Single molecule linear analysis of DNA in nano-channel labeled with sequence specific fluorescent probes.

An array of nano-channels was fabricated from silicon based semiconductor materials to stretch long, native dsDNA. Here we present a labeling scheme in which it is possible to identify the location of specific sequences along the stretched DNA molecules. The scheme proceeds by first using the strand displacement activity of the Vent (exo-) polymerase to generate single strand flaps on nicked dsDNA. These single strand flaps are hybridized with sequence specific fluorophore-labeled probes. Subsequent imaging of the DNA molecules inside a nano-channel array device allows for quantitative identification of the location of probes. The highly efficient DNA hybridization on the ss-DNA flaps is an excellent method to identify the sequence motifs of dsDNA as it gives us unique ability to control the length of the probe sequence and thus the frequency of hybridization sites on the DNA. We have also shown that this technique can be extended to a multi color labeling scheme by using different dye labeled probes or by combining with a DNA- polymerase-mediated incorporation of fluorophore-labeled nucleotides on nicking sites. Thus this labeling chemistry in conjunction with the nano-channel platform can be a powerful tool to solve complex structural variations in DNA which is of importance for both research and clinical diagnostics of genetic diseases.

Molecular detection of SARS-COV-2 in exhaled breath at the point-of-need.

The SARS-CoV-2 pandemic has highlighted the need for improved technologies to help control the spread of contagious pathogens. While rapid point-of-need testing plays a key role in strategies to rapidly identify and isolate infectious patients, current test approaches have significant shortcomings related to assay limitations and sample type. Direct quantification of viral shedding in exhaled particles may offer a better rapid testing approach, since SARS-CoV-2 is believed to spread mainly by aerosols. It assesses contagiousness directly, the sample is easy and comfortable to obtain, sampling can be standardized, and the limited sample volume lends itself to a fast and sensitive analysis. In view of these benefits, we developed and tested an approach where exhaled particles are efficiently sampled using inertial impaction in a micromachined silicon chip, followed by an RT-qPCR molecular assay to detect SARS-CoV-2 shedding. Our portable, silicon impactor allowed for the efficient capture (>85%) of respiratory particles down to 300 nm without the need for additional equipment. We demonstrate using both conventional off-chip and in-situ PCR directly on the silicon chip that sampling subjects' breath in less than a minute yields sufficient viral RNA to detect infections as early as standard sampling methods. A longitudinal study revealed clear differences in the temporal dynamics of viral load for nasopharyngeal swab, saliva, breath, and antigen tests. Overall, after an infection, the breath-based test remains positive during the first week but is the first to consistently report a negative result, putatively signalling the end of contagiousness and further emphasizing the potential of this tool to help manage the spread of airborne respiratory infections.

Cylindrically symmetric electrohydrodynamic patterning.

Cylindrically symmetric structures such as concentric rings and rosettes arise out of thin polymeric films subjected to strong electric fields. Experiments that formed concentric rings and theory capable of explaining these and other cylindrical structures are presented. These rings represent an additional member of a class of structures, including pillars and holes, formed by electrohydrodynamic patterning of thin films, occasionally referred to as lithographically induced self-assembly. Fabrication of a set of concentric rings begins by spin coating a thin poly(methyl methacrylate) film onto a silicon wafer. A mask is superimposed parallel to the film leaving a similarly thin air gap. Electric fields, acting in opposition to surface tension, destabilize the free interface when raised above the glass transition temperature. Central pillars nucleate under small cylindrical protrusions patterned on the mask. Rings then emerge sequentially, with larger systems having as many as 10 fully formed rings. Ring-to-ring spacings and annular widths, typically on the order of a micron, are approximately constant within a concentric cluster. The formation rate is proportional to the viscosity and, consequently, has the expected Williams-Landel-Ferry dependence on temperature. In light of these developments we have undertaken a linear stability analysis in cylindrical coordinates to describe these rings and ringlike structures. The salient feature of this analysis is the use of perturbations that incorporate their radial dependence in terms of Bessel functions as opposed to the traditional sinusoids of Cartesian coordinates. The theory predicts approximately constant ring-to-ring spacings, constant annular widths, and growth rates that agree with experiment. A secondary instability is observed at higher temperatures, which causes the rings to segment into arcs or pillar arrays. The cylindrical theory may be generalized to describe hexagonal pillar/hole packing, gratings, and rosettes with the first being of particular importance given the ubiquitous observation of hexagonal packing. The perturbation analysis presented here is relevant to any system with cylindrical symmetry, for which the radial dependence can be described in terms of Bessel functions.

Genome mapping on nanochannel arrays for structural variation analysis and sequence assembly.

We describe genome mapping on nanochannel arrays. In this approach, specific sequence motifs in single DNA molecules are fluorescently labeled, and the DNA molecules are uniformly stretched in thousands of silicon channels on a nanofluidic device. Fluorescence imaging allows the construction of maps of the physical distances between occurrences of the sequence motifs. We demonstrate the analysis, individually and as mixtures, of 95 bacterial artificial chromosome (BAC) clones that cover the 4.7-Mb human major histocompatibility complex region. We obtain accurate, haplotype-resolved, sequence motif maps hundreds of kilobases in length, resulting in a median coverage of 114× for the BACs. The final sequence motif map assembly contains three contigs. With an average distance of 9 kb between labels, we detect 22 haplotype differences. We also use the sequence motif maps to provide scaffolds for de novo assembly of sequencing data. Nanochannel genome mapping should facilitate de novo assembly of sequencing reads from complex regions in diploid organisms, haplotype and structural variation analysis and comparative genomics.

Self-formation of sub-60-nm half-pitch gratings with large areas through fracturing.

Periodic micro- and nanostructures (gratings) have many significant applications in electronic, optical, magnetic, chemical and biological devices and materials. Traditional methods for fabricating gratings by writing with electrons, ions or a mechanical tip are limited to very small areas and suffer from extremely low throughput. Interference lithography can achieve relatively large fabrication areas, but has a low yield for small-period gratings. Photolithography, nanoimprint lithography, soft lithography and lithographically induced self-construction all require a prefabricated mask, and although electrohydrodynamic instabilities can self-produce periodic dots without a mask, gratings remain challenging. Here, we report a new low-cost maskless method to self-generate nano- and microgratings from an initially featureless polymer thin film sandwiched between two relatively rigid flat plates. By simply prising apart the plates, the film fractures into two complementary sets of nonsymmetrical gratings, one on each plate, of the same period. The grating period is always four times the thickness of the glassy film, regardless of its molecular weight and chemical composition. Periods from 120 nm to 200 microm have been demonstrated across areas as large as two square centimetres.

Novel polymer patterns formed by lithographically induced self-assembly (LISA).

A variety of self-assembly patterns, e.g., concentric rings, rods, and pillars, in polymer thin film have been achieved by lithographically induced self-assembly (LISA) in this study. The variations of the LISA patterns are controlled by many operation factors, such as the choice of the polymers, mask topology, process temperatures, surface tension, and so forth. It was found that as the inter- and intramolecular hydrogen bond interactions were incorporated into the polymer [poly(methyl methacrylate-co-methacrylic acid)], novel LISA patterns such as rods and corresponding arrays (concentric ring, triangle, hexagonal, etc.) were formed, in addition to the pillar arrays formed in poly(methyl methacrylate) under the same experimental conditions. The origins of the rod array are determined by the topology of the masks. Under a plain mask, the patterns developed from any nonuniform defects (spots) on the mask or polymer thin film and propagated outward. However, under a mask with protruding flat patterns, the rod patterns started along the edge of the protrusions and propagated inward. By increasing the process temperature, those novel rods and corresponding array patterns could transform back to pillar or pillar arrays.