11 %. On the other hand, negligible effects are found if DNA is visualized with the minor groove-binding probe 4′,6-diamidino-2-phenylindole. The apparent drop in extension under nanoconfinement is attributed to an interaction of the dye and ATP, and the resulting expulsion of YOYO-1 from the double helix. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

We present an analytic technique for probing protein-catalyzed transient DNA loops that is based on nanofluidic channels. In these nanochannels, DNA is forced in a linear configuration that makes loops appear as folds whose size can easily be quantified. Using this technique, we study the interaction between T4 DNA ligase and DNA. We find that T4 DNA ligase binding changes the physical characteristics of the DNA polymer, in particular persistence length and effective width. We find that the rate of DNA fold unrolling is significantly reduced when T4 DNA ligase and ATP are applied to bare DNA. Together with evidence of T4 DNA ligase bridging two different segments of DNA based on AFM imaging, we thus conclude that ligase can transiently stabilize folded DNA configurations by coordinating genetically distant DNA stretches. [ABSTRACT FROM AUTHOR]

We report the simultaneous mapping of multiple histone tail modifications on chromatin that has been confined to nanofluidic channels. In these channels, chromatin is elongated, and histone modification can be detected using fluorescently tagged monoclonal antibodies. Using reconstituted chromatin with three distinct histone sources and two histone tail modification probes (H3K4me3 and H3K9ac), we were able to distinguish chromatin from the different sources. Determined ratios of the two modifications were consistent with the bulk composition of histone mixtures. We determined that the major difficulty in transitioning the mapping method to site-specific profiling within single genomic molecules is the interference of naturally aggregating, off-the shelf antibodies with the internal structure of chromatin. [ABSTRACT FROM AUTHOR]

An aluminum bow-tie nano-antenna is combined with the resonance Raman effect in the deep ultraviolet to dramatically increase the sensitivity of Raman spectra to a small volume of material, such as benzene used here. We further demonstrate gradient-field Raman peaks for several strong infrared modes. We achieve a gain of ∼105 in signal intensity from the near field enhancement due to the surface plasmon resonance in the aluminum nanostructure. The on-line resonance enhancement contributes another factor of several thousands, limited by the laser line width. Thus, an overall gain of hundreds of million is achieved. [ABSTRACT FROM AUTHOR]

We report the profiling of the 5-methyl cytosine distribution within single genomic-sized DNA molecules at a gene-relevant resolution. This method linearizes and stretches DNA molecules by confinement to channels with a dimension of about 250×200nm2. The methylation state is detected using fluorescently labeled methyl-CpG binding domain proteins (MBD), with high signal contrast and low background. DNA barcodes consisting of methylated and non-methylated segments are generated, with both short and long concatemers demonstrating spatially resolved MBD binding. The resolution of the technique is better than 10 kbp, and single-molecule read-lengths exceeding 140 kbp have been achieved. [ABSTRACT FROM AUTHOR]

Nanochannels offer a way to align and analyze long biopolymer molecules such as DNA with high precision at potentially single basepair resolution, especially if a means to detect biomolecules in nanochannels electronically can be developed. Integration of nanochannels with electronics will require the development of nanochannel fabrication procedures that will not damage sensitive electronics previously constructed on the device. We present here a near-room-temperature fabrication technology involving parylene-C conformal deposition that is compatible with complementary metal oxide semiconductor electronic devices and present an analysis of the initial impedance measurements of conformally parylene-C coated nanochannels with integrated gold nanoelectrodes. [ABSTRACT FROM AUTHOR]

A nanopore-based device provides single-molecule detection and analytical capabilities that are achieved by electrophoretically driving molecules in solution through a nano-scale pore. The nanopore provides a highly confined space within which single nucleic acid polymers can be analyzed at high throughput by one of a variety of means, and the perfect processivity that can be enforced in a narrow pore ensures that the native order of the nucleobases in a polynucleotide is reflected in the sequence of signals that is detected. Kilobase length polymers (single-stranded genomic DNA or RNA) or small molecules (e.g., nucleosides) can be identified and characterized without amplification or labeling, a unique analytical capability that makes inexpensive, rapid DNA sequencing a possibility. Further research and development to overcome current challenges to nanopore identification of each successive nucleotide in a DNA strand offers the prospect of 'third generation' instruments that will sequence a diploid mammalian genome for ∼$1,000 in ∼24 h. [ABSTRACT FROM AUTHOR]

We have performed restriction mapping of DNA molecules using restriction endonucleases in nanochannels with diameters of 100-200 nm. The location of the restriction reaction within the device is controlled by electrophoresis and diffusion of Mg2+ and EDTA. We have successfully used the restriction enzymes Smal, Sacl, and Pacl, and have been able to measure the positions of restriction sites with a precision of ≈1.5 kbp in I mm using single DNA molecules. [ABSTRACT FROM AUTHOR]

We show that genomic-length DNA molecules imaged in nano-channels have an extension along the channel that scales linearly with the contour length of the polymer, in agreement with the scaling arguments developed by de Gennes for self-avoiding confined polymers. This fundamental relationship allows us to measure directly the contour length of single DNA molecules confined in the channels, and the statistical analysis of the dynamics of the polymer in the nanochannel allows us to compute the SD of the mean of the extension. This statistical analysis allows us to mea- sure the extension of A DNA multimers with a 130-nm SD in 1 min. [ABSTRACT FROM AUTHOR]

DNA confined to rigid nanotubes shows density fluctuations around its stretched equilibrium conformation. We report an experimental investigation of the length-scale dependent dynamics of these density fluctuations. We find that for highly elongated molecules a Rouse description is consistent with observations at sufficiently large length scales. We further find that for strongly fluctuating molecules, or short length scales, such Rouse modes cannot be detected due to strong mixing of fluctuation modes. [ABSTRACT FROM AUTHOR]

We report the fabrication of poly(p-phenylene vinylene) nanostructures by direct scanning near-field lithography of its soluble precursor. The technique is based on the spatially selective inhibition of the precursor solubility by exposure to the ultraviolet optical field present at the apex of scanning near-field fiber probes with aperture diameters between 40 and 80 nm (±5 nm). After development in methanol and thermal conversion under vacuum we obtain features with a minimum dimension of 160 nm. We demonstrate the use of the technique for the direct writing of two-dimensional photonic crystals with intentional defects and a periodicity relevant to applications in the visible range. Using a Bethe-Bouwkamp model, we then discuss the influence of probe size, tip-sample distance, and film thickness on the resolution of the lithographic process. We also discuss limitations to the resolution that can arise from physical properties of the lithographic medium. [ABSTRACT FROM AUTHOR]