This invention relates to an improved plasmonic device suitable for detecting and identifying small quantities of a target molecule in a gaseous or liquid sample. The device is especially suitable for detecting trace amounts of noxious substances such as biohazards, toxic chemicals, poisonous gases, narcotics or traces of explosive material or residues.

The detection of noxious substances in the environment has long been a concern; indeed in recent years it has become more so with a growing appreciation of the long-term health risks to people exposed to chemicals and the rise of international terrorism and the consequential possibilities of the unlawful use of explosives, poisons and materials which are biohazards. There therefore remains a pressing technical need to improve the performance and efficiency of those devices performing these duties.

Detectors having many different designs have been developed over the years and some are readily available on the market. Those available include devices in which the method of detecting the noxious target is based on chromatography or some form of spectroscopic method for example fluorescence spectroscopy, infrared spectroscopy, Raman spectroscopy and mass spectrometry. However, in many instances these devices have relatively high detection-limits meaning that, in order to reliably detect trace contaminants, a considerable volume of sample has to be analysed over a significant period of time. In doing so, the risk of a false reading becomes more likely. These problems are exacerbated because those analysers on the market typically operate passively; in other words whilst they deliver the sample to a zone adjacent the detector they thereafter rely on natural processes, such as the Brownian motion of the target molecules, to cause final migration onto the detector itself.

US 20110279817, for example, discloses an optical device and associated analysing apparatus for detecting the presence of a target molecule in a gaseous sample. In this device, a gas containing the target is caused to flow over a detector comprising a striated dielectric substrate having metal film elements deposited on its ridges. At the same time, the substrate is illuminated with incident light and Raman-scattered light, emitted by the target absorbed onto the substrate, detected. The benefit of using a striated substrate is that surface plasmon polaritons can be induced in the metal film elements causing the Raman emissions of the target to be enhanced. However no provisions are made for driving the target onto the detector from the sample. A similar passive device is taught in US 20120162640 where the substrate comprises an array of nanoparticles arranged on a substrate whose surface is provided with two sets of ridges orthogonal to each other. In use, localised surface plasmons are induced in the various metal nanoparticles to enhance Raman-scattering of the target molecule.

US 2009273779 also describes an optical device for detecting a foreign object in a sample using conventional Raman or surface-enhanced Raman spectroscopy. This device is characterised by the presence of a Raman-active substrate consisting of a plasmonic band structure region which can be coupled to optical radiation, the plasmonic band structure region comprising a layer of a first material patterned with an array of sub-regions of a second material, the first material having a first refractive index and the second material having a second refractive index, a side- wall of each sub-region being coated with a metallic layer, wherein the array of sub-regions give rise to a plasmonic band structure, and each sub-region is configured to confine at least one plasmon resonance excited by optical radiation coupled into the plasmonic band structure region. This then gives rise to a Raman signal output from a foreign object placed proximate the plasmonic band structure region. Typically, the substrate comprises a regular tiled array in which the sub-regions are located at the vertices of pyramidal nano-voids. We are aware that such substrates are available commercially under the trade name Klarite ^® . Again no provision is made to drive the foreign object to the platform.

US 8129676 describes a device for detecting ions in an analyte in which a stream of ions generated in a separate ionisation stage is directed towards a detector by an electric field. In this case, the detector comprises a surface-enhanced Raman spectroscopy system comprised of an array of detector elements each comprising one or more metallic segments separated by an insulator. However, the patent gives no information about the dimensions of these segments, in particular whether they are comprised of nanostructures, or indeed whether they are intentionally stimulated to undergo plasmon resonance at an optimum frequency.

US 2008/0239307 discloses a surface-enhanced Raman-scattering method and apparatus for sequencing polymeric biomolecules such as DNA, RNA and proteins. In one of a number of embodiments of the method, shown in Figure la, a DNA analyte is schematically shown to translocate by electrophoresis through a nanopore in a substrate and between nanostructures capable of undergoing plasmonic resonance. Here, Raman-scattered light emitted by the constituent nucleotide bases as they pass in order between the nanostructures is investigated so that the sequence of the analyte can be reconstructed. Whilst this device is directed to a different duty from that foreseen by the present invention this patent application does at paragraph 18 speculate that the method generally can be implemented in applications including environmental monitoring and national security. However, no guidance is given as to exactly what is envisaged. Other nanopore sequencing devices are disclosed in WO 2007/011389, US 2012/0258544 and WO 2009/030953.

US 2003/0036204 teaches methods and apparatuses for producing small bright nanometric light sources from apertures that are smaller than the wavelength of the emitted light. In one embodiment this is achieved by coating the nanopores and the surface of a nanoperforated substrate with a metal film and inducing surface plasmon polaritons therein by the application of a beam of incident light. Configurations of the film are exemplified which limit the propagation of these polaritons so that the emissions are confined to small target areas. The method and apparatus is said to have application in microscopy, photolithographic processes, optical storage devices and to analyse the properties of small objects such as protein and nucleic acid molecules and single cells.

In a similar vein, WO 2007/011876 teaches an apparatus comprising a metal film and at least one resonance configuration formed therein. The configuration itself comprises a pore extending through the film and a single non-annular feature that causes a variation in a dielectric function along a first surface proximate to the aperture. This feature may be a second aperture, protrusion or a depression. The apparatus however is not designed to detect hazardous materials; rather it is orientated towards solving a different technical problem in a completely different technical field; achieving sub- ayleigh criterion resolution in optical microscopy.

EP 1650550 describes a surface plasmon detector in which the analyte (here present in solution) is driven by electrophoresis between a pair of electrodes to a detection zone. The detection zone comprises one of the electrodes attached to the reverse side of which is a prism through which a beam of irradiating light is caused to pass. The device further comprises a photodetector for detecting the intensity of light which has been reflected back out of the prism as a function of various angles of incidence around resonance conditions. The electrode itself is neither nanoporous nor provided with nanostructures. A similar device is taught in JP 1078393.

US 2007/0252982 describes a SERS analyser including a tunable resonant cavity in a substrate comprising reflective members and an electro-optic material disposed therebetween. Coupled to the cavity is a Raman signal-enhancing structure. This device is also not provided with a gradient for driving an analyte to the cavity/structure arrangement. Furthermore the substrate does not appear to be nanoporous. Finally, US 2005/084912 discloses a device comprising a microscope assembly for sequencing the successive bases in a nucleic acid such as DNA. It comprises a substrate having a plasmon resonant surface (here a metal film coating in which can be generated surface plasmon polaritons) and a separate lens assembly comprising a tip region and nanolens comprised of plasmon resonance particles. This lens assembly can be moved towards and away from the substrate to produce near-field electromagnetic gap modes in the intervening space.

We have now developed an improved nanopore plasmonic detection device which can be applied to both liquid and gaseous samples and enables the target molecule in the analyte to be selectively driven to the detector without the need for ionisation which in the case of application to larger molecules can lead to significant degradation and possibly misleading results when complex molecules are being detected. Thus, according to the present invention, there is provided a device for detecting the presence of a hazardous target molecule in a sample characterised in that it comprises:

• a chamber for containing the sample;

• a substrate, located in the chamber, which is provided with at least one nanopore therethrough;

• at least one nanostructure, juxtaposed within the nanopore or on a surface of the substrate adjacent thereto, each nanostructure being capable of generating an associated detection window comprising an electromagnetic field by having induced therein localised surface plasmons;

• a source of incident electromagnetic radiation for inducing said localised surface plasmons in said nanostructure(s);

• at least one means for establishing a gradient between the sample and the substrate for driving the target molecule from the sample into the detection window and

• a detector for detecting electromagnetic radiation emitted by the target molecule as it passes though the electromagnetic field.

The detection device of the present invention can be used to detect the presence or indeed absence of charged and polarisable target molecules present in both gaseous and liquid samples; especially target molecules having a characteristic spectroscopic fingerprint which is significantly different from the other major constituents of the sample. Whilst the device can in principle be used for any detection, as opposed to sequencing, duty, the device is especially useful for the detection of biological pathogens or poisons in drinking water, cooling fluids or air; the detection of residual pesticides and pollutants in aqueous extracts taken from soils and foodstuffs and the detection of narcotics, explosives or their residua on hard surfaces, clothing or the like.

The substrate employed in the device of the invention is suitably made of a dielectric material such as glass, silicon or silicon nitride and in one embodiment is a sheet of such material. However the use of a composite sheet for example in which the dielectric material provides the surface(s) bearing the nanostructure(s) is also envisaged. The substrate is perforated with one or more nanopores which may disposed in any arrangement including in a regular array across the whole or a part thereof. Typically, each nanopore has a diameter in the range 0.5 to 100 nm, preferably from 1 to 50nm most preferably from 1 to 20nm. Useful results can be obtained using nanopores whose diameters lie in the range 5 to 25nm. In addition to being nanoporous one or more surfaces of the substrate can be further provided with wells, cavities or channels of nanodimensions.

Turning to the nanostructures, these are juxtaposed within the nanopore and/or on at least one surface of the substrate and are sized and arranged so that localised surface plasmons, as opposed to surface plasmon polaritons, can be induced therein. An advantage of this arrangement is that, by judicious choice of size and shape, the nanostructures can be tuned to produce optimum plasmonic field intensity and density for a given wavelength of the incident electromagnetic radiation. In practice, this means that each nanostructure should have a maximum dimension of greater than 1 micron, preferably in the range 1 to 500 microns, most preferably in the range 1 to 150 microns. In one embodiment, these nanostructures may be organised as a plurality of pairs, preferably in a regular disposition on a surface of the substrate and about the inlets and/or outlets of the nanopores. In this embodiment, at least some of the nanostructures are spaced apart from their pair by greater than 10 nanometres, preferably from 10 to lOOnm, most preferably from 10 to 30nm. As a consequence of this pairing, the space between the two nanostructures can be made subject to a strong induced electromagnetic field which causes enhancement of any spectroscopic emissions from target molecules present therein. Hereinafter the space which this electromagnetic field occupies is referred to as the 'detection window'. The nanostructures can be disposed on the surface of the substrate adjacent the inlet side of the nanopore, on the surface of the substrate adjacent the outlet side of the nanopore or both. Nanostructures in each pair can be disposed on the same surface or opposite surfaces.

In another embodiment, the nanostructures are located on the internal surface of each nanopore so that the internal space within the nanopore comprises the detection window. In yet another embodiment, local 'hotspots' on the substrate, comprising a relatively high density of nanostructure pairs and their associated nanopores are employed. This arrangement then provides a target area to which the target molecule can be driven to in the first instance by the gradient described below. Such an arrangement is especially advantageous when the gross concentration of the target molecule in the sample is anticipated to be low.

The nanostructures themselves are typically fabricated from metals or dielectric materials coated with metal. Metals which can be employed are those capable of undergoing plasmon resonance to a significant extent, for example, gold, silver, copper, aluminium, platinum, palladium, molybdenum and chromium and alloys thereof. Preferably, the metal used will be gold, silver, copper or an alloy thereof. To enhance the performance of the device further, the nanostructure may have attached to its surface binding sites which are specifically adapted to capture the particular target molecule and enhance the characteristic spectroscopic emissions being sought. Such reactive groups can work by any chemical or physical means; for example when detecting say a signal characteristic of a bacterial pathogen, e.g. the Legionella bacterium, the reactive group can comprise a polynucleotide probe adapted to bind to certain unique base- pair sequences in the bacterium DNA by hybridisation.

In the case where the detection window does not comprise the inside of the nanopores, it can be generated by any suitable arrangement of nanostructures on the surface of the substrate. One simple embodiment, for example, comprises a regular disposition of the nanostructures on an otherwise substantially smooth nanoperforated dielectric substrate. Such an arrangement has the advantage that it is easy to make; for example by first coating the substrate with a metallic film, then masking up the product and finally etching away the remaining exposed metal and substrate with a chemical or ion beam to leave discrete nanostructures arranged about the nanopores. Methods for carrying out such a method are well known in the art. In one preferred arrangement, pairs of triangular nanostructures are employed in a 'bow tie' configuration to reduce the size of the detection window adjacent the inlets and/or outlets nanopores and increase the density of the electromagnetic field generated from a given degree of plasmon resonance. In another embodiment, the two nanostructures can be two half annuli juxtaposed to create a 'doughnut' configuration around a substantially nanopore. In yet another embodiment the two nanostructures comprise a pair of complete annuli stacked on top of each other over the nanopore and separated from each other by a dielectric layer.

In one working form, the device comprises one or more nanoperforated substrates and their associated nanostructures arranged in a chamber for containing it and the sample. Suitably the chamber is provided with a means, such as a pump, fan or the like for passing the sample over the substrate and nanostructures; preferably in a direction parallel or substantially parallel to a surface of the substrate. The chamber may contain different substrates arranged at different locations. This arrangement can advantageously be used to detect different target molecules in a sample having a complex composition. It is especially useful when the means for establishing the gradient is used to selectively direct different target molecules of differing masses to different substrates or regions of the same substrate.

The device is further provided with a means to establish a gradient between the sample and at least one surface of the substrate. An effect of this is that the target molecule is thereby induced to flow along the gradient from the sample bulk to the detection window(s). In an embodiment of the invention, the means to establish the gradient may also cause translocation of the target molecule into or through the nanopore. This active, as opposed to passive, transfer of the target molecule to the detection window(s) significantly improves the capture efficiency and therefore the sensitivity of the device. This is very important where the level of target molecule in the sample is very small and it has the further advantages that (a) the time to a meaningful result is considerably shortened and (b) the signal to noise ratio of the output signal from the device is improved. The gradient established in the device can include for example gradients caused by variations in osmotic pressure, magnetic field or by using chemical reactions to generate a thermodynamic or entropic gradient. However, in one suitable embodiment the gradient is created by an electric field produced by applying a potential difference between the sample bulk and a point of opposite polarity located on the opposite side of the substrate. The target molecule is then caused to move towards the detection window and optionally into and through the nanopores by electrophoresis, in the case of charged target molecules, or by dielectrophoresis, in the case of uncharged, polarisable species. In one embodiment, the direction of this electrical field can be reversed; for example to remove the target molecule from the substrate after detection so that in effect the device can be cleaned. In an embodiment, the means for establishing this potential difference and the detector are connected via a feedback loop so that, once the detector detects a threshold level of the target molecule, the direction of the electric field can be varied with time to hold the target molecule in the detection window.

The potential difference referred to above can be achieved by locating a first electrode in that part of the chamber designed to hold the sample bulk, locating a second electrode, of opposite polarity, on the opposite side of the substrate and then applying a voltage therebetween. In one embodiment of this design, the nanostructures are arranged around the inlet of the nanopores; in another they are arranged around the outlets of the nanopores; in yet another they comprise a film within the nanopores and in yet another a combination or two or more of these arrangements is employed. It is possible to locate the second electrodes in the nanopores if so desired. Finally, in another embodiment, the side of the substrate opposite to that of the first electrode is coated with a layer of metal or semiconductor which is able to act as the second electrode.

The remainder of the device comprises a high-intensity source of incident electromagnetic radiation, for example a laser, a detector for detecting the characteristic electromagnetic radiation emitted by those target molecules in the detection window and any necessary ancillary optics and electrical circuitry. Either or both of the source of incident electromagnetic radiation and the detector can be continuously associated with some or all of the nanostructures and the detection windows. Alternatively, the nanostructures and the detection windows can be scanned using for example a movable microscope or raster arrangement. The electromagnetic radiation detected by the detector can in principle be any spectroscopic emission which is characteristic of the target molecule when it is present in the detection window as opposed to a spectral emission or shifts characteristic of the nanostructures themselves. Suitably, this spectroscopic emission is either fluorescence radiation or Raman-scattered radiation. In the case where fluorescence emissions are being detected, it may be useful to chemically or biologically label (as the case may be) the target molecule with a dye. However where the device is adapted to detect Raman- scattered light from the target molecule, labelling will not normally be required and rather wavelengths characteristic of one or more vibrational modes of the target will be monitored by the detector in order to generate a signal characteristic of the wavelength being observed or of a ratio of such wavelengths. When Raman spectroscopy is employed the scattering can be derived from single- or multi-photon events.

The nature of the detector employed in the device is not critical and can suitably comprise for example a photodetector, a single photon avalanche diode, an electron-multiplying charge- coupled device or a complementary metal oxide semiconductor device. The detector can additionally be attached to a microprocessor, PC and/or an alarm to process the signal derived therefrom.

Whilst it is envisaged that the whole device can be of unitary design it can also advantageously consist of two components for example a permanent housing in which the source of incident electromagnetic radiation, the detector and optics are located and a disposable cartridge comprising a shell containing the chamber, substrate and nanostructures linked together by microfluidic pathways. In this case the shell then can be made of for example glass, silicon or plastic. If the device is of unitary design and is used for guard duties, it may be provided with a wireless transmitter to send the signal from the detector back to a central location. The device can then be positioned remotely in which case it may employ a stand-alone power source such as battery if so desired.

Finally there is provided a method for using the device of the present invention to detect the presence of a hazardous target molecule in a sample. It is characterised by the steps of (a) providing a nanoperforated substrate comprising at least one nanostructure juxtaposed within the nanopore(s) or on a surface of the substrate adjacent thereto; said nanostructure being adapted to generate an associated detection window comprising an electromagnetic field by having induced therein localised surface plasmons; (b) bringing the sample into contact with the surface whilst maintaining a gradient between the sample and the surface so as to drive the target molecule from the sample into the detection window and (c) detecting electromagnetic radiation characteristic of and emitted by those target molecules in the detection window.

In this method the field gradient is preferably an electrophoretic or dielectrophoretic field gradient as described above and the electromagnetic radiation detected is either fluorescence or Raman-scattered radiation.

The present invention is now illustrated with reference to the following Example in which: Figure 1 shows a cartridge containing a detection device according to the invention;

Figure 2 shows a sectional view of the device and

Figure 3 illustrates various arrangements of nanopores and nanostructures suitable for use in connection with the device

Referring to Figure 1, a cartridge 1 embodying the detection device is fabricated from polydimethylsiloxane and comprises sample reservoir 2 (to be filled by the user) and an associated valve 4; a reservoir 3 for containing a flushing fluid such as water (suitably prefilled at the source of manufacture) and associated valve 5; a detection device according to the invention 7 and a reservoir for waste 9. 1 further comprises various ancillary valves and pneumatic inlets (labelled v and p respectively) to enable the sample, flushing fluid and effluent to be pumped around the system. In use, 7 and its associated microfluidic pipework is flushed out by opening 5 and pumping the fluid around the system. 5 is then closed and the sample contained in 2 is pumped through 7 and microfluidic lines 6 and 8 into 9 after opening 4. Once the sample has been discharged through 7, 9 can be isolated and emptied by means of valve 11a and lib and pumping if so desired. Thereafter 9 can be flushed out by 3 via valve 12. In Figure 2, 7 is provided with a transparent glass window 10 in its upper face through which the assembly 13 can be observed. 13 comprises a plinth 14 on which a dielectric sheet of silicon nitride 15 is mounted. The exposed surface of 15 is covered with a regular array of gold nanostructures 16 in a bow-tie configuration (see Figures 3A and 3B). Between the jaws of each bow-tie is positioned a nanopore 17 which connects upper and lower chambers 18 and 19. 18 and 19 contain annular electrodes 20 and 21 of opposite polarity connected by control box 26. As the sample is caused to flow from 6 through 7 and out of 8, the target molecules 22 in the sample are driven by the potential difference between 20 and 21 into the detection window between the jaws of the bow-tie and through 17. 15 is continuously illuminated through 10 by a laser 23 and its associated optics. Raman-scattered radiation emitted by 22 and passing back out through 10 and via dichroic mirror 24 is detected by one or more photodetectors 25 tuned to two different characteristic Stokes frequencies of the target molecule being sought. A signal generated by each of 25 is then passed to a common microprocessor (not shown) for ratio analysis.

Various configurations of nanopores and nanostructures are shown schematically in Figure 3. Figures 3A and 3B illustrate in plan and section an array of bow-tie nanostructures corresponding to that used in Figure 2. Figures 3C and 3D illustrate alternative designs in which respectively the nanostructures 16 sit on the side of 15 opposite 18 and where each of 16 extends through and coats the internal surfaces of the nanopores 17.