WO2013088098A2 - Efficient ultra-long dna analysis in compact nanofluidic channels - Google Patents

Abstract

The present application relates to nanochips, which can be used to visualise long molecules in a single field of view, and methods using the nanochips. These methods include mapping/barcoding and sequencing of DNA.

Description

Efficient Ultra-long DNA Analysis in Compact Nanofluidic Channels

The present application relates to nanochips, which can be used to visualise long molecules in a single field of view, and methods using the nanochips. These methods include sequencing, mapping or barcoding nucleotides.

Direct visualization of individual DNA molecules by stretching in nanochannels allows the acquisition of contextual information along the DNA. It also allows organisms, in particular disease-causing microorganisms, to be identified. By identifying the microorganisms on the single-molecule level there is no need for culturing of the samples, any DNA amplification, or procedures that constitute important bottlenecks during conventional diagnostics extending the. time between sampling and diagnosis to several days or even weeks. This may give opportunities to identify the microorganisms within a day or even shorter time spans, thereby offering the patient faster identification of optimal treatment. The ability to obtain contextual information along very long individual lengths of chromosomal DNA has important implications for genomics [see Mir, KU. Sequencing Genomes: From Individuals to Populations, Briefings in Functional Genomics and Proteomics, 8: 367-378 (2009)] Direct visualization of DNA single genomic DNA molecules has been demonstrated for molecules of the size of several 10s of kbps to 100s of kbps. Labeling strategies involve intercalating dyes such as YOYO™-l and TOTO™-l, restriction enzymes, melting-induced barcodes, and single strand flap hybridization labeling. This is described in Neely, R.K., J. Deen, and J. Hofkens, Optical Mapping of DNA: Single- Molecule-Based Methods for Mapping Genomes. Biopolymers, 201 1. 95(5): p. 298- 31 1.

Stretching of nucleotides on surfaces has also been reported by Aaron Bensimon [Alignment And Sensitive Detection Of DNA By A Moving Interface. Science, 1994. 265(5181): p. 2096-2098] and David Schwartz [Ordered Restriction Maps Of Saccharomyces-cerevisiae Chromosomes Constructed By Optical Mapping. Science, 1993. 262(5130): p. 1 10- 1 14]. Micro or nanofluidic devices have been described for handling nucleic acids. For example see: US 2008/273918; WO 209/113010; WO 2008/056160; US2005/074900; US2009/12336; US2002/ 127740; EP 1221617, US5744366

Recently, the dissolution of condensed human chromosomes and the subsequent stretching of the chromosomal DNA in a slit was demonstrated [Rasmussen KH et al. A Device for Extraction, Manipulation and Stretching of DNA from Single Human Chromosomes. Lab on a Chip, 11: 1431-3 (2011). However the stretching can also be done based on elongational flow and improved stretching can be introduced by utilizing hydrodynamic drag [see WO2012056192].

A common issue in all the above DNA stretching methods is that an individual DNA molecule is stretched in one single line or row. This makes acquisition inefficient since for each molecule only one row in the imaging device such as a change coupled device (CCD) is used. Multiple images need to be taken and stitched together to visualise the whole, length of the molecule. This increases the time required to image the molecule and can also lead to inaccuracies at the boundaries of the images. The stretching and imaging could take place in parallel, so that several identical molecule each travel through a nanochannel which is adjacent to another nanochannel through which another molecule is passing. However, it is difficult to handle several DNA molecules in synchrony and to ensure that each parallel nanochannel is occupied by a nucleotide molecule. Specifically for the elongational-flow approach, due to its particular geometry, it is difficult to obtain strands of long DNA stretched in parallel in a controllable manner without overlap.

However, due to the large length of biologically relevant DNA an important challenge arises. To keep the number of frames necessary to image the DNA to a reasonable number, the DNA needs to be folded within the field of view so that, for example the pixels of the CCD are used efficiently and so that the identification process can take place within a reasonable time. Convoluted nanochannels have been disclosed in patent application US2008242556A1 (Han Cao et al, Bionanomatrix). The nanochannels disclosed have curves with a large radius and are not densely packed in one area. Therefore the problem associated with viewing a large portion of a single nanochannel at one time remains.

This problem has been solved by designing channels that are convoluted in a systematic arrangement, _. thereby enabling the fitting of up to 1mm of DNA into a 50μπι x 50μπι field of view. This would allow a human chromosome to be imaged with less than 100 frames corresponding to a few minutes with typical acquisition times.

In one embodiment of the invention, chips are designed to contain meandering channels of the dimensions necessary to display each of a set of chromosomes (e.g. 23 human pairs). Machine vision and chromosome-specific labeling can be used to send the chromosomal DNA from a particular chromosome to the portion of the device containing meandering channels of matching dimensions. A major advantage of the approach is that whole chromosomes can be handled and visualized .individually. Even if breaks occur in the chromosomes, at the minimum the majority of DNA from each chromosome will be kept together and preferably in a contiguous or ordered arrangement. Chips can be made targeting only specific chromosomes of interest, e.g. human chromosome 6 containing the HLA region. The methods of this invention would be suitable for analyzing 1 -3 Mbp stretch of human DNA suitable for HLA analysis. The present invention relates to a nanochannel or a nanogroove that is convoluted in a systematic arrangement so that within a given field of view a large amount of DNA is visualized. The arrangement maximises the length of a single nanochannel that can be seen within the field of view of an imaging device, it also allows the image to be optimised so that the imaging device is used as efficiently as possible whilst maintaining the required level of magnification and resolution to visualise the desired details. In one aspect the application provides a nanofluidic device comprising a substrate comprising one or more nanochannels wherein a single continuous portion of a nanochannel is systematically arranged in a single plane to optimise the length of nanochannel that can be visualised within a single field of view in an imaging device. The nanofluidic device of the invention contains portions of a nanochannel which are arranged so that the maximum length of nanochannel can be visualised at one time at the desired magnification which allows the contents of one section of the nanochannel to be resolved from the adjacent section of the nanochannel. Preferably each single continuous portion of a nanochannel which is systematically arranged comprises a plurality of sub portions or lengths. Each subportion or length is adjacent to another subportion or length so that they are placed alongside one another. The space between the adjacent subportions or lengths in minimised to maximise the length of the single continuous portion of the nanochannel which can be visualised in a single field of view. Preferably the subportions or lengths are relatively elongated with respect to the spacing between adjacent subportions or lengths, i.e the length of the subportion or length is long as compared to the space between subportions of the nanochannel. The continuous portion of the nanochannel is preferably in the form of a labyrinth, i.e. it forms a tortuous path. The arrangement can be coiled for example as a spiral, or as a back and forth serpentine form, to maximise the length of the nanochannel that can be viewed at a single time at the optimal resolution and magnification required. Examples of suitable arrangements are shown in the figures..

,

The term "nanochannel" as used herein refers to a tube-like structure or a nanogroove. A "nanogroove" refers to a trench in a substrate with a cover that leaves a small space above the bottom of the trench. A tube-like structure can have a cylindrical cross-section, or can have planar walls so that it has for example a square or rectangular cross-section. The trench in the nanogroove can have a semi-circular cross-section or have walls with a semi-circular bottom. Alternatively, the bottom of the trench can be of a V shape, or square cross-section. The DNA is prevented from escaping from the nanogroove due to an entropic barrier. The diameter of the nanochannei is preferably 50-500nm, typically 200nm.

The term "systematically arranged" as used herein means that the pattern formed by the continuous section of the nanochannel is such that the subportions or rows are close enough so that they can be resolved by the optical system of the imaging device used to visualise the contents of the nanochannel. Preferably the nanochannel forms a pattern wherein the centre-to-centre distance between adjacent subportions of the nanochannel is uniform. Preferably the overall dimensions of the systematically arranged portion of the nanochannel should match the dimensions of field of view of the imaging means. Alternatively the overall dimensions of the systematically arranged portion of the nanochannels is smaller than the dimensions of the field of view of the imaging means. This enables the entire systematically arranged portion can be^" visualised in a single field of view. Preferably the systematically arranged portion of the nanochannel should be the same overall shape as the field of view of the imaging device e.g. circular, square or rectangular.

In a preferred embodiment the arrangement of the nanochannel is designed such that the molecule within the nanochannel is mostly aligned with the rows of the pixels in the imaging device.

In a preferred embodiment the centre-to-centre distance between adjacent subportions of the systematically arranged single continuous portion of the nanochannel is between 1.5-20 times the width of the nanochannel. As used herein the term "adjacent subportions " refers to the part of the continuous portion of the nanochannel which is next to another subportion of the continuous portion. The centre-to-centre distance is the maximum centre-to-centre distance between different subportions of the continuous portion. For example, if the single continuous portion of the nanochannel is arranged so that it forms parallel lines of the nanochannels linked at each end by a curved section i.e. a back and forth serpentine form, then the centre-to-centre distance is the distance between the nanochannel in one row and the parallel nanochannel on the subsequent row. If the nanochannel takes the form of the spiral then the centre-to-centre distance is measured between one subportions of the continuous portion and the subportions of the nanochannel to the inside or to the outside. The optimal centre-to-centre distance can be calculated based on the imaging means used as discussed below, to obtain optimal resolution. Preferably the centre-to-centre distance is as small as possible while maintaining the possibility to distinguish adjacent subportions. In practice, for common optical microscopes, a preferred centre - to - centre distance is 260nm - 4μπι.

In the preferred embodiment the systematically arranged single continuous portion of the nanochannel takes the form of a spiral or a serpentine. As used herein the term "spiral" refers not only to a curve on a plane that winds around a fixed centre point at a continuously increasing distance from the centre i.e. a circular spiral, but also to other shapes or spirals. For example, the spiral can take the shape of a square or a triangle or hexagon, or other regular shape and form a concentric path emanating from the centre of the spiral. The input and output for the spiral can be either at the periphery and. the centre of the spiral, or both the input and output can be at the periphery. The input and output can also be both at the centre. Examples of suitable arrangements are shown in the figures. As used herein a "serpentine" refers to the arrangement wherein the continuous portion of the nanochannel forms a series of parallel lines which are connected at each end by a curved section i.e. a serpentine form. Such an arrangement is shown in Figure 4. The turns of the serpentine can be made angular or rounded or square. The turns in the serpentine may. create problems during the analysis of the DNA due to different degrees of stretching. To avoid this problem a spiral design can be used. Here the channel is curved with a radius of curvature which is large compared to the cross section of the nanochannel and compared to the persistence length of the DNA. The challenge with this design is that the image analysis is more difficult.

Preferably the total length of the systematically arranged single continuous portions of the nanochannel is up to l OOrrun.The length of each segment can be less than or equal to the size of the field of view. It can also be larger, in which case several images need to be acquired for each molecule. One example is a total length of 1mm with each segment being ΙΟΟμηι in length. Another example is a total length of 10mm with each segment being 200μηι in length.

It is possible to have longer lengths if nanogroove structures as opposed to nanotube structures are used, as the introduction of the DNA into the "serpentine" structure is easier for a groove geometry. In that case the DNA can first be introduced into a slit structure and subsequently transferred to the groove.

The nucleic acid can pass through a serpentine channel so that it is aligned to the rows of pixels in the imaging device such as a CCD. This results in the imaging means being used efficiently and the number of frames necessary to image a long molecule is minimized. For maximum information density each row on the imaging means (such as a CCD) should correspond to one stretch of the molecule e.g. DNA. However to minimize cross talk and sensitivity to misalignment, it may be necessary to have at least one buffer row between each DNA molecule. The adjacent sections of the continuous portion should be sufficiently spaced to avoid blooming and/or bleed through of the fluorescent signal to adjacent pixels in the imaging device.

In order for the nucleic acid to be optically observed in a nanofluidic channel, the channels should be sealed. In addition, at least one side i.e. the substrate or the lid must be optically transparent. The surface of the channels may be negatively charged with a minimal roughness to prevent sticking and entanglement of the nucleic acid. Methods of creating a suitable surface are described in the prior art and below and are known to the skilled person. Ideally the material used to create the channels should be hydrophilic to allow for easy wetting.

The nanochannel device of the invention can contain a plurality of systematically arranged continuous portions. These can be arranged in a suitable design on the nanochip. For example, Figure 2. shows a number of portions which are arranged in a back and forth meander or serpentine form. A plurality of systematically arranged continuous portions can be present on different parallel planes throughout the device. These planes are horizontal when the device is being viewed by the imaging means. The focus of the imaging means can be altered to view each plane.

As used herein the term "imaging device", and "imaging means" refers to any device which can be used to visualise the nucleic acid molecule. This includes charge coupled devices (CCD) cameras, electron multiplying charge couple device (EMCCD), a complementary metal oxide semiconductor (CMOS) detector, intensified CCD, silicon intensified target. (SIT) camera, a 2-D array of point detectors (e.g. Avalanche photodiode (APD) or photomultiplier tube (PMT)) or a scanner. The detectors are typically cooled to -50°C to minimize thermal noise. The imaging devices are conventionally used with a microscope such as an optical microscope or confocal microscope. The images of the nucleotides can be projected on to the imaging device such as the CCD camera. The images are then digitised and stored in the memory. The stored images can then be subjected to image analysis algorithms. These algorithms can distinguish signal from backgrounds, monitor changes in signal characteristics, and perform other signal processing functions! The memory and signal processing may be performed offline on a computer, or in specialised digital processing (DSP) circuits controlled by a microprocessor. The "field of view" as used herein of an imaging device refers to the area which is visible at one time using the imaging means. The dimensions of the field of view, and its size depends on the level of magnification used by the imaging means. With a typically used 512 pixel EMCCD (8.2x8.2 mm^Λ2 detector array), the field of view is 165 μm x 165μιη with 60x magnification, and it scales linearly with the magnification. In a field of view of 165μπι x 165μιη, one can fit nanochannels of 20mm total length, if the nanochannels have a center - to center separation of 1 μηι.

As used herein the term "nucleic acid" refers to DNA as deoxyribonucleic acid and ribonucleic acids (RNA). The molecules can be single stranded, or double stranded. The molecules are preferably double stranded. The examples used herein refer to DNA, but can equally apply to RNA. The term "ultra long " as used herein with reference to nucleic acid molecules refers to a molecule of 0.5-500Mbp. Preferably, this term refers to nucleic acid molecules of l -200Mbp, more preferably 5-100Mbp. For example the molecules are 1Mb, 2Mb, 4Mb, 10Mb, 20Mb, 40Mb, 100Mb, 200Mb or 500Mb long.

In a preferred embodiment the entrance to the systematically arranged single continuous portion of the nanochannel is preceded by one or more obstacles. The nanofluidic device of the invention has a micro-channel i.e. a channel which has a diameter of 1 μιη or more. A large number of nanochannels can be bundled together and formed at the end of the micro-channel. At the end of the micro-channel, a number of obstacles to the flow can be present. These obstacles are, for example, pillars, within the micro-channel, allow the nucleic acid molecule to become disentangled and partially stretched before it reaches the entrance to the nanochannel. Figure 3 shows an example of an array of pillars used to disentangle the nucleic acid molecules and decrease the entropic barrier to entry into the nanochannels. The decrease in the entropic barrier to entry thereby facilitates entry of the nucleic acid molecule into the nanochannel. It also decreases the probability of looped molecules forming and entering the nanochannel. This helps to ensure that the molecules within the nanochannels are linear.

In addition the device may contain a number of obstacles set up in an array in order to direct the flow of fluid, especially those containing agarose beads within the device. For example a "bumper" type array can be used to direct the flow towards on wall of a microchannel from which the nanochannels extend. Suitable arrangements are well known to the person skilled in the art and are described for example in Morton, K.J ., et al., Hydrodynamic metamaterials: Microfabricated arrays to steer, refract, and focus streams of biomaterials. Proceedings of the National Academy of Sciences of the United States of America, 2008. 105(21): p. 7434-7438. The device may also contain obstacles arranged to trap for example an agarose bead containing a nucleic acid molecule and maintain it in a position at or near the entrance to a nanochannel. Suitable trap arrangements are well known to the person skilled in the art and are described for example in Di Carlo, D., L.Y. Wu, and L.P. Lee, Dynamic single cell culture array. Lab on a Chip, 2006. 6(11): p. 1445-1449. Figure 12 shows an example of such traps and "bumper" arrays. The invention provides a system for extracting biomplecules on chip from small amounts of sample material, single cells, nuclei or chromosome in a manner that keeps the biomolecules or biomolecular complexes substantially intact. In particular, in an embodiment applied to genomic DNA, megabase lengths of DNA can be kept intact. Following extraction, the ultra-long DNA is linearly elongated and displayed for detection in the meandering channels. In a further embodiment the chip design allows reagents to be flushed over the DNA and allows features of interest to be labelled and then mapped. Events along the span of the DNA region being imaged can be followed in real-time. The invention provides an unprecedentedly long-range view of the genome, which encompasses the haplotype blocks as well as the structural organization of the genome including repetitive regions. The long-range view will facilitate the de novo identification of a significant amount of previously characterized and uncharacterised copy number/ structural variation.

The major inventive part of this application is that channels are laid out and coupled to the means of detection, in a more efficient way for detecting ultra-long or whole chromosome lengths of DNA than prior approaches. In the prior art there is no suggestion of fitting long lengths of stretched DNA into the field of view of a CCD chip by folding it via a convoluted nanochannel. This not only increases efficiency and throughput of visualizing ultra-long DNA but also enables reactions and interactions on the ultra-long DNA to be monitored in real-time. In addition we describe curvilinear/meandering nanogooves within micro/nano slits, which the prior art has not discussed. These have a particular advantage of facilitating reagent exchange. In alternative embodiments, the nanochannels or grooves can be structured as a spiral or a circle (concentric rings) enabling the following approach. The circle or spiral can be spread over 2-dimensions. The shape, arrangement and dimensions of the circles or spirals are matched to the means for detection. In this case, preferably the method for reading is a compact disc player (CD, DVD, or Blu-ray) or any kind of instrument that operates in a similar manner. The point of this approach is that the use of the method of detection analyses DNA efficiently because the DNA is layed out along a path that the means of read-out can follow rapidly and efficiently, without needing to stop and start many times (although some degree of stop and start can be tolerated). Once the DNA has entered the nanochannels, the disc can be spun and the DNA and any patterning thereon can be detected. Typically, detection is by means of a point detector (e.g. APD, PMT). The point detector may have a means of being translated with respect to the disc. Preferably this translation is from the inside to the outside of the disc or vice versa.

A commercial disc player can be used or adapted to perform the read-out. An advantage of this approach, that the reader is inexpensive and potentially portable. A further advantage is that the spinning of the disc can be used, not only to detect/image the DNA but optionally also to stretch it. The disc can be rotated in one direction only or rotation can be switched back and forth. An overall rotation in one direction can be complemented by back and forth short range rotation. DNA can also be stretched by the application of an electric field. Optionally alternating electric fields can be applied. _. Alternatively, stretching can be achieved by inducing a flow using any means known in the art, including pressure-driven flow. Existing approaches for inducing pressure drive flow can simply be applied at distinct points along the circle or radius. The pressure points can be arranged such that flow induced on a manner that leads to hydrodynamic drag acting on the DNA to ficillate stretching of the DNA. Similarly, the electrical contacts can be arranged to induce electrophoretic stretching of the DNA, which my complemented by hydrodynamic drag acting on the DNA. Existing methods for using hyrdrodynamic drag to facilitate better elongation of the DNA can be adapted (see WO2012056192)

A further advantage of having channels on a spinning disc for stretching of long DNA is that the spinning of the disc can accommodate further functionalities e.g. the extraction and processing of DNA. For example, a 2-dimensional fluidic architecture can be patterned on the disc. Cells can be loaded onto the outer part of the disc. On spinning of the disc at a sufficient speed, centripetal force can push the material inward. Material comprising cells, chromosomes or DNA can, for example be forced through narrow aperture which has a physical effect on said material. In the case of cells, under appropriate conditions the physical effect can facilitate or allow the bursting of the cell and extraction of DNA.

Spinning the disc to different speeds can serve to separate subcomponents of a biological material according to size. For example, chromosomes from the human set of 46 can be separated to distinct radial sites on the spinning disc. DNA from an , individual chromosome then enters into a radially arranged nanocharinel in which it is . stretched/elongated. A chromosome may be accommodated within a single turn of circle or in .multiple turns, depending on the radius of the disc.

Complex nanofluidic architectures can be patterned on to a disc by any means for making micro and nanofluidic structures. This includes injection molding and soft lithography as well as traditional micro and nano lithography approaches. For simple concentric channels, a disc writer can be used, as found on personal computers. For the mode of operation described in the preceding few paragraphs, a shape of substrate, other than a disc can be optionally used as long as it can be rotated.

Fabrication of Nanochips

There are a multitude of ways to fabricate nanostructured Chips depending on the facilities and equipment available. One common fabrication method uses nanoimprint lithography (NIL). This has the benefit that it is possible to order finished master stamps commercially (for example available from NIL Technology, Denmark), thus eliminating the need for an electron-beam lithography system. A common mass production technique, capable of defining nanostructures, is injection molding. With suitable choice of low-fluorescence polymer matrix it may prove useful for large series of devices. Although focused ion beam (FIB) milling is a slow linear technique, it may find use for creating complicated three-dimensional structures with resolution comparable to that of electron-beam lithography.. Direct laser writing systems (for example available from Nanoscribe GmbH, Germany) are now also capable of creating complex three-dimensional structures with feature sizes below 100 nm.

A multitude of more exotic alternative fabrication techniques are described in the literature. A fabrication scheme based on fused silica wafers is described below, for which the following is needed:

• Fused silica wafers. (Available from Hoya)

• Ι ΙΟμιη thick fused silica coverslipsTor sealing of the chips. The thickness is optimized for compatibility with oil immersion objectives. (Available from Valley Design)

• Access to cleanroom equipment for photo (tJV) and electron- beam (e-beam) lithography and reactive ion etching (RIE) as well as standard resists (e.g. AZ (photo lithography) and ZEP (e-beam lithography) resists) and chemicals from any large supplier.

A commonly used fabrication process based on fused silica is outlined below.

A full-scale cleanroom, with spinners for resist deposition, mask aligners for exposure of micron scale patterns and an electron-beam writer for definition of nanoscale structures, is required. Even without an e-beam writer, slit-like channels (depth in the nanometer range and widths larger than 0.5 μιη) can readily be defined with UV lithography and carefully tuned RIE etching. Reactive-ion etchers are used for etching channels with straight walls.

In order to align the nanostructures and the rnicrochannels it is useful to first define alignment marks in the wafer periphery. This can be done by either etching or depositing metals on the wafer, the latter described below. Standard processes can be used for the following steps.

Definition, of alignment marfa 1. Treat the fused silica wafers with HMDS (hexamethyldisiiazane) to increase resist adhesion.

2. Spincoat and bake a combination of resists used for liftoff, e.g.. a LOR/AZ or other similar sandwich constructs, to enable a pattern with an undercut.

3. Expose and develop the resist to create the undercut structure.

4. Run a low-power oxygen descum plasma to remove remaining resist residues.

5. Evaporate a 5 nm Cr (or Ti) adhesion layer and subsequently a 50-80 nm thick Au layer.

6. Strip the resist using a chemical stripper, e.g. Microposit Remover 1165 or acetone. Instead of using chemicals, the resist can be stripped by an oxygen plasma treatment. However, this is not recommended since it can burn the resist,, making it very hard to remove, and also induces roughness on the sample surface.

Alignment marks can alternatively be formed by anisotropic RIE etching, and in the case of silicon also through anisotropic wet etching using e.g. KOH. If etching is used to define the alignment marks it is important that they provide a sufficient contrast for the alignment in the mask aligner, an etch depth of at least 200 nm is recommended.

For metal alignment marks it is also possible to first deposit a layer of metal and subsequently spin on and pattern a photoresist and in a last step etch away the exposed metal. Al is commonly etched using either a wet etch using phosphoric acid or a dry etch containing chlorine chemistry. Au is commonly etched by using wet etches of either potassium iodine or aqua regia (1 :3 HN(¾:HO).

Definition of nanochannels

1. Treat the fused silica wafers with HMDS to increase resist adhesion.

2. Spincoat and bake a 150-250 nm thick layer of ZEP520A e-beam. resist. ZEP is chosen because of its good dry-etch resistance. Other resists can be used but they often require deposition of an extra metallic etch mask.

3. Thermally evaporate 15 nm Ai on top as a discharge layer. This is only needed ,. ^· when working with isolating substrates such as fused silica.

4. Expose the resist (exposure dose approximately 280 μθ/αιι² at 100 kV). 5. Remove the Al layer using a suitable developer e.g. MF322 developer.

6. Develop the resist using a suitable developer e.g. ZED N50 developer.

7. Run a low-power oxygen descum plasma in order to remove remaining resist residues.

8. Etch the nanochannels into the fused silica using RIE with CHF3/CF₄ chemistry.

9. Strip the resist using a chemical stripper, e.g. Microposit Remover 1165. Definition of microchannels

Standard processes can be used to carry out the following steps.

1. Treat the fused silica wafers with HMDS to increase resist adhesion.

2. Spincoat and bake a 2-5 pm thick layer of photoresist, e.g. an AZ resist, that has relatively high etch resistance.

3. Expose and develop the resist.

4. Run a low-power oxygen descum plasma in order to remove, remaining resist residues.

5. Etch the microchannels (approximately 1 pm deep) using RIE with CHF₃/CF₄ chemistry.

6. Strip the resist using a chemical stripper, e.g. Microposit Remover 1165 or acetone.

Drilling of access holes There is a multitude of ways of producing access holes through a wafer. Examples include micromilling, deep reactive ion etching (DRIE) Or ultrasonic drilling. However, these techniques often demand some specialized equipment, which is very expensive compared to that needed for powder blasting. A setup based on powder blasting is described below.

1. Spincoat at least 5 pm photoresist on both sides of the wafer.

2. Cover the backside (i.e. the non-structured side) with an adhesive plastic film, e.g. 70 pm thick Nitto SWT 20 film. Instead of using a soft film, in order to mask the wafers/chips during powder blasting, a metal mask, defined in a thick brass plate, can be used. The chip is then attached to the metal mask using reversible thermal glue. It should be noted that since the metal mask is hard, it wiil also be degraded by the powder blasting, which attacks hard surfaces.

3. Make holes through the film over the reservoir structures using a scalpel or e.g. laser ablation.

4. Powderblast using 50-1 10 μτη sized A1₂0₃ particles from the backside of the wafer (i.e. the non-structured side). A small powder-blasting tool and the powder can be obtained from Danville Eng.

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5. Remove the film, strip the resist in a chemical stripper and/or acetone and carefully clean the wafers in an ultrasonic bath.

Sealing of the chips The last step in the production of the chips is sealing. This can be done in several different ways depending on the material of the chips. Polymer-based devices are generally sealed using polymer fusion bonding. The device is bonded to a lid with a polymer film by heating until the polymer layers on the chip and lid intermix. The combination of polymer compositions and temperatures must be carefully chosen to create a sufficiently strong bond while maintaining the structural integrity of the micro- and nanochannels. Anodic bonding is the standard technique to bond borosilicate glass to silicon, also for silicon with a hydrophilic oxide layer, but it might cause wide nanochannels (nanoslits) to collapse. Fused silica can be bonded covalently via condensation of hydroxyl groups when two surfaces are brought together. Table 1 summarizes two standard ways of creating a high density of the necessary hydroxyl groups, involving thorough cleaning to remove organic residues and subsequent surface activation.

Table 1 : Two fusion-bonding protocols for fused silica. The Piranha-based protocol can be used to bond silicon with a thin layer of oxide (< 150 nm) and borosilicate glass. However, the final annealing should in this case be done at 400-450°C to avoid excessive strain due to the difference in thermal expansion coefficient between silicon and glass.

For the RCA-based method the hydrogen peroxide should be added after the mixture has reached the correct temperature to avoid disintegration of the hydrogen peroxide.

Spacing of nanochannels

The nanochannels should be positioned so as to minimise the space between adjacent subportions or lengths of the nanochannel. However, they need to be far enough from each other that they can be resolved by the optical system in use, otherwise cross talk might contaminate the signal. For standard optical microscopy the resolution is given by the Abbe limit ^{o i>6}^^NA which translates into about 235nm for a microscope with the wavelength of the observed light

1.40.

For example, a typical camera is the Andor iXon with a 512x512 pixel electron multiplying EMCCD chip. The physical pixel size on the charged couple device (CCD) is l 6Mm. In the ideal case with an ultrastable stage, an ultra-dense nanochannel array and with an ideally working optical microscope, it is possible to align each row in the systematically arranged portion of the nanochannel to a row in the CCD. To utilize all pixels the magnification must be chosen such that the center-to-center distance of neighboring pixels is greater than the optical resolution given by ^NA (~ 235nm in the above example). Otherwise there will be cross-talk between the pixels, i.e. each pixel will contain significant information from the two neighboring strands of DNA. This means that the magnification must be less than A standard

60x objective is therefore be adequate giving a pixel separation of 267nm. In this case the center-center distance between the nanochannels must be 267nm and with a typical width of lOOnm of the nanochannels, we need a channel separation of 167nm.

In another example assuming a less stable microscope, the channels may be separated ^' so that they are Ιμιη apart. With a 60x objective each DNA then has a little less than 4 pixels in width making the setup more robust to drift and misalignment.

The CCD chip is typically 8.2 x 8.2 mm² in size. With a 60x objective and channels separated by 1 μηι this gives us a total length of DNA per frame corresponding to 8200μm /60 x '8200/60 = 19mm. With typical buffer conditions of 0.5xTBE and a typical, channel effective diameter of lOOnm we have a relative extension of -50%. Working with DNA stained with for example a typical 1 YOYO™-l dye per 10 bp, the contour length is 0.4 lnm per bp. The 19mm therefore corresponds to a DNA of length of 46Mbp. .

A bacterial genome is typically in the range 1-lOMbp. This makes it possible to have more than one genome per field of view ensuring a high throughput in applications where the bacteria are identified by for example a barcode pattern that provides a means for identification on a single molecule level without cell growth or DNA amplification. DNA barcoding uses short genetic markers in an organism's DNA to identify the species. Applications may include .diagnostics of infectious disease and mapping of the (human) microbiome. Apart from bacteria, relevant microorganisms may include yeast and archaea.

To image the entire human chromosome number 1 (size 247Mbp) in a nanochannel arrangement of the invention one would therefore need 247/46=5.4 field of views, each field of view acquired during at least 100 frames each 100ms. The total time for acquiring an image of the chromosome 1 is thus on the order of 1 minute. For the entire human genome which comprises

bp, the required number of fields of view is

taking on the order often minutes to complete.

The acquisition can be sped up at least a factor of ten by increasing the light intensity to decrease the total acquisition time necessary for an adequate signal to noise ratio. The acquisition can also be sped up by using larger channels, or higher salt concentration in the buffer used resulting in a smaller degree of relative extension of the molecules. The degree of stretching can be decreased at least a factor of ten. However, this also decreases the attainable resolution in terms of basepairs correspondingly, so a balance has to be made to optimise results.

The acquisition can also be sped up by using lower magnification thereby creating a larger field of view, possibly combined with a CCD with more pixels (such as a 1024x1024 EMCCD also available from Andor, Photometries, Hamamatsu and other camera manufacturers) to compensate for the lower resolution.

Using the recent development in super-resolution microscopy the Abbe limit is broken and the resolution is improved an order of magnitude. This allows for much more densely packed channels. Given a certain size of the CCD, a balance must be struck between desired throughput (number of bp per unit time) and resolution (in terms of number of bp). To facilitate the imaging of the DNA, the output channels are typically connected to the outside on the trans side from the microscope objective. Stretching DNA

The polynucleotide molecules can be stretched to increase the spacing between individual nucleotides and so improve resolution. However stretching the molecule decreases the number of nucleotides which can be seen in the nanochannel at one time. Therefore a balance heeds to be made between the two parameters. This can be determined by the person skilled in the art. The molecules can be stretched by the nucleotides entering and being confined in the nanochannels. The diameter of the nanochannel affects the amount the molecule is stretched. For example a relatively small diameter will cause the molecule to be stretched more than a nanochannel with a relatively large diameter through which the molecule can pass more freely. The molecules can also be stretched by using forces acting on the ends of the molecule such as fluid flow, tethering and laser trapping. Alternatively or in addition to salt concentration can be manipulated to alter the degree of stretching. Preferably the nucleotides are stretched by 25%, 40%, , 75%, 100%, 150% or 200%. of the crystallographic DNA length. Stretching 100% refers to the crystallographic length of the DNA which for unstained nucleic acid, which for DNA of B-form is 0.34nm per base pair; 100% stretching corresponds to the contour length of the nucleic acid, i.e. the extension of the molecule when it is completely aligned along a straight line. A molecule cannot be stretched by confinement only to more than 100%. However, during the introduction of the DNA into the nanochannels it will experience forces that may . stretch it to more than 100%. The DNA may relax to its equilibrium extension once it is located inside the nanochannel. The polynucleotides can comprise chromatin i.e. proteins associated with the nucleotides or it can be substantially free of proteins.

If the polynucleotide breaks during its progress through the nanochannel the order of the fragments is preserved and so can still be analysed.

An entire polynucleotide can be visualised by taking several images of one systematically arranged portion as the molecule passes through the nanochannel. This is shown in Figures 8 and 9. This enables the whole length of the molecule to be visualised. If the molecule is shorter than the length of the systematically arranged portion then the entire length can be visualised in a single imageThe DNA molecule can be moved through the channel by for example pressure driven flow of the liquid, by electrophoresis or by electroendosmosis.

Background fluorescence in all its forms, including light scattering or ^' intrinsic fluorescence of any of the materials used in the analysis and from contamination of particulate and molecular matter, should be minimised during methods of the invention.

In a further aspect the application relates to a method of visualising a single molecule comprising introducing said molecule into a systematically arranged single continuous portion of a nanochannel of a nanofluidic device of the invention, and viewing said molecule on an imaging device. Preferably, the molecule is exposed to a suitable dye prior to application to the nanofluidic device. In a preferred embodiment the molecule is a nucleic acid molecule, more preferably an ultra-long nucleic acid molecule. A "dye" as used herein preferably relates to an intercalating dye i.e. a dye which sticks between two nucleic acid strands of a double stranded molecule. Examples of these dyes include the dimeric cyanine (normally intercalating) dyes POPO-1, BOBO- 1 , YOYO- 1, TOTO-l , JOJO-1, POPO-3, LOLO-1 , BOBO-3, YOYO-3, TOTO-3. There are also normally groove binding dyes such as DAPI and SYBR green 1. Oligreen dye can be used to label ssDNA.

The target DNA is preferably double stranded but single stranded (ss) DNA can also be analysed. Denaturation conditions (including, formamide, urea, DMSO and/or single-strand binding proteins etc) will preferably be used with ssDNA. In. a further aspect the present invention provides a method of detecting the presence of a micro-organism comprising obtaining a nucleic acid from said micro-organism, introducing said nucleic acid into a systematically arranged single continuous portion of a nanocharuiei of a nanofluidic device and viewing said nucleic acid using an imaging device. A nucleic acid molecule can be obtained from a micro-organism using any standard technique available to the skilled person. Preferably the nucleic acid is contacted with a suitable binding agent prior to introducing the nucleic acid into the nanofluidic device. As used herein a "binding agent" refers to any molecule which binds to the nucleic acid. This includes nucleic acid dyes, such as intercalating dyes discussed above. The term "binding agent" also refers to molecules that bind sequence specifically to the nucleic acid such as non-cutting restrictions enzymes, other nucleic acid binding proteins, DNA analogues such as PNA (peptide nucleic acid), gamma PNA (with or without G-clamps) & LNA (locked nucleic acid), specific constructs such as molecular beacons (hairpin loops), padlock probes. These binding agents are preferably labeled with a fluorescent marker, for example a quantum dot. The presence of a micro-organism may be detected by a specific "bar code" created by |the binding agent. This would allow the identity of any micro-organisms present to be analysed. This technology would be useful in diagnosing infection without the need for culturing any micro-organisms present in a sample prior to analysis. Microorganisms include viruses, bacteria, fungi, protozoa, algae and other single celled organisms. These methods can also be used to analyse nucleic acids from any organism. For example structural variations such as copy number variations, translocations, and inversion can be detected along chromosomal DNA. In addition these methods can be used to identify the presence of. contaminating nucleic acid, or to determine the origin of unknown tissue samples. For example, the presence of a contaminant in a food product can be identified.

The nanochips of the invention can be used to map interactions between polynucleotides and other molecules. This can be used to carry out real-time imaging of multiple signals. The DNA can be fingerprinted, barcoded or mapped. This mapping typically identifies feature that occur at a plurality of sites along the elongated polynucleotide. The mapping may comprise peptide, protein and antibody binding. Sites of DNA modification,, such as methylation, hydroxymethylation, DNA breakage or nicking, depurination, adduct formation and drug binding can be mapped. The following can also be mapped: binding of transcription factors or polymerase loading- binding of RNA, to define regions of the genome containing genes; repetitive regions of the genome, which may particularly be useful for DNA fingerprinting for forensic and other applications.

In a further aspect of the invention, it relates to a method of sequencing a nucleic acid molecule comprising introducing said nucleic acid into a systematically arranged single continuous portion of a nanochannel of a nanofluidic device, adding suitable reagents to initiate the sequencing reaction, and monitoring the sequencing reaction using an imaging device. Preferably, the nucleic acid molecule is fixed once it has entered the systematically arranged portion of the nanochannel. This will prevent the nucleic acid molecule moving through the channel whilst the sequencing reaction takes place. This will allow the reaction to be monitored more easily. The reaction can be monitored in real-time. Suitable sequencing reagents are well known to the person skilled in the art. The addition of a specific base can be monitored, for example by adding a specific dye to each of the bases, e.g. A, G, T, C. Methods for carrying out sequencing in suitable reagents are well known to the person skilled in the art.

The nucleic acid can be fixed in space in various ways. The electrostatic interaction between the nucleic acid and the walls can be tuned by external electrodes and by changing the internal chemistry of the channels as described in US2008242556A1. The DNA can also be embedded in a dynamic gel structure that can be controlled by external means such as light, change in temperature and change in. local chemistry. .

In another aspect the present invention provides a kit comprising the device of the invention together with suitable reagents, and optionally instructions. For example the suitable reagents can be selected from buffers, dyes, binding agents, anti-fade reagents, additives to prevent breakage of the nucleic acid, sequencing reagents. Suitable sequencing reagents include but are not limited to fluorescent nucleotides, dyes, polymerase enzymes, quantum dots. In a further aspect of the method, DNA can be delived to the nanofluidic structures of the device by encapsulation droplets or agarose beads. Agarose bead methods for delivering DNA are described in the Figures 12 and 13. Chromosomes prepared by .the polyamine method can also be encapsulated in agarose beads, wherein they can be . biochemically manipulated (e.g. addition of protease to degrade histone proteins etc, binding chromosome-specific probes) and washed (e.g. by transferring the beads into different wash buffers, before or after loading onto the chip). The chromosomal DNA in the beads can then be. delivered to the nanofluidic part of the device as shown in Figs 12 and 13.

In a further aspect, superresolution as known in the art, can be used to resolved between DNA in closely spaced nanochannels or to resolve features matched along the DNA. See the following references for superresolution methods that can be applied to stained DNA: Ingmar Schoen, Jonas Ries, Enrico Klotzsch, Helge Ewers, Viola 201 1 Nano letters vol. 1 1 (9) pp. 4008-1; Flors, C. Biopolymers 2010, 95 (5), 290-297; Flors, C. Photochem. Photobiol. Sci. 2010, 9 (5), 643-648; Flors, C; Ravarani, C. N. J.; Dryden, D. T. F. ChemPhysChem 2009, 10 (13), 2201-2204; Persson, F.; Bingen, P.; Staudt, T.; Engelhardt, J.; Tegenfeldt, J. O.; Hell, S. W. Angew. Chem., Int. Ed. , 201 1 , 50 (24), 5581-5583; Jungmarui,R.;Steinhauer,C.;Scheibie,M.;Kuzyk,A.;Tirmefeid, P.; Sirnmel, F. C. Nano Lett. 2010, 10 (1 1), 4756-4761.

In a further aspect the present invention provides a mould for use in the prepartion of a device of any one of claims 1 to 6 by injection moulding.

In another aspect the present invention provides a master stamp for use in the prepartion of a device of any one of claims 1 to 6 by nanoimprint lithography.

In a further aspect, to facilitate the study in nanochannels of the interaction of ultra- long linear DNA with proteins a highly effective passivation scheme based on lipid bilayers can be implemented [see Persson et al. Lipid Passivation in Nanofluidics. Nanoletters, DOI: 10.1021/nl204535h (2012)]. This can enable virtually complete long-term passivation of nanochannel surfaces to a range of relevant reagents including streptavidin-coated quantum dots, RecA proteins and RecA-DNA complexes. The performance of the lipid bilayer is significantly better than that of standard BSA-based passivation. The passivated devices allow us to monitor single DNA cleavage events during enzymatic degradation by DNAse I. We expect effective passication of the meandering channels described in the invention will open up the detailed, systematic study of a wide range of protein- DNA interactions with high spatial and temporal resolution. Other surface coatings or passivation additives can be tested, using labeled proteins and coatings to which proteins of various charges stick the least can be chosen for implementation in the device. See Banerjee et al

[Advanced Materials 23: 690-718, 201 1] for various anti-fouling methods that can be tested.

The invention will now be described with reference to the non-limiting examples below which refer to the following figures:

Figures

The invention will be described with reference to the following examples which refer to the figures as described below.

Figure la shows the design of a "meander" or "serpentine"chip.

Figure lb shows the design of a portion of a "meander" or "serpentine" chip. The continuous nanochannel contains a plurality of subportions or lengths (1 ,3,5,7) which are placed alongside one another. The subportions or lengths are relatively elongate with respect to the spacing between adjacent subportions or lengths, i.e the length of the subportion or length (a) is long as compared to the space between subportions of the nanochannel (b).

Figure, lc shows the design of a portion of a circular "spiral" chip. The continuous nanochannel contains a plurality of subportions or lengths (1 1 ,13,15), which are placed alongside one another.

Figures Id and l e show spiral arrangements of the nanochannel: Figure I d shows a spiral arrangement where both the input ad output are located at the periphery. Figure le shows an arrangement where the input is located on the periphery whilst the output is located centrally. The input and output could also be reversed.

Figure I f shows an alternative arrangement for the continuous portion of the nanochannel.

Figure 2 shows the overall design of a meander or "serpentine" channel. Arrays of. posts are made close to the entry zones to help disentangle long nucleic acid molecules. The entry channels are placed close together so that the local flow is enhanced.

Figure 3 shows details of the entry zone. A pillar array is used to disentangle the nucleic acid molecules to effectively decrease the entropic barrier to entry into the nanochannels.

Figure 4 shows details of the serpentine (back and forth meander) pattern. The turns are defined so that most of the nucleic acid is aligned in parallel lines. The turns are circular with a radius of curvature much greater than the persistence length of the nucleic acid molecules.

Figure 5 shows an actual device depicted using standard brightfield microscopy. This is an image of 200nm x 200nm channels.

Figure 6 shows Brightfield optical microscopy image of 200nm x 200nm channels.

Figure 7 shows yeast. chromosomes in serpentine channel (200nm x 200nm). A single 90μιη molecule (left). Two molecules, 55μηι and 120μηι. The longer molecule is folded within the channel or co-localised with a smaller molecule as indicated by the increased intensity. The serpentine is 50μπι wide.

Figure 8 shows the average of multiple sequential fluorescence images of DNA in the serpentine channel-(200nm x 200nm). The serpentine is 50μιη wide.

Figure 9 shows a sequence of frames of yeast DNA in serpentine channel (200nm x 200nm). The exposure time is 100MS. The serpentine is 50μιη wide.

Figure 10 shows chromosomal DNA from Schizosacharomyces pombe. To the left the DNA is extended due to the forces exerted during the introduction of the DNA into the nanochannel. After 10 minutes the DNA relaxed towards its equilibrium length as shown on the right.

Figure 11 shows a chromosomal length of DNA from Schizosaccharomyces pombe. Figure 11a shows the chromosome stained with YOYO-1. Figure 1 lb shows the melt pattern at 45 °C

Figure 12 shows the key steps for using agarose beads to introduce nucleic acid molecules into the nanochannels of the device

Figure 13 shows the bumper array, trapping and escape mechanisms in ,the device for use with agarose beads.

Examples

A person skilled in the art will be able to practice the invention. The literature and patents cited in this specification can be used for guidance. Furthermore, a number of examples are given below to facilitate the person skilled in the art to carry out the invention. A device was designed with a standard layout consisting of fairly large micron channels for bulk transport of sample and nanochannels connecting the micron channels, as shown Figure la. . Two considerations were made for the design of the entry areas of the nanochannels. To ensure a reasonable flow rate towards the nanochannels in the micronchannel, a large number of nanochannels were bundled together (Figure 2). In practice this ensures that the capture area in the micronchannel is sufficiently large for a reasonable number of DNA molecules to be captured at typical DNA concentrations (~0.5pg/mL).

The second consideration concerns limiting the entropic barrier to entry of the DNA into the nanochannels. By defining pillars in the microchannel (Figure 3), the DNA is disentangled and partly stretched before it reaches the nanocharmel. This decreases the entropic barrier to entry thereby facilitating entry of the DNA into the nanochannels. It also decreases the probability of loops entering the nanocharmel.

The serpentine (Figure 4) is defined so that most of the DNA is aligned in parallel lines. The turns are circular with a radius of curvature much greater than the persistence length of the DNA.

Actual devices are shown using standard brightfield microscopy (Figure 5 and Figure 6). DNA from lambda-phage virus (48.5kbp) and from Saccharomyces cerevisiae (from 225kb to 1900kbp) were stained with YOYO™-l (Invitrogen) according to standard protocols and diluted in 0.05x TBE buffer + lOmM NaCl containing 3% beta- mercaptoethanol to minimize photobleaching. The base-dye ratio was 10.T . This causes the contour length of the DNA to increase 20%. The yeast DNA was obtained from New England Biolabs in a gel plug and extracted from the gel plug by dissolving the gel plug in an _.eppendorf test tube. The gel plug is optionally melted by heating (e.g between 45 and 65 degrees C). The gel is optionally digested using Agarase enzyme.

Both lambda phage and yeast DNA was introduced into the nanochannels (Figure 7). The channel cross section is 200nm x 200nm so that the extension is expected to be ~0.25 (25%) for the buffer conditions used. The DNA molecule in the left frame of Figure 7 is 90μηι long. With an estimated extension of 0.25, this indicates a contour length of 360u^'m, which in turn corresponds to a length of 880kbp. The longer molecule in the right frame of Figure 7 is at least IMbp long.

To give an impression of the DNA moving in the serpentine, an average was made of a molecule moving from one end to the other (Figure 8). Another example is given in Figure 9. Here sequential frames, separated by I s, are shown.

Chromosomal DNA from Schizosacharomyces pombe was obtained using standard extraction techniques, It was diluted in a buffer containing 5mMNaCl and stained with YOYOl . The DNA was introduced into a serpentine nanocharmel device of the invention, and maintained at 31°C. As shown in Figure 10 the molecule was initially extended due to the forces exterted when the molecule entered the nanocharmel. After 10 minutes the molecule had relaxed and shortened towards its equilibrium length.

Chromosomal DNA from Schizosacharomyces pombe was obtained using standard extraction techniques, It was diluted in a buffer containing 5mMNaCl and stained with YOYO1. The DNA was introduced into a serpentine nanocharmel device of the invention, and maintained at 45°C. A DNA melting pattern was obtained using standard methods. The dye is released as the DNA denatures. This produces a pattern where single stranded regions, are seen as darker areas compared to double stranded regions. The pattern obtained for the chromosomal DNA is shown in Figure 1 lb.

As an alternative to using DNA in a gel plug, chromosomes suitable for loading onto the chip can be prepared by the poly amine method as described by Cram et al. [L. S. Cram, C. S. Bell and J. J. Fawcett, Methods Cell Sci., 2002, 24, 27-35] and pipetted directly into the device. The proteins binding to DNA in a chromosome can be digested using a protease to release substantially naked DNA.

Melting mapping methods described in WO2012056192 can be implemented before DNA is passed into the meandering channels.

An example of a superresolution switching buffer comprises: 10 mM phosphate buffered saline (PBS, pH 7.4, Sigma P3813) with an oxygen scavenger (0.5 mg ml-1 glucose oxidase (Sigma), 40 mg ml-1 catalase (Sigma) and 10% w/v glucose (Fischer Scientific)) and 50 mM b-mercaptoethylamine (MEA, Fluka). Replacement of MEA with b-mercaptoethanol confers anti-fade, and anti -light-induced breaking of the DNA.

A number of different approaches area available for extracting nucleic acids from single cells or nuclei isolated can be integrated with the devices of this invention. A number of suitable methods are reviewed in Kim et al. Integr Biol 2009 vol. 1 (10) pp. 574-86. Cells can be treated with KCL to remove cell membranes. Cells can be burst by adding a hypotonic solution. A variety of different chemical and physical lysis methods can be implemented as known in the art and previously tested in microfluidics.

Lipid Passivation

For the creation. of lipid bilayers (LBLs) on the surface of nanofluidic channels we used zwitterionic POPC (l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) lipids with 1% Lissamine™ rhodamine B l ,2-dihexadecanoyl-s«-gtycero-3- phosphoethanolamine, triethylammonium salt (rhodamine-DHPE) lipids added to enable observation of the LBL formation with fluorescence microscopy. Prior to each coating procedure, lipid vesicles of approximately 70 nm diameter were created by extrusion (see ESI). The extruded vesicle solution was flushed through one of the microchannels of the fluidic system. Subsequently, the lipid vesicles settle down on the surface, rupture and form patches of LBL that connect within a few minutes to a continuous LBL, coating the entire microchannel. The LBL is subsequently allowed to spread spontaneously into the nanochannels while the flow of lipid vesicles is sustained in the coated microchannel to ensure a steady supply of vesicles. During the coating process a counter flow (~80μιη/$) through the nanochannels is imposed into the coated microchannel to avoid any debris or vesicles in the nanochannels. An alternative' slightly quicker method was also tested involving flushing lipid vesicles from the LBL-coated microchannel through the nanochannels results in deposition and rupture of lipid vesicles inside the nanochannels. However, with this method care needs to be taken to prevent vesicles and other residues from getting deposited and potentially, blocking the nanochannels.

Capturing nucleic acid molecules within the nanochannels using beads.

Figure 12 shows the basic steps for capturing nucleic acid molecules' in the nanochannel of the device by using beads, such as agarose beads. Beads containing a nucleic acid molecule are introduced into the device via a microchannel. The beads are small enough to freely pass through the microchannel but are too large to enter the nanochannel. The nanochannels are arranged to extended from the walls of a microchannel. A trap arrangement is present in the microchannel adjacent to the entrance of each nanochannel. The trap arrangement holds the bead in position adjacent to the nanochannel entrance. A gentle flow is applied to cause the beads to pass through the microchannel and enter the trap. The beads are maintained in the trap by the gentle flow. The nucleic acid molecule can be extracted from the agarose bead and enters the nanochannel by applying an electric field. The obstacles or pillars which make up the trap arrangement are placed so that a bead can be captured and maintained in a position at the nanochannel entrance whilst a gentle flow is maintained. However, they are positioned far enough apart that a bead can pass through on application of sufficient pressure. Once the molecule has entered the nanochannel a strong flow can be used within the microchannel to force the empty bead out of the trap, thus clearing it for a subsequent molecule if desired.

The beads can be encouraged to enter the traps by using a "bumper" arrangement, for example as shown in Figure 13. Obstacles such as pillars are placed within the microchannel of the device to direct flow and^' the beads towards the wall of the microchannel where the trap arrangements are placed. Preferably an obstacle is placed adjacent to the trap arrangement. The spacing between the obstacle and the trap is such that a bead will pass through if the trap already contains another bead. If the trap is empty the obstacle is arranged so that the bead will enter. These arrangements help to improve the efficiency of molecule capture in the nanochannels.

Sequencing reactions

Sequencing reactions are carried out inside the chip, preferably upon DNA or a nucleic acid molecule which is elongated in the serpentine nanochannel. There are a substantial number of cyclical sequencing by synthesis biochemistries which have been described that require a exchange of reagents after each base incorporation. Coupling an efficient reagent exchange system to the nanochannel enables this approach to be carried out. The reagent exchange can be carried out by accommodating the DNA in a nanogroove within a slit and then iteratively flushing through the desired reagents through the slit. The reagents are able to diffuse into the nanogrooves from the overlying slit, without dislodging the DNA.

The system is particularly suited to real-time sequencing, where little or no iterative reagent exchange is needed. In this, the DNA or nucleic acid molecule is elongated in the nanochannel alongside reagents that are required to carry out a DNA synthesis reaction. The synthesis reaction is comprised of components that can make up FRET donor pairs. In one example the polymerase is labeled with a Quantum Dot and the base is read by detection of FRET from the labeled polymerase and one of 4 dyes that are attached to each of the four bases; a typical Qdot donor may be Qdot 605 and the acceptor may be cy5.5. When the nucleotide is being incorporated FRET can be detected. Once incorporation is complete, the label is free to diffuse away. See for example, WO/2005/040425, WO2010002939, and WO/2010/11 1674. Also see http://www3.appliedbiosystems.corn/cms/groups/global_marketing_group/documents/ generaldocuments/cms_091831.pdf.

Alternatively the FRET donor can be an intercalating dye and the acceptors can be a label on the nucleotides (see WO/2005/040425). It is preferable to passivate the inner surface of the nanochip. Nodes of DNA synthesis can be initiated at multiple points along the polynucleotide in the nanochannel. The nodes of synthesis can be initiated from primers that have bound to the target or from nicks created in the DNA. The nicks can be created randomly by DNAse 1. Standard nick translation reactions can be implemented with regular nucleotides being replaced with fluorescently labeled nucleotides. Nicks can also be created in a more controlled and sequence specific manner by one or more Nickas^'e enzymes. DNA synthesis can also be initiated from the ends of molecules. Preferably antioxidants are included in the reaction mix. Where nodes of synthesis are initiated at multiple locations along the molecule then to add redundancy, synthesis from a 5' node overlaps with a node that has started further downstream. A strand displacing polymerase such as Phi29 can be used. A 5 '3 ' exonuclease activity possessing enzyme such as Taq DNA polymerase can also be used; here rather than displacing the existing strand, the existing strand is digested away. Preferably the nucleotides contain a label at a terminal phosphate position. It is preferable that extra phosphates beyond the three normal ones are added in a modified nucleotide. Substituting manganese for magnesium improves the incorporation of terminal phosphate modified nucleotides. Engineering the polymerase improves the incorporation of nucleotides. Specifically, the polymerase can be engineered or evolved to minimize insertion errors and if required to increase processivity, fidelity and to remove 3 '5 exonuclease activity. Preferred dyes that label the nucleotides include Cy3B and Atto647N. Where the polymerase is labeled with a dye or nanoparticle , it may be preferable to label the polymerase with more than one dye and said dyes may interact by FRET.

Claims

Claims

1. A nanofluidic device comprising

a substrate with a surface comprising one or more nanochannels wherein a single continuous portion of a nanochannel is systematically arranged to optimise the length of nanochannel that can be visualised within a single field of view of an imaging device.

2. The nanofluidic device of claim 1 wherein the single continuous portion of a nanochannel which is systematically arranged comprises a plurality of sub portions or lengths wherein each subportion or length is adjacent to another subportion or length.

3. The nanofluidic device of claim 1 or claim 2 wherein the centre to centre distance between adjacent sub portions or lengths of the systematically arranged single continuous portion of a nanochannel is between 1.5-20 times the width of the nanochannel.

4. The nanofluidic device of any one of claims 1 to 3 wherein the systematically arranged single continuous portion of a nanochannel takes the form of a labyrinth.

5. The nanofluidic device of any one of claims 1 to 3 wherein the systematically arranged single continuous portion of a nanochannel takes the form of a spiral or serpentine.

6. The nanofluidic device of any one of claims 1 to 5 wherein the entrance to the

systematically arranged single continuous portion of a nanochannel is defined by one or more obstacles.

7. A method of visualising a single nucleic acid molecule comprising introducing said molecule into a systematically arranged single continuous portion of a nanochannel of a nanofluidic device of any one of claims 1 to 6 and viewing said molecule on an imaging device.

8. The method of claim 7 wherein said molecule is a nucleic acid molecule.

9. The method of claim 7 or claim 8 wherein the molecule is contacted with a dye or a binding agent.

10. The method of claim 9 wherein said nucleic acid molecule is an ultra-long nucleic acid.

11. A method of sequencing a nucleic acid molecule comprising introducing said nucleic acid into a systematically arranged single continuous portion of a nanochannel of a nanofluidic device of any one of claims I to 6, adding suitable reagents to initiate the sequencing reaction and monitoring the sequencing reaction using an imaging device.

12. A method of detecting the presence of a microorganism comprising obtaining a nucleic acid molecule from said microorganism; introducing said nucleic acid into a systematically arranged single continuous portion of a nanochannel of a nanofluidic device of any one of claims 1 to 6 and viewing said nucleic acid using an imaging device.

13. A method of detecting structural variations in chromosomal DNA comprising introducing said chromosomal DNA into a systematically arranged single continuous portion of a nanochannel of a nariofluidic device of any one of claims 1 to 6 and viewing said nucleic acid using an imaging device.

14. The method as claimed in claim 12 or claim 13 wherein said nucleic acid molecule is contacted with a dye and/or an binding agent prior to introducing said nucleic acid to said nanofluidic device to enable the nucleic acid or chromosomal to be analysed to be determined using a barcoding method.

15. A kit comprising the device of any one of claims 1 to 6 and optionally suitable reagents.

16. A mould for use in the preparation of a device, of any one of claims 1 to 6 by injection moulding.

17. A master stamp for use in the preparation of a device of any one of claims 1 to 6 by nanoimprint lithography.

18. The method of claim 9 wherein said nucleic acid molecule is double- stranded DNA with length lOOkb-lMb.

19. The method. of claim 9 wherein said nucleic acid molecule is double-stranded DNA with length IMbp-lOMbp.

20. The method of claim 9 wherein said nucleic acid molecule is double-stranded DNA with length 10Mb- lOOMbp.

21. The method of claim 9 wherein said nucleic acid molecule is double-stranded DNA with length 100Mbp-1 OOOMbp.

22. A method according to claim 11 where said suitable reagents comprise one or more- from the group comprising polymerase, nucleotides, labelled nucleotides, oligonucleotides, labelled oligonucleotides, monovalent cations, divalent cations, buffer, antioxidants, DNA modifying enzymes, nickase, DNAse, single-strand binding protein, helicase, FRET donor, FRET acceptor, Fluorescent nanoparticles, Quantum Dots, intercalating dye, DNA binding dye, surface passivation reagents.

23. A method scaffolding comprising introducing said chromosomal DNA into a

systematically arranged single continuous portion of a nanochannel of a nanofluidic device of any one of claims 1 to 6 and viewing said nucleic acid using an imaging device.

24. A method according to the previous claims comprising analysing substantially the complete length of DNA from a single chromosome in a few fields of view of a 2-D detector.

25. A method according to the previous claims comprising analysing the substantially the complete length of DNA from a single chromosome in a single field of view of a 2-D detector.

26. A device according to the previous claims comprising nanochannels for analysing substantially the complete length of DNA from a single chromosome in a few fields of view of a 2-D detector.

27. A device according to the previous claims comprising nanochannels for analysing the substantially the complete length of DNA from a single chromosome in a single field of view of a 2-D detector.

28. Use according to the previous claims for analysing substantially an entire chromosome, genome or other complete DNA set.

28. Use of claim 23 in combination with high-throughput DNA sequencing to assemble contigs or substantially complete genomes.

29. A nanofluidic device comprising

a substrate with a surface comprising one or more nanochannels wherein a single continuous portion of a nanochannel is systematically arranged to optimise the length of nanochannel and DNA therein that can be visualised within a single field of view of an imaging device.

30. A device according to 29 where the length of nanochannel corresponds to the length of DNA under analysis, from a particular chromosome, at 25%- 100% stretching.