This invention relates to a device for investigating a nucleic acid molecule; in particular a device for sequencing DNA or RNA either of natural or synthetic origin.

In our previous applications WO 2014/053853, WO 2014/053854, WO2014/167323, WO2014/167324 and WO2014/111723, we have described a new sequencing method which involves progressive digestion of a polynucleotide analyte to generate an ordered stream of single nucleotides, preferably a stream of single nucleoside triphosphates, each of which can be captured one-by-one into corresponding microdroplets in a microdroplet stream. Thereafter, each droplet can be chemically and/or enzymatically manipulated to reveal the particular single nucleotide it originally contained. In one embodiment, these chemical and/or enzymatic manipulations comprise a method involving the use of one or more two-component oligonucleotide probe types each of which is adapted to be able to selectively capture one of the single nucleotide types from which the analyte is constituted. Typically, in each of such probe types, one of the two oligonucleotide components comprises characteristic fluorophores and in the probe's unused state the ability of these fluorophores to fluoresce remains extinguished by virtue of the presence of quenchers located close-by or by self-quenching. In use, when the probe has captured its corresponding single nucleotide, it is rendered susceptible to subsequent exonucleolysis or endonucleolysis thereby liberating the fluorophores from the quenchers and/or each other enabling them to fluoresce freely. By this means, the original single nucleotide present in each droplet can be inferred indirectly by spectroscopic means.

In designing sequencing devices it is necessary to manipulate many thousands of microdroplets ensuring for example that they can be reliably delivered to locations where they can be merged with other and/or their contents analysed for the presence or absence of fluorescence.

One possible way in doing this is to establish pathways on a substrate along which the microdroplets can be propelled by electrowetting forces. Devices for manipulating relatively large droplets in this way have been previously described in the art; see for example U.S. Pat. No. 6,565,727, US20130233425 and US20150027889. Typically, this is achieved by causing the droplets, for example in the presence of an immiscible carrier fluid, to travel through a microfluidic channel defined by two opposed walls of a cartridge or tubing. Embedded in the walls of the cartridge or tubing are electrodes covered with a dielectric layer each of which are connected to an A/C biasing circuit capably of being switched on and off rapidly at intervals to modify the electrowetting characteristics of the layer. This gives rise to localised directional capillary forces that can be used to steer the droplet along a given path.

A variant of this approach, based on optically-mediated electrowetting, has been taught in for example US20030224528, US20150298125 and US20160158748. In particular, the first of these three patent applications discloses various microfluidic devices which include a microfluidic cavity defined by first and second walls and wherein the first wall is of composite design and comprised of substrate, photoconductive and insulating (dielectric) layers. Between the photoconductive and insulating layers is disposed an array of conductive cells which are electrically isolated from one another and coupled to the photoactive layer and whose functions are to generate corresponding discrete droplet-receiving locations on the insulating layer. At these locations the surface tension properties of the droplets can be modified by means of electrowetting forces. The conductive cells may then switched on and off by light impinging on the photoconductive layer. This approach has the advantage that switching is made much easier and quicker although its utility is to some extent still limited by the arrangement of the electrodes. Furthermore, there is a limitation to the speed at which droplets can be moved and the extent to which the actual droplet pathway can be varied.

A double-walled embodiment of this latter approach has been disclosed in University of California at Berkeley thesis UCB/EECS-2015-119 by Pei. Here, a cell is described which allows the manipulation of relatively large droplets in the size range 100-500 μm using optical electrowetting across a surface of Teflon AF deposited over a dielectric layer. A light-pattern over un-patterned electrically biased amorphous silicon is described in schematic form. However, in the scheme shown the dielectric layer is thin (100 nm) and disposed only on the wall bearing the photoactive layer.

We have now developed a nucleic acid investigative device which is capable of manipulating many thousands of microdroplets simultaneously in accordance with the requirements of a reliable single nucleotide detection method. It has the advantage of being easily reconfigurable by the application of computer software making it very versatile; for example by allowing the user to readily adapt it for optimum accuracy, throughput or detecting particular epigenetic modifications. Thus according to the present invention there is provided a device for investigating a nucleic acid analyte characterised by comprising:a first zone comprising an attachment site to which the analyte is attached; a first pathway for causing a first fluid medium to flow over the attachment site thereby allowing the nucleic acid to be progressively pyrophosphorolysed into its constituent nucleoside triphosphates; a second pathway for removing the nucleoside triphosphates from around the attachment site and a means for creating in the second pathway a second medium comprised of aqueous microdroplets in a water-immiscible carrier;a microdroplet manipulation zone for manipulating the microdroplets using optically-mediated electrowetting comprised of:a first composite wall comprised ofa first transparent substrate;a first transparent conductor layer on the substrate having a thickness in the range 70 to 250 nm;a photoactive layer activated by electromagnetic radiation in the wavelength range 400-1000 nm on the conductor layer having a thickness in the range 300-1000 nm anda first dielectric layer on the conductor layer having a thickness in the range 120 to 160 nm;a second composite wall comprised ofa second substrate;a second transparent conductor layer on the substrate having a thickness in the range 70 to 250 nm andoptionally a second dielectric layer on the conductor layer having a thickness in the range 25 to 50 nm;wherein the exposed surfaces of the first and second dielectric layers are disposed less than 10 μm apart to define a microfluidic space adapted to contain microdroplets;an A/C source to provide a potential difference across the first and second composite walls connecting the first and second conductor layers;at least one source of first electromagnetic radiation having an energy higher than the bandgap of the photoexcitable layer adapted to impinge on the photoactive layer to induce corresponding ephemeral first electrowetting locations on the surface of the first dielectric layer;means for manipulating the points of impingement of the electromagnetic radiation on the photoactive layer so as to vary the disposition of the ephemeral electrowetting locations thereby creating at least one first electrowetting pathway along which the microdroplets may be caused to move;an detection zone disposed downstream of the microdroplet manipulation zone or integral therewith anda fluorescence or Raman-scattering detection system comprising a source of second electromagnetic radiation adapted to impinge on the microdroplets in the detection zone and a detector for detecting fluorescence or Raman-scattering emitted therefrom.

