Single-molecule DNA sequencing of widely varying GC-content using nucleotide release, capture and detection in microdroplets.

Despite remarkable progress in DNA sequencing technologies there remains a trade-off between short-read platforms, having limited ability to sequence homopolymers, repeated motifs or long-range structural variation, and long-read platforms, which tend to have lower accuracy and/or throughput. Moreover, current methods do not allow direct readout of epigenetic modifications from a single read. With the aim of addressing these limitations, we have developed an optical electrowetting sequencing platform that uses step-wise nucleotide triphosphate (dNTP) release, capture and detection in microdroplets from single DNA molecules. Each microdroplet serves as a reaction vessel that identifies an individual dNTP based on a robust fluorescence signal, with the detection chemistry extended to enable detection of 5-methylcytosine. Our platform uses small reagent volumes and inexpensive equipment, paving the way to cost-effective single-molecule DNA sequencing, capable of handling widely varying GC-bias, and demonstrating direct detection of epigenetic modifications.

Single-molecule detection of deoxyribonucleoside triphosphates in microdroplets.

A new approach to single-molecule DNA sequencing in which dNTPs, released by pyrophosphorolysis from the strand to be sequenced, are captured in microdroplets and read directly could have substantial advantages over current sequence-by-synthesis methods; however, there is no existing method sensitive enough to detect a single nucleotide in a microdroplet. We have developed a method for dNTP detection based on an enzymatic two-stage reaction which produces a robust fluorescent signal that is easy to detect and process. By taking advantage of the inherent specificity of DNA polymerases and ligases, coupled with volume restriction in microdroplets, this method allows us to simultaneously detect the presence of and distinguish between, the four natural dNTPs at the single-molecule level, with negligible cross-talk.

Fabricating large-area metallic woodpile photonic crystals using stacking and rolling.

Stacking thin polymer films supporting metal nanowire gratings provides a simple route, demonstrated here, to producing large-area metallic woodpile structures with high throughput. Under appropriate conditions the grating films can spontaneously roll up, giving a rapid and controllable method of creating multilayers. The resulting three-dimensional (3D) wire structures are flexible and potentially stretchable. Since this process can be extended to include a wide variety of functional materials, it opens up the manufacture of many tailored 3D optical metamaterials.

Optical nano-woodpiles: large-area metallic photonic crystals and metamaterials.

Metallic woodpile photonic crystals and metamaterials operating across the visible spectrum are extremely difficult to construct over large areas, because of the intricate three-dimensional nanostructures and sub-50 nm features demanded. Previous routes use electron-beam lithography or direct laser writing but widespread application is restricted by their expense and low throughput. Scalable approaches including soft lithography, colloidal self-assembly, and interference holography, produce structures limited in feature size, material durability, or geometry. By multiply stacking gold nanowire flexible gratings, we demonstrate a scalable high-fidelity approach for fabricating flexible metallic woodpile photonic crystals, with features down to 10 nm produced in bulk and at low cost. Control of stacking sequence, asymmetry, and orientation elicits great control, with visible-wavelength band-gap reflections exceeding 60%, and with strong induced chirality. Such flexible and stretchable architectures can produce metamaterials with refractive index near zero, and are easily tuned across the IR and visible ranges.