WO2014167322A1

WO2014167322A1 - Plasmonic detector

Info

Publication number: WO2014167322A1
Application number: PCT/GB2014/051104
Authority: WO
Inventors: Cameron Alexander FRAYLING; Bruno Flavio Nogueira de Sousa SOARES
Original assignee: Base4 Innovation Ltd
Priority date: 2013-04-09
Filing date: 2014-04-09
Publication date: 2014-10-16
Also published as: GB201306447D0

Abstract

A device (7) for detecting the presence of a target molecule in a sample characterised in that it comprises: • a chamber for containing the sample; • an essentially non-perforated substrate (15) located in the chamber; • at least one nanostructure (16), juxtaposed on a surface of the substrate (15) each nanostructure (16) 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 (22) for inducing said localised surface plasmons in said nanostructure(s) (16); • at least one means (17,18,19) for establishing a gradient between the sample and the substrate (15) for driving the target molecule from the sample into the detection window and • a detector (24) for detecting electromagnetic radiation characteristic of and emitted by those target molecules in the detection window. Preferably the gradient is an electrophoretic or dielectrophoretic field gradient. The device (7) is especially suitable for detecting trace amounts of noxious substances such as toxic chemicals, poisonous gases, narcotics or traces of explosive material or residues.

Description

PLASMONIC DETECTOR

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 methods for example fluorescence spectroscopy, infrared spectroscopy, Raman spectroscopy and mass spectrometry. However, in many instance 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. And 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 perpendicular to each other. In use, localised surface plasmons are induced in the various metal nanoparticles to enhance Raman-scattering of the target.

US 2009273779 also describes an optical device for detecting a foreign object in a sample using conventional Raman or surface-enhanced Raman spectroscopy. The 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 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.

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-Rayleigh 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. However this device is also not provided with a gradient means for driving the analyte to the cavity/structure arrangement. The same is true of WO 2010/066727, WO 03/027619, WO 2013/060989, US 2009/045351, JP 2005/337771, GB 2419940, US 2011/166405 and WO 2006/118337 all of which are further illustrative of detectors which exhibit improved sensitivity by virtue of the presence of nanostructures capable of undergoing enhanced localised surface plasmon resonance.

Finally, 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 nanostructures, or indeed whether they are intentionally stimulated to undergo plasmon resonance at an optimum frequency.

We have now developed an improved 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 target molecule in a sample characterised in that it comprises:

• a chamber for containing the sample;

• an essentially non-perforated substrate located in the chamber;

• at least one nanostructure, juxtaposed on a surface of the substrate 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 characteristic of and emitted by those target molecules in the detection window. The detection device of the present invention can be used to detect the presence 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 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 in aqueous extracts taken from soils and foodstuffs; the identification of bacterial pathogens in liquids swabbed or taken from human beings, hospital equipment or the like and the detection of narcotics, explosives or their residua on hard surfaces, clothing or the like.

The essentially non-perforated 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 fabricated from a sheet of such material. However the use of a composite sheet for example in which the dielectric material provides the surface bearing the nanostructure(s) is also envisaged. In the context of this patent, the term 'essentially non-perforated' is used to mean that, whilst the substrate may be provided with structures such as wells, channels, cavities and the like, themselves of nano-dimensions, these structures either do not extend completely therethrough or, to the extent they do, they have, at at least one critical point, a narrowing which renders translocation of the target molecule through the substrate impossible. In one embodiment however these structures do not extend completely therethrough; in other words the substrate is non-perforated. In another embodiment described below, the substrate further comprises a metal or semiconductor layer attached to the surface of the substrate opposite to that bearing the nanostructures (hereinafter referred to as the 'operative surface'). In this embodiment, the structures referred to above may completely penetrate the dielectric material to the layer providing that they do not completely perforate it.

Turning to the nanostructures, these are juxtaposed on the operative surface, and are sized and arranged so that localised surface plasmons, as opposed to surface plasmon polaritons, can be induced therein. An advantage of this 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 from 1 to 500 microns, most preferably 1 to 150 microns. In one embodiment, these nanostructures may be organised as a plurality of pairs, preferably a regular disposition of such pairs on the operative surface, in which at least some of the nanostructures are spaced apart from their pair by greater than 10 nanometres preferably in the range 10 to lOOnm most preferably in the range 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'. In yet another embodiment, local 'hotspots' on the operative surface, comprising a relatively high density of the nanostructure pairs and hence detection windows, are employed to facilitate the driving of the target molecule generally towards the detection window.

The nanostructures themselves are typically fabricated from metals or dielectric materials coated with metals. 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 emissions being sought. Such reactive groups can work by any chemical or physical means; for example when detecting say genetic material 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 by hybridisation.

The detection window can be generated by any suitable arrangement of nanostructures on the operative surface. One simple embodiment comprises a regular disposition of the nanostructures on an otherwise substantially smooth dielectric surface. 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 with a chemical or ion beam to leave the discrete nanostructures. Methods for carrying out such a method are well known in the art. However more complex structures can be fabricated, for example the nanoparticle array shown in US 20120162640. In one preferred arrangement, pairs of triangular nanostructures are employed in a 'bow tie' configuration to reduce the size of the detection window 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 circular orifice giving access to the substrate. This configuration is especially useful where the orifice is coincident with a well in the substrate immediately below. In its working form, the device comprises one or more substrates and their associated nanostructures arranged in a chamber for containing it and the sample. Suitably the chamber is provide with a means, such as a pump, fan or the like for passing the sample over the substrate and nanostructures; preferably in a parallel or substantially parallel direction. 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 the substrate. An effect of this is that the target is thereby induced to flow along the gradient from the sample bulk to the detection window(s) on the substrate. 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 levels of target molecule in the sample are 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 the electrical field produced by applying a potential difference between the sample bulk and a position on the substrate in close proximity to the detection window. The target molecule is then caused to move towards the detection window by electrophoresis, in the case of charged target molecules, or 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 operative surface after detection so that in effect the substrate 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 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 the chamber, locating a second electrode, of opposite polarity, on the substrate between the nanostructures and then applying a voltage therebetween. In one embodiment, this configuration can be modified so that at least part of the substrate region in close proximity or between the nanostructure(s) comprises the types of well, channel or cavity mentioned above. In this configuration, it is preferred that the second electrode sits in or at the bottom of the well, cavity or channel in order to cause the target molecule to pass through the detection window towards its ultimate destination.

In another embodiment, the side of the substrate opposite to the operative surface is coated with a layer of metal or semiconductor which is able to act as the second electrode. It will be appreciated that there will be many designs employing this feature which can achieve the desired result; in one example the layer comprises the bottom of or is substantially adjacent to the bottom of the well, channel or cavity referred to above and is made by perforating or etching the substrate towards the internal surface of the layer. Alternatively the metal or semiconductor layer can be bonded to a previously nano-perforated dielectric substrate precursor. Finally, the metal or semiconductor layer can be brought into close proximity with the operative surface by means of wells, cavities or channels of nano-dimensions arranged on a different, suitably opposite, surface of the substrate. In this embodiment, the different surface is first patterned with the required wells, cavities and channels and then coated or filled with the metal or semiconductor using known deposition methods.

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 will often 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 foe 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 is preferred that it is provided with a wireless transmitter to send the signal from the detector back to a central location. The device can thus 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 a sample molecule in a sample. It is characterised by the steps of (a) providing an essentially non- perforated substrate comprising a surface having at least one nanostructure; 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 substrate, nanostructures and electrodes suitable for use in 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 by peristalsis. 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 by means of valve 10 and emptied via valve 11 and by 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 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 an electrode 17 connected to a power source 18. 18 is also connected to an annular electrode 19 of opposite polarity located within chamber 20. As the sample is caused to flow from 6 through 7 and out of 8, the target molecules 21 in the sample are driven by the potential difference between 17 and 19 into the detection window between the jaws of the bow-tie. 16 are continuously illuminated through 10 by laser 22 and its associated optics. Raman-scattered radiation emitted by 20 and passing back out through 10 and via dichroic mirror 23 is detected by photodetector 24 tuned to at least one characteristic Stokes frequency of the target molecule being sought. A signal generated by 24 is then passed to a microprocessor (not shown) for analysis.

Various detector designs are shown in Figure 3. Figures 3A and 3B illustrate in plan and section an array of bow-tie nanostructures that can be used in the device shown in Figure 2. Figures 3C to 3E illustrate alternative designs in which respectively the electrode 17 sits in a well of nano-dimensions 25; the dielectric sheet 15 is perforated and wherein 17 comprises a layer of gold and wherein 17 extends into wells of nano-dimensions 26 fabricated in the surface of 15 opposite that bearing the nanostructures 16.

Claims

Claims:

1. A device for detecting the presence of a target molecule in a sample characterised in that it comprises:

• a chamber for containing the sample;

· an essentially non-perforated substrate located in the chamber;

• a detector for detecting electromagnetic radiation characteristic of and emitted by those target molecules in the detection window.

2. A device as claimed in claim 1 characterised in that the means for establishing the gradient comprises at least one pair of electrodes arranged so as to drive target molecules from the sample into the detection window by electrophoresis or dielectrophoresis.

3. A device as claimed in claim 1 or claim 2 characterised in that the essentially non-perforated substrate is provided with at least one well, cavity or channel of nano dimensions in close proximity to the nanostructure(s).

4. A device as claimed in claim 3 characterised in that the well(s), cavity(s) and channel(s) and the nanostructure(s) are arranged on the same surface of the substrate.

5. A device as claimed in claim 3 characterised in that the well(s), cavity(s) and channel(s) and the nanostructures are arranged on opposite surfaces of the substrate.

6. A device as claimed in claim 2 to claim 5 characterised in that at least one electrode is located in close proximity to the detection window.

7. A device as claimed in any of the preceding claims characterised in that the nanostructures are made of a material selected from the group consisting of gold, silver, copper, aluminium, platinum, palladium, molybdenum and chromium and alloys or complexes thereof

8. A device as claimed in any one of the preceding claims characterised in that substrate is made of a dielectric and that at least one of the electrodes comprises an electrically-conducting layer attached to a second surface of the substrate.

9. A device as claimed in claim 8 characterised in that the electrically-conducting layer comprises a layer of metal or a semiconductor.

10. A device as claimed in claim 8 or claim 9 characterised in that the nanocavity extends from the dielectric layer to the metallic layer but not therethrough.

11. A device as claimed in any of the preceding claims characterised in that the chamber is adapted to allow the sample to flow therethrough.

12. A device as claimed in claim 11 characterised in that the flow of sample through the chamber is substantially parallel to the surface of the substrate.

13. A device as claimed in any of the preceding claims characterised in that the chamber comprises part of a microfluidic path.

14. A device as claimed in any of the preceding claims characterised in that the detector is arranged to detect Raman-scattered radiation.

15. A device as claimed in any one of claims 1 to 13 characterised in that the detector is arranged to detect fluorescence.

16. A device as claimed in any of the preceding claims characterised in that it further comprises a wireless transmitter.

17. A device as claimed in any of the preceding claims characterised in that it comprises a chip consisting of the chamber and the substrate and housing adapted to receive the chip and comprising the source of electromagnetic radiation and the detector.

18. A chip for use in the device claimed in claim 17.

19. Use of the device claimed in any of claims 1 to 16 to detect a target molecule

20. Use of the device claimed in claim 19 characterised in that the target molecule is a biological pathogen.

21. Use of the device claimed in claim 19 characterised in that the target molecule is a toxic substance, explosive substance or a narcotic.

22. A method for detecting a target molecule in a sample characterised by the steps of (a) providing an essentially non-perforated substrate comprising a surface having at least one nanostructure; 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.

23. A method as claimed in claim 22 characterised in that the gradient is an electrophoretic or dielectrophoretic field gradient.

24. A method as claimed in either claim 22 or 23 characterised in that the electromagnetic radiation emitted by the target molecules is fluorescence.

25. A method as claimed in either claim 22 or 23 characterised in that the electromagnetic radiation emitted by the target molecules is Raman-scattered radiation.