WO2005033685A2 - Sensor platforms utilising nanoporous membranes - Google Patents

Abstract

The invention relates to the use of nanoporous membranes (1, 14) and in particular polymer track etched membranes to develop novel electrode surfaces for use in sensor devices. The first type of novel electrode comprises a nanoporous, thin layer of conductive material (2) formed integrally on the surface of a nanoporous membrane. The membrane can be used in combination with one or more nanoporous electrodes to make a conductivity sensor for example. Alternatively, the membrane may then be partially or fully removed before the resultant electrode is put to use to produce an electrode surface comprising a generally planar conducting electrode element the surface of which is covered with one or more projecting nanowires (84). Methods of fabrication are also described.

Description

SENSOR PLATFORMS UTILISING NANOPOROUS MEMBRANES

The invention relates to electrodes for sensor applications, to sensors incorporating such electrodes, and to methods for the fabrication of the same, which make use of certain properties of nanoporous membranes in fabrication and/ or in use. The invention in a first particular expression relates to an electrode formed integral with a nanoporous membrane, for example as a conductivity sensor, and to methods for the fabrication of the same. The invention in a second particular expression relates to a nanowire electrode surface formed by making use of a nanoporous membrane and to methods for the fabrication of the same.

In this context the term "nanoporous membrane" (NPM) is used to refer to a thin film of a material such as a polymer or ceramic that contains pores or voids such that a liquid solution may permeate from one side of the membrane to the other. Examples of specific technologies that have been used or proposed for the fabrication of such nanoporous membranes are; particle track etching of polymers by heavy ion bombardment, anodic treatment of aluminium oxide, electron beam writing of photoresist, self-assembly of block copolymers or amphiphilic liquid crystal materials with vertically aligned cylindrical phases and nano-imprint lithography. Other technologies may also be used.

A specific class of "nanoporous membranes" is polymer track etched membranes (PTM). The method of fabrication of these membranes is described by R. Legras, Y. Jongen in US patent 4956219. The method is based on irradiating thin polymer films with energetic ions and then chemically etching them to produce cylindrically shaped pores or voids running from one side of the membrane to the other. The diameter, density and shape of these pores depends on the irradiation conditions and on the type and length of chemical etching. The positions of the pores can be controlled by the methods described for example in WOO 149402 and US2003020024. Specific polymer materials in which PTM have been produced include polycarbonate, polyimide, PET, PEEK and polyvinylidenefluoride (PVDF).

PTMs are used as support membranes for microscopy, as filter membranes and as a support for cell culture (US patent 6120875). PTMs can be used as a platform on which to make biosensors. WO0204918, Lee and Yanavich, for example, describes the use of nanoporous membranes in binding assays such as immunoassays. The function of the membrane is to increase the local concentration of the analyte and thereby improve the sensitivity of the assay. The membrane surface is chemically modified by attachment of modifiers to which label binding ligands bind in the presence of the target analyte. The pore diameter and pore density are chosen so as to concentrate the analyte on the membrane surface or to selectively filter out the species of interest from a mixture. This illustrates one benefit of using a nanoporous membrane. Another benefit is disclosed in WO03016040, Brinker et al. They describe the use of inorganic nanoporous membranes as the solid support onto which are supported membrane mimetic architectures such as bilayers, tethered bilayers and hybrid bilayers. They cite the advantage of using a nanoporous solid support as being its ability to directly hydrate the bilayers through its water- filled channels. They suggest that the nanoporous membrane could be made on top of an electrode so as to form an impedance or electrochemical sensor. However the optimum design of such a sensor, the form of the electrode or its operation is not discussed. PTM can also be used as templates to create nanowires or nanotubes of metals or polymers by electroplating {Realisation of supported track-etched templates and their use for the synthesis of metallic and organic nanostructures. L. Dauginet-De Pra, E. Ferain, R. Legras, S. Demoustier-Champagne, Nuclear Instruments and Methods in Physics Research B, 196, 81-88 (2002). Conducting polymer nanotubes prepared in PTMs have been used as gas sensors (Elaboration of conducting polymer nanostructures. applications as responsive materials in gas sensors and biosensors, 220th ACS Fall National Meeting, Washington, USA, August 2000, S. Demoustier-Champagne, P. Y. Stavaux, M. Delvaux). In this type of sensor, the conducting polymer tubes connect a top and bottom electrode on the membrane. Gas permeates the membrane and changes the resistance of the nanotubes which then changes the resistance of the cell.

Similarly, PTMs containing gold nanotubes have been used to make sensors (Yoshio Kobayashi and Charles R Martin, "Highly sensitive methods for electroanalytical chemistry based on nanotube membranes", Analytical Chemistry, Vol 71, No 17, September 1, 1999, 3665-3672; Erich D Steinle, David T Mitchell, Marc Wirtz, Sang Bok Lee, Vaneica Y Young and Charles R Martin, "Ion channel mimetic micropore and nanotube membrane sensors", Analytical Chemistry, Vol 74, No.10, May 15, 2002, 2416-2422). In both these cases however, the membrane is acting to separate the target species from solution and external electrodes are used to measure the current or voltage in the cell.

The nanoparticles produced in NPMs can be used as sensors in their own right. US patent 2002187504, Chien et al describes the use of individual magnetic nanowires for probing and manipulation of molecules. They also describe ways of functionalising the surface of the nanowires with different chemical consituents. However, the nanowires are not used collectively as a sensor surface, as described in this invention.

Conductivity sensors for general laboratory use generally consist of two parallel plate electrodes with a fixed gap between. The electrodes are immersed directly in the solution of interest and a small voltage is applied between the plates. The resulting current flow is measured and by calibrating the cell against standard solutions of known conductivity, the conductivity of the solution can be measured. Miniaturised conductivity sensors are required in microfluid devices such as those designed to perform capillary electrophoresis (CE) or isotachophoresis (ITP) for example. Making the sensor smaller improves the resolution and sensitivity of the separation device. Standard designs, unlike the design proposed in this patent, use in-plane rather than through-plane electrode geometries where the electrodes either face each other tip to tip (opposing), are placed side by side (parallel) or are interdigitated. Reducing the separation between the two electrodes allows the range of the sensor to be extended to lower conductivity but to do this using the in-plane geometries requires a fabrication process for making the electrodes with a higher resolution. When the desired separation is in the range from 0.5 to 20 micron such a fabrication process is hard. The through- plane geometry has the further advantage that only one of the electrodes needs to be made small so as to have a small area of overlap. Reducing the area of overlap of the electrodes improves the spatial resolution and sensitivity of the sensor. Another advantage is that the NPM, rather than the electrodes can be patterned so as to define the active area of the sensor.

It is an object of the invention to provide novel electrodes for sensor applications, sensors fabricated therefrom, and fabrication methods therefor which mitigate some or all of the above disadvantages by making use of nanoporous membranes.

It is an object of the invention in a first expression of this concept to provide novel electrodes for sensor applications, sensors fabricated therefrom, and fabrication methods therefor that is formed integral with a PTM, thereby eliminating the need to use external electrodes to measure the electrical properties of what ever sample permeates through the PTM.

It is an object of the invention in a second expression of this concept to provide novel electrodes for sensor applications, sensors fabricated therefrom, and fabrication methods therefor, making use of PTMs during fabrication to provide electrodes with enhanced surface functionality.

The underlying concept of the invention lies in the use of the nanopores in a PTM to develop electrode surfaces with additional functionality. The PTM may then in a first expression of this inventive concept be retained integrally as part of the system to give yet further functionality, or in a second expression of this inventive concept be partially or fully removed before the resultant electrode is put to use.

Thus in accordance with the invention in the first expression in a first aspect an electrode for sensor application comprises an electrode element formed as a thin layer of conductive material integrally on the surface of a nanoporous membrane.

In a first principal embodiment of the first expression of the invention the electrode is incorporated into a novel conductivity cell. Preferred features are discussed hereinbelow in the context of this embodiment, but the skilled person will appreciate where such features have general applicability to other embodiments of the invention.

In accordance with this embodiment, a conductivity cell comprises a first electrode element and a second electrode element together disposed to form a conductivity cell, and a nanoporous membrane disposed to separate the two electrode elements that form the conductivity cell, at least one of the electrode elements being formed as a thin conductive layer integrally on the surface of the nanoporous membrane as above described.

In particular, each electrode element comprises a thin film such as a thin conducting metal film disposed upon a surface of the nanoporous membrane, preferably such that the said first and second electrode elements are disposed upon opposite surfaces of the nanoporous membrane so as to be spaced apart thereby.

The novel conductivity sensor of this embodiment of the invention thus comprises a nanoporous membrane (NPM) supporting layer with a thin conducting metal film on at least one surface. The arrangement of the thin metal film on at least one surface is crucial to the invention. It is arranged so that the film is continuous but does not bridge or block the nanopores in the membrane. With this arrangement liquids and gases may still permeate freely into or through the NPM whilst measurements of the impedance are being made. Thus the NPM may continue to function as a support membrane for microscopy or a filtration membrane or a cell culture surface while sensing is taking place.

The sensor platform described above can be used to measure the electrical impedance of any liquid or gas that permeates through the NPM. The measured impedance between the two electrodes, within the linear range of the sensor, is proportional to the impedance of the liquid or gas permeating the membrane. In a conventional impedance sensor, the magnitude of the measured impedance for any given impedance of the liquid or gas being sensed is given by the separation between the electrodes (d) divided by the area of overlap of the electrodes (A). This parameter is known as the cell constant. Therefore it is desirable to have both a large area of overlap and a small separation between the two electrodes when trying to measure high impedances such as for a non-conducting liquid with only a small quantity of an ionic species mixed in.

Frequently, it is desirable to minimise the width of the electrodes and their separation so as to reduce the sensing volume and space occupied by the sensor. Making the sensor smaller improves the spatial resolution and sensitivity of the separation device. Standard designs use in-plane rather than through-plane electrode geometries where the electrodes either face each other tip to tip (opposing), are placed side by side (parallel) or are inter-digitated. Reducing the separation between the two electrodes allows the range of the sensor to be extended to lower conductivity but to do this using the in-plane geometries requires a fabrication process for making the electrodes with a higher resolution. When the desired separation is less than 0.05 mm a relatively expensive photolithography fabrication process is required.

In contrast, by using a NPM sensor in accordance with the invention as described above, the electrode separation is determined by the thickness of the membrane and this thickness is usually less than 0.05 mm anyway. Therefore comparatively inexpensive methods of fabricating the sensor can be used. . The through-plane geometry has the further advantage that only one of the electrodes needs to be made small so as to have a small area of overlap. Reducing the area of overlap of the electrodes improves the spatial resolution and sensitivity of the sensor.

In applications where it is desirable to distribute the sensing function over a large sensing area (such as the base of a well in a microtitre plate), using the through-plane geometry, a smaller total number of electrodes is required since the electrode on one side can be common to all the electrodes on the other side of the membrane.

A further key aspect of the invention is that the PTM can be patterned according to the methods described for example in WOO 149402 and US2003020024. This means for example that the working and counter electrodes of the sensor can be relatively large, with a large overlap area, while the active area of the sensor is small, because the polymer has only been made nanoporous in a small part of the centre of the overlap area.

A further related advantage is that the PTM can be patterned in such a way as to precisely define the positions on the membrane where the solution under test contacts with both electrodes. At other positions, the test solution contacts the working electrode on one side of the membrane but is blocked from reaching the counter electrode on the opposite side.

The pore size and pore density of PTMs can be controlled and this fact can be employed to extend the range of the sensor to lower or higher conductivity.

Taking these considerations into account, pore diameters are preferably in the range 1-500 nm, and for example 25-250 nm. Pore densities are to some extent functionally related to pore sizes, but for these size ranges little effect can be expected at densities below 10⁶cm^"2 and in practice densities of at least 10 cm^" and more preferably 10 cm^" or greater are likely to be suitable.

The nanoporous membrane making up the device is fabricated by any suitable method, including those above described. The nanoporous membrane comprises a thin sheet of suitable dielectric material and conveniently comprises suitable polymeric material. In particular the nanoporous membrane is a polymer track etched membrane. Suitable polymeric materials include polyesters such as polyethylene terephthalate, wholly aromatic polymers including but not limited to polycarbonate, polyethers, polyesters, polysulphones, polyethersulphones, polyetherketones and polyetheretherketones. Also polyolefins, cellulose acetates, cellulose nitrates, polyimides and polyvinylidenefluoride (PVDF).

The membranes in a first alternative may be essentially self-supporting and able to act as a substrate for the electrodes, having a thickness of the order of 10-25 μm for example. In a second alternative thinner nanoporous membranes, for example with thickness in the range 0.1 μm to 10 μm, may be supported on a suitable substrate.

At least one of the electrodes comprises a thin conductive film laid down on a membrane surface so that the film is continuous but does not bridge or block the nanopores in the membrane. Film thicknesses of 10 to 200 nm are likely to be suitable. Any conducting material that may be deposited as a thin film may be used. High-conductivity and/or noble metals suitable for thin film deposition, such as gold, platinum, titanium, palladium, silver, nickel copper and alloys thereof, and most preferably gold and alloys thereof are preferred. Carbon may also be used. Conveniently each sensor may comprise a plurality of paired electrodes. In a first alternative this might be formed as a plurality of electrode pairs. More conveniently for many applications, the sensor comprises a larger first electrode on a first surface of the membrane, and a plurality of smaller second electrodes on the second surface of the membrane laterally spaced over an area generally coextensive with the said first electrode so as to form a plurality of sub cells, one in each overlap region. This arrangement can give greater sensitivity for a given sensor footprint area.

One of the electrodes may be patterned to provide a plurality of sensors.

A further advantage of the embodiment is that the liquid or gas being measured may be flowed through or within the nanoporous membrane.

In the field of separation science, conductivity sensors are often used in combination with separation techniques such as capillary electrophoresis (CE) or isotachophoresis (ITP) that act so as to spatially separate different chemical species (cations or anions) in a solution according to their mobility in the presence of a high external electric field. Such devices will be familiar to those skilled in the art.

The present embodiment can further be comprised as a separation device having a separation zone, a means to apply a high external potential to a solution in the separation zone, and a conductivity sensor as hereinabove described in fluid connection with the separation zone. The separation zone provides a fluid conduit and is for example a capillary.

The conductivity sensor is placed usually immediately after the separation zone and gives a change in impedance with time as the different species pass through the sensing zone. Constituents with very similar mobilities travel at similar rates through the separation zone and so may not be resolved from one another by the conductivity sensor if the area of the sensor is large. Frequently in this application therefore the conductivity sensor is designed to minimise the sensing volume (for example a pair of planar micro-electrodes of about 0.1 mm width and with a separation of about 0.1 mm).

The novel membrane sensor of the above described embodiment of the invention has the further advantage in this case that, since the electrodes are on top of each other rather than side by side, the spatial resolution of the membrane sensor is twice as good as the equivalent planar design.

The novel membrane sensor allows design of a device for performing separations on polarisable liquids that gives improved resolution of the constituent species for any design of cell length, separation voltage.

A further optional refinement of this embodiment of the invention allows for a sensor to be made that measures the electrical properties of bio-molecules growing on top of the NPM sensor platform. The sensor is constructed as described before with a NPM membrane and a thin nanoporous metal electrode on one side. The metal electrode in this case is patterned into a plurality of individually connected electrodes. The sensor measures the impedance between any two of these electrodes on the surface of the NPM. The novel feature of this sensor platform lies in the nanoporous nature of the metal surface. Biological materials such as cells or bacteria for example can grow on top of the sensor yet still receive nutrients such as water, oxygen, carbon dioxide, nitrogen etc through the NPM. The nanoporous nature of the electrode surface can also be used to locate or hold cells or other bio-species or particles that have a larger size than the membrane pores onto the region of the electrode. This can be done for example by applying a pressure drop through the membrane or by differential wetting caused by the porosity of the membrane.

In a further embodiment of the first expression of the invention, the sensor platform comprises a nanoporous membrane with at least one and preferably with a plurality of nanoporous conducting film electrode elements disposed on a surface thereof. Each electrode element can be individually addressable. The nanoporous conducting film electrode elements can be used to apply electrical fields to biological species such as cells or tissue that are in contact with, or in near proximity to, the surface of the NPM. Such fields can be used for example to manipulate the position of cells on the surface of the NPM or to electroporate cells so as to change the transport of materials in and out of the cells or tissue or to study the electrophysiology of the cells or tissue.

In a further embodiment of the first expression of the invention, the sensor platform comprises a nanoporous membrane with at least one and preferably with a plurality of nanoporous conducting film electrode elements disposed on a surface thereof, wherein one or more of the electrode elements are additionally functionalised by provision of one or more functionalising layers on each electrode element. Different electrode elements may be functionalised differently and may be made using different thin, conducting materials.

For example, some of the nanoporous conducting film electrode elements may be treated in such a way as to bind amino acids, proteins, enzymes, antibodies, antigens or DNA molecules to the electrode element surface. Methods of functionalising the surface in this way are well known in the art (see for example "Catechol Amperometric biosenor based on mix self assembled monolayers (SAM) gold electrodes", Marty, J.L. et al, PI -154, Euroanalysis- 12, Dortmund 2002, also CD Bain et al, J.Am.Chem.Soc, Vol 111, 1989, p321). Methods of applying the functional material to the electrode surface are for example spotting, droplet dispensing or nanoimprinting.

In a first alternative a plurality of electrode elements, at least some of which are functionalised, are provided on one surface of the membrane. Conveniently, the electrode elements are arranged in an array of size as befits the end application. For example in the case of a DNA or antibody spot array there would be a rectangular array of 12 x 5 electrode elements, each of size 100 micron with a pitch of 1 mm. For example in the case of a 96 well microtitre plate there would be a rectangular array of 12 x 8 elements, each of size approximately 1 mm with a 9 mm pitch.

The electrode elements can be used to make an amperometric biosensor. In this type of bio-sensor the working electrode has the amino acid, protein, enzyme, antigen, anibody or DNA probe immobilised onto it and this electrode is biased at a constant potential relative to an untreated nanoporous counter electrode. A reference electrode (e.g silver/silver chloride) is used to calibrate the magnitude of the potential bias. The advantages of this sensor platform are similar to the previous case - namely that the NPM provides a water and gas permeable platform on which biological species such as cells or bacteria grow more easily or which can be used as a filter.

In a further alternative, the sensor platform comprises a nanoporous membrane with a plurality of nanoporous conducting film electrode elements disposed on either side, wherein one or more of the electrode elements are additionally functionalised by provision of a functionalising layer on the conducting film surface. This allows for example that only electrode elements on one side are functionalised, or that electrode elements on one side are differently functionalised from electrode elements on another side.

In a further embodiment of the invention a supported NPM covers the surface of the working electrode and is intimately bonded to it. The NPM may be patterned so as to define the position of the nanoporous areas relative to the underlying electrodes. The properties of the NPM such as pore diameter, surface energy, pore density, packing arrangement or surface chemistry are such that the NPM is more permeable to one type of chemical species than to another. The NPM therefore acts so as to make the sensor more selective to the detection of one chemical species compared to another. This arrangement of patterned NPMS can also be used to ensure that drops of solution (sample or reagents) placed on top of the working area of the electrodes wet only a well defined area.

In a further aspect of the invention in its first expression a method of fabrication of an electrode for a sensor comprises the steps of preparing a nanoporous membrane by any suitable method and disposing an electrode element comprising a thin conductive layer integrally on the surface of the nanoporous membrane, for example by deposition.

In a particular embodiment, a method of fabrication of a conductivity cell comprises the steps of preparing a nanoporous membrane by any suitable method, disposing a first electrode and a second electrode together thereupon to form a conductivity cell such that the nanoporous membrane serves to separate the two electrodes that form the conductivity cell.

In particular, each electrode element comprises a thin film such as a thin conducting metal film and is deposited upon a surface of the nanoporous membrane, preferably such that the said first and second electrodes are disposed upon opposite surfaces of the nanoporous membrane so as to be spaced apart thereby.

The thin metal film is deposited to such an extent that the film is continuous but does not bridge or block the nanopores in the membrane. Within this general requirement any suitable deposition technique may be used, for example thermal or electron beam evaporation, metal sputtering, electroless plating or electroplating.

The nanoporous membrane is prepared by any suitable technique suitable to the preferred pore densities as above described. In particular, a particle track etching method is used, wherein the membrane comprises a polymeric sheet material which has been first damaged through particle beam bombardment and subsequently made porous by selective attack (eg chemical etching) of the damaged areas of the material to open nanopores to the desired extent.

Other preferred features of the method will be understood by analogy with the foregoing.

In accordance with the invention in the second expression in a first aspect a novel electrode surface comprises a generally planar conducting electrode element the surface of which is covered with one or more projecting nanowires or nanotubes. A nanowire or nanotube for the purpose of this description is a wire or rod-like structure whose diameter is less than 1 micron, preferably less than 300 nm and particularly less than 100 nm and whose length is greater than or equal to its diameter. The nanowires or nanotubes are intimately connected to the electrode surface, and preferably integrally formed therewith. It is intended that the invention should apply equally to generally solid elongate structures, which might more readily be referred to as nanowires, and which are typically formed from metallic material, and to hollow elongate structures, which might more readily be referred to as nanotubes, and which are typically formed from non-metallic material, such as conducting polymeric material or carbon. Where examples herein relate to nanowires, it will be understood that the concept will readily be adapted to nanotubes unless the context clearly prohibits, and the two should generally be considered interchangeable in the context of the invention. In the following description, we will for convenience generally use the term 'nanowire' to refer to both solid or hollow rod-like structures. The exact morphology will depend on the particular material they are made from.

A first advantage of this type of electrode is that the nanowires are of a size close to that of biological molecules such as amino acids, proteins, enzymes, antibodies, antigens or DNA. Therefore these species may interact more strongly with the nanowire electrode surface compared to an electrode surface without the nanowires.

A second advantage of this type of electrode is that the nanowires are smaller in size than cells and so can be used to sense, manipulate or apply fields to parts of a cell on a length scale that could not otherwise be easily achieved.

A third advantage of this type of electrode is its increased surface area compared to a planar electrode of the same size. The peak current response per unit area of a gold nanowire electrode of this type has been shown to be larger than that of an equivalent planar gold electrode. Preferably, the nanowires or nanotubes are electrically conducting. Suitable materials are selected from, but not limited to; metals such as gold, platinum, titanium, palladium, silver, nickel, cobalt, iron, copper and alloys thereof; conducting polymers such as polythiophene, polyaniline, polypyrrole and other such electrodepositable polymers; carbon. Generally, metallic materials are typically deposited as solid structures and non-metallic materials as hollow structures, but this is not pertinent to the invention.

In accordance with the invention in the second expression in a further embodiment a novel electrode surface comprises a generally planar conducting electrode element, a NPM on top and one or more nanowires/ tubes connected to the planar electrode and embedded within the NPM and/or projecting above the surface of the NPM.

In accordance with the invention in the second expression in a further embodiment a novel electrode surface comprises a NPM contained within the electrode surfaces described above the first and second aspects. The advantage of this arrangement is that the NPM part of the surface can be used to locate or hold cells or other bio-species or particles that have a larger size than the membrane pores onto the region of the electrode. This can be done for example by applying a pressure drop through the nanoporous part of the membrane or by differential wetting caused by the porosity of the membrane or by using the fact that water, oxygen or other nutrients can permeate the membrane only in the nanoporous part.

In a further embodiment of the second expression of the invention, the electrode platform comprises a plurality of electrode elements. Conveniently, the electrode elements are arranged in an array of size as befits the end application. For example in the case of a DNA or antibody spot array there would be a rectangular array of 12 x 5 electrode elements, each of size 100 micron with a pitch of 1 mm. For example in the case of a 96 well microtitre plate there would be a rectangular array of 12 x 8 elements, each of size approximately 1 mm with a 9 mm pitch.

In accordance with the invention in the second expression in a second aspect suitable methods are described for making the nanowire or nanotube electrodes. The methods are as follows.

A suitable supporting substrate is prepared. This comprises a flexible or rigid supporting substrate (e.g. silicon, glass, polyimide, polyethersulphone (PES), polyethylene napthalate (PEN, Kaladex), polyester (PET, Melinex)) onto the surface of which has been defined a pattern of bottom metal electrodes. Optionally, a thin (<100 nm) cross-linked polymer resin layer may be deposited on top, in order to protect the substrate material from the solvent in which the next polymer layer is dissolved. On top of this substrate, a layer of a track-etchable polymer (e.g. polycarbonate, polyimide, PVDF or other materials known in the art) is deposited. Spin coating may be used for example. The thickness of this film is in the range 0.1 to 50 micron but most preferrably in the range 0.5 to 10 micron. The polymer layer is made nanoporous by the methods described previously.

Nanowires or nanotubes are grown into the nanoporous regions of the membrane by electroplating from a suitable solution. The polymer membrane is totally or partially removed by dissolving it in a suitable solvent or by plasma etching, leaving the nanowires standing on the surface of the bottom electrodes or protruding above the surface of the partially removed NPM or embedded within the NPM. The position of the nanowires or nanotubes can be controlled by patterning the membrane according to the methods referred to previously. The positions where the membrane is removed can be defined photolithographically or by other types of masking processes.

A variation of this method is where a self-supported nanoporous polymer membrane is used to prepare the nanowires. A NPM is coated one side with a thin (50 to 100 nm thickness) metal film. An electrical contact is made to the thin metal electrode and further metal electroplated on top so as to form a continuous (non-porous) bottom metal film on one of the NPM. A supporting substrate is optionally added to adhere to the bottom electrode for example by using an adhesive layer. The NPM is turned over and metal nanowires grown into the NPM by electroplating. The length of the nanowires can be controlled by the plating current density and the plating time. The NPM is then removed by dissolving it in a suitable solvent or by plasma etching for example, leaving the nanowires on the bottom electrode surface, or partly removed to leave the nanowires protruding above the surface of the partially removed NPM or embedded within the NPM.

Preferably, the nanowires occupy a significant fraction of the exposed surface area of the electrode.

The invention will now be described by way of example only with reference to figures 1 to 17 in which:

Figure 1 illustrates the material used for the invention in perspective view and cross section;

Figure 2 is a schematic representation of the electrode layout of a first example of a conductivity sensor;

Figures 3 to 6 are graphical representations of results obtained for the sensor of the first example; Figure 7a is a cross section of a second example of a conductivity sensor;

Figure 7b is schematic representation of the electrode layout of the second example of a conductivity sensor;

Figures 8 and 9 show graphical representations of results obtained for the sensor of the second example;

Figure 10 shows a plan view of a microtitre well plate with bottom and top electrode layers separated by a NPM. The NPM has been pattered so as to accurately define the sensing area.

Figure 11 shows the current-voltage response of different gold electrodes to ImM FcCOOH (Ferrocene monocarboxylic acid) in 50mM phosphate buffer

(pH 7.0, 0.1M potassium perchlorate). (a) solid gold electrode, 1.6 mm diameter, (b) polycarbonate (PC) nanoporous membrane with no nanowires,

(c) Whatman Nucleopore PC membrane with pore diameter 150 nm, (d) PC

NPM with pore diameter 101 nm and pore density 1.51xl0⁹ cm^"2; Figure 12 shows the peak current density from the CV experiments shown in

Figure 11 ;

Figure 13a shows a plan view of a microtire well plate with nanowire electrodes fabricated in each well. Figure 13b shows the cross-section view of the same; Figure 14 shows a cross-section through a novel nanowire electrode;

Figure 15 shows a cross-section through a novel nanowire electrode;

Figure 16 shows a plan view of a circular planar electrode element;

Figure 17 shows the cumulative response of nanoporous gold electrode fabricated on nanoporous polycarbonate membrane, functionalised on one side with glucose oxidase, to 10 uM glutamate additions.

The basic material for all examples is shown on figure 1, which illustrates a small portion of nanoporous membrane in the sensor region with electrodes on either surface. A thin layer polymeric material (1) is provided with pores (3) to a suitable pore density, and a conductive metal layer (2) deposited thereupon so as to form a continuous layer of conductor but to leave the pores (3) open and unoccluded.

Example 1 describes the fabrication and use of a conductivity sensor made using a 0.02 mm thick polycarbonate nanoporous membrane fabricated by the polymer track etching process. The polycarbonate membrane is thick enough to be self supporting, and will therefore resemble figure 1 in the sensor region. Figure 2 gives a schematic of the sensor area, with a single upper electrode (12) overlapping a lower electrode (11) to from the sensor, separated by the dielectric nanoporous membrane layer therebetween (14).

Example 1 illustrates this point with reference to figures 3 to 6. The performance of sensors with increasing pore density and pore diameter is presented.

Example 1 describes the fabrication of a free-standing conductivity sensor using a 0.02 mm thick polycarbonate NPM. Four different polycarbonate nanoporous membranes (hereinafter examples 1A to ID) were made by the method as described in US 4956219. The pore diameter of the membranes was measured to be 85 nm for example 1 A, 96 nm for example IB, 100 nm for example 1C and 200 nm for example ID. The pore density for each was measured to be

' respectively. A 50 nm thick gold film was thermally evaporated onto each side of the membranes using a shadow mask (one for each side) to pattern the gold. The area of overlap of the electrodes on either side of the membranes was fixed to 0.06 cm². Electrical connections were made to the thin gold layer on each side of the membrane. The electrodes were connected to a spectrometer and the impedance spectrum of each membrane was recorded over the frequency range from 20Hz to 1 OKHz. The area of overlap of the electrodes was immersed with the solution to be analysed and the impedance spectrum recorded. This was repeated for 0.1M KC1 solution and de-ionised water and repeated for all the 4 different membranes. The results are shown in Figures 3 to 6 for examples 1A to ID respectively.

The data shows that the measured resistance falls as the conductivity of the solution is increased. The membrane with the largest pore diameter and highest pore density gives the largest change in resistance with conductivity of the solution while the membrane with the smallest pore density gives the smallest response. Increasing the pore diameter for a fixed pore density also increases the response of the sensor.

In order to maximise the performance of the sensor, it is desirable to make the NPM so thin that it becomes too fragile to support itself. In this case the sensor platform may be fabricated by forming the NPM on top of patterned metal electrodes supported on a suitable carrier substrate. A suitable carrier substrate might be a simple structural carrier, or might be a flexible circuit board or the like. This format of the sensor is especially suitable for use in micro-fluid capillary separation devices (CE and ITP chips).

Example 2 describes the fabrication and use of a miniaturised novel conductivity sensor based on a supported nanoporous membrane. A 1.1 mm thick borosilicate glass substrate (31) was coated with 10 nm of chromium (32) followed by 50 nm of gold using thermal evaporation. A positive working photoresist (Shipley SI 818-28) was spin coated onto the gold surface. The resist was exposed and developed using a photomask so as to define microelectrodes of width 0.05 and 0.1 mm (33). The resist pattern was transferred into the gold layer by wet etching the gold using a potassium iodide based etchant solution. The resist layer was then removed using a photoresist stripper solution.

A 4 μm thick layer of polycarbonate (34) was spin coated onto the substrate from a 9 wt% solution of polycarbonate in chloroform. The sample was baked for 4 hours at 180 °C. The polycarbonate layer was made nanoporous only over the top of each microelectrode by the methods described in US 4956219 and E. Ferain, R. Legras, K. Ounadjela, WO0149402 and E. Ferain, R. Legras, H. Hanot, US2003020024. The resultant membrane had a 96 nm pore diameter and 1x10 cm^" pore density. The outline of the nanoporous part is shown as (36) in figure 7b. A top electrode (35) was deposited on the surface to complete the cell. The resultant electrode arrangement is shown schematically in figure 7b.

The overlap between the top and bottom electrodes was 5 mm long by the width of the electrode (50 or 100 micron) wide. Electrical connections were made using small wires bonded on to the top and bottom electrodes by conducting epoxy.

The response of the device was measured by placing drops of aqueous KC1 solution of different concentration (ranging from 0.001M to 1M) on top of the top electrode and recording the impedance spectrum of the sensor in the range 20 Hz to lOOKHz. De-ionised water was also tested. The results for a bottom micro-electrode of width 50 micron (overlap area of 0.0025 cm ) are shown in figure 8. The impedance of the sensor reduced with increasing conductivity of the KC1 solution. The response of one of the 100 micron wide electrodes was compared to the response of one of the 50 micron wide electrodes. Both sensors had the same top electrode and so only differed by their area of overlap. Figure 9 shows the change in impedance of each sensor in response to 0.1 M KC1 solution. The sensor with the smaller area of overlap has the larger change in impedance.

Example 3

This example describes a generic design for an array of NPM sensors of type described in this patent, integrated with a microtitre well plate. Figure 10 shows part of the well plate with 9 wells. The outline of the micro-fluid wells on the plate is shown as layer 4. The design shows 2 bottom (working) electrodes per well (layer 1) and one common top (counter) electrode per well (layer 3). In order to address wells near the centre of the plate, electrodes have to run under some of the outer wells without being effected. This is done by patterning the membrane as shown (layer 2).

Example 4

Free-standing Polycarbonate (PC) nanoporous membranes (NPM) with different pore diameter and pore density were obtained. The NPM had a thickness of approx 20 micron. The membranes were dried in the oven at 120 °C for 1 hour, taped to a 0.175 mm thick PET film support piece and then a 50 nm thick gold film was thermally evaporated onto one side through a shadow mask, so as to define a series of electrodes of size approximately 10 mm diameter. The membranes were then electroplated in a solution of gold cyanide (Pur-A-Gold 401, Enthone OMI (UK) Ltd of Woking, Surrey) at 55°C for 10 mins at a constant current of 5 mA/cm² so as to seal up one side with gold. The thickness of the gold layer after plating was approximately 1 micron. The membrane was then turned over and gold nanowires were grown into the membrane by plating at a constant current of 5 mA/cm² for 5 mins. After plating, the PC membrane was dissolved away by immersing and rinsing it in chloroform. A copper strip was attached to the back of the nanowire electrode using conducting epoxy adhesive. Finally, protective tape was placed over and around the electrode so as to define the working area of nanowires that would be exposed to the test solution.

For comparison, one of the electrodes was prepared identically to the above procedure but the final step of electroplating of the nanowire was omitted. This sample is referred to as the "bare Au" in Figure 11 or "bare nP" in Figure 12.

A standard 3 electrode system was employed for cyclic voltametry (CV). Nanowire (nP) electrodes were used as working electrodes, an external micro Ag/AgCl electrode (OD=lmm) served as the reference electrode and a Pt wire was used as an external counter electrode. CV measurements were carried out in one well of 24-well template and nP electrodes were then connected to an electrochemical workstation (Autolab). A conventional solid gold electrode (1.6mm O.D. encased in Kel-F plastic from BAS) was used for the comparison with nP electrodes.

I mM FcCOOH (Ferrocene monocarboxylic acid, Sigma) was prepared in 50mM phosphate buffer (pH 7.0, 0.1M potassium perchlorate) and all CV experiments were carried out using this solution. Each experiment was performed using 1ml of ImM FcCOOH.

Figure 11 shows the CVs obtained using different nP electrodes and the solid gold electrode. ). Potential was cycled between -0.2 V and 0.4 with a scan rate from 10 to lOOmV/s. The response of all the electrodes is shown to be reversible. Figure 12 shows the peak current density obtained for each different electrode. Generally, the increase of peak height is proportional to the size of working electrode. Consequently, working electrodes with larger sizes show a larger peak current. As can be seen in Figure 12 it is apparent that the nanowire electrodes (which have a working area of approximately 10- 14 mm ) show higher responses to FcCOOH than the solid gold electrode (which has an area of approximately 2 mm ) and the control electrode prepared on a NPM but without nanowires.

Example 5

This example describes a generic design for an array of nanowire sensors of type described in this patent, integrated with a microtitre well plate. Figure 13a shows part of the well plate with 9 wells. The outline of the micro-fluid wells on the plate is shown as layer 4. The design shows 2 nanowire electrodes per well. The nanowires are shown as layer 3. Figure 13b shows a cross- section through the plate. A solid thin film metal electrode (82) is formed on top of a supporting substrate (83). Nanowires (84) are formed on top of the electrode. A bottomless microtire well plate (80) is attached by means of an adhesive (81) to form a fluid-tight seal around each well.

Figures 14 to 16 show alternative designs of novel nanowire electrode. Figure 14 shows nanowires (20) projecting through a NPM (21) on top of a planar conducting bottom electrode (22). Figure 15 shows nanowires (20) embedded in a NPM (21) on top of a planar conducting bottom electrode (22). Figure 16 shows a plan view of a circular planar electrode element (22) with nanowires (23) surrounding a region of nanoporous membrane (21) all surrounded by a non-porous part of the same membrane (24). Example 6

This example describes the fabrication and testing of an amperometric biosensor made using a functionalised thin gold electrode deposited on one side of a nanoporous membrane.

Free-standing polycarbonate (PC) nanoporous membranes purchased from Whatmann (Nucleopore brand) were used. The pore diameter of these membranes was approximately 150 nm, as determined by SEM examination. A 50 nm thick gold film was thermally evaporated onto one side through a shadow mask, so as to define a series of circular electrodes of diameter approx 15 mm. A copper strip was attached to the electrode using conducting epoxy adhesive.

Black, polystyrene, bottomless 96 well microtitre plates were purchased from Greiner Bio-One Ltd (part number 655000-06). The bottom of the microtitre plate was coated with a 50 micron thick pressure sensitive adhesive film (Adhesives Research Inc ARClad 8102). A 3 mm hole was drilled through the adhesive film in the centre of each well. The membranes were immersed in surfactant solution (composition - BAS commercial product - WENZ surfactant ,product code: CF-1075) for 20 mins at room temperature. Then a 1 micro litre drop of horse radish peroxidase redox polymer solution (BAS, no. CF - 1070 or in kit with sufactant as MF-2096) was deposited onto the gold surface. Finally, a solution containing the enzyme glutamate oxidase (200 units per ml from YAMASA in Tokyo, Japan) was pipetted on top of the redox polymer layer and allowed to dry to from a film. The electrode with redox polymer and enzyme layer on was fixed to the adhesive tape underneath one well of the microtitre plate, centred on the 3 mm hole. In order to test the linearity and reproducibility of the sensor, 10 micro molar aliquots of glutamate were added stepwise to a buffer solution in contact with the sensor surface. The response of the sensor is shown in Figure 17. The graph shows that the sensor responded linearly. The gradient of this graph 1 9 gives the responsivity of the sensor as 52 nA(μM)^" cm^" .

Claims

1. An electrode for sensor application comprising an electrode element formed as a thin layer of conductive material integrally on the surface of a nanoporous membrane.

2. An electrode in accordance with claim 1 incorporated as the working electrode of an amperometric biosensor.

3. An electrode in accordance with claim 1 incorporated to form a conductivity cell.

4. A conductivity sensor cell in accordance with claim 3 comprising a first electrode element and a second electrode element together disposed to form a conductivity cell, and a nanoporous membrane disposed to separate the two electrode elements that form the conductivity cell, at least one of the electrode elements being formed as a thin conductive layer integrally on the surface of the nanoporous membrane in accordance with claim 1.

5. A conductivity sensor cell in accordance with claim 4 wherein each electrode element comprises a thin film such as a thin conducting metal film disposed upon a surface of the nanoporous membrane, preferably such that the said first and second electrode elements are disposed upon opposite surfaces of the nanoporous membrane so as to be spaced apart thereby.

6. A cell in accordance with claim 5 having a through-plane electrode geometry wherein generally parallel electrodes are at an electrode separation determined by the thickness of the membrane as less than 0.05 mm.

A cell in accordance with one of claims 4 to 6 wherein the membrane is patterned to precisely define the positions on the membrane where the solution under test contacts with both electrodes, such that at other positions, the test solution contacts a working electrode on one side of the membrane but is blocked from reaching a counter electrode on the opposite side.

8. An electrode or cell in accordance with any preceding claim wherein pore diameters are in the range 25-250 nm.

9. An electrode or cell in accordance with any preceding claim wherein pore densities are at least 10⁶cm^"2.

10. An electrode or cell in accordance with any preceding claim wherein the nanoporous membrane comprises a thin sheet of dielectric polymeric material comprising a polymer track membrane selected from polyesters such as polyethylene terephthalate, wholly aromatic polymers including but not limited to polycarbonate, polyethers, polyesters, polysulphones, ethersulphones, polyetherketones, polyetheretherketones, polyolefins, cellulose acetates, cellulose nitrates, polyimides and polyvinylidenefluoride (PVDF).

11. An electrode or cell in accordance with any preceding claim wherein the thin conductive film laid down on a membrane surface is disposed to a thicknesses of 10 to 200 nm so that the film is continuous but does not bridge or block the nanopores in the membrane.

12. An electrode or cell in accordance with any preceding claim wherein the thin conductive film comprises a thin film deposit of high-conductivity and/or noble metals selected from the group comprising gold, platinum, titanium, palladium, silver, nickel copper and alloys thereof, and most preferably gold and alloys thereof.

13. A conductivity sensor cell in accordance with any one of claims 4 to 12 comprising a plurality of electrodes including at least a larger first electrode on a first surface of the membrane, and a plurality of smaller second electrodes on the second surface of the membrane laterally spaced over an area generally coextensive with the said first electrode so as to form a plurality of sub cells, one in each overlap region.

14. A separation device having a separation zone, a means to apply a high external potential to a solution in the separation zone, and a conductivity sensor in accordance with one of claims 4 to 13 in fluid connection with the separation zone.

15. An electrode in accordance with claim 1 incorporated to form a sensor platform comprises a nanoporous membrane with a plurality of nanoporous conducting film electrode elements disposed on a surface thereof.

16. A sensor platform in accordance with claim 15 wherein each electrode element is individually addressable.

17. A sensor platform in accordance with claim 15 or 16 so arranged as to be adapted in use to apply electrical fields to biological species such as cells or tissue that are in contact with, or in near proximity to, the surface of the nanoporous membrane.

18. A sensor platform in accordance with any one of claims 15 to 17, wherein one or more of the electrode elements are additionally functionalised by provision of one or more functionalising layers on each electrode element.

19. A sensor platform in accordance with claim 18 comprising a nanoporous membrane with a plurality of nanoporous conducting film electrode elements disposed on either side, wherein one or more of the electrode elements are additionally functionalised by provision of a functionalising layer on the conducting film surface.

20. A solid electrode surface comprising a substrate with a nanoporous membrane in accordance with any preceding claim intimately bonded to it, the nanoporous membrane being patterned so that only selected regions of the electrode can contact with liquids or gases in contact with the top surface of the membrane.

21. An electrode surface comprising a generally planar conducting electrode element the surface of which is covered with one or more projecting nanowires or nanotubes, each nanowire or nanotube comprising a solid or hollow wire or rod-like structure whose diameter is less than 1 micron and whose length is greater than or equal to its diameter.

22. An electrode surface in accordance with claim 21 comprising electrically conducting nanowires selected from metals such as gold, platinum, titanium, palladium, silver, nickel, cobalt, iron, copper and alloys thereof;

23. An electrode surface in accordance with claim 21 comprising electrically conducting nanotubes selected from conducting polymers such as polythiophene, polyaniline, polypyrrole and other such electrodepositable polymers.

24. An electrode surface in accordance with claim 21 comprising electrically conducting nanotubes fabricated from carbon.

25. An electrode surface in accordance with one of claims 21 to 24 comprising a generally planar conducting electrode element, a nanoporous membrane disposed on the surface thereof, and one or more nanowires or nanotubes connected to the planar electrode and embedded within the nanoporous membrane and/or projecting above the surface of the nanoporous membrane.

26. An electrode surface in accordance with claim 21 wherein the nanoporous membrane includes electrode surfaces in accordance with claim 1.

27. An electrode surface in accordance with one of claims 21 to 25 composed as an electrode platform comprising a plurality of electrode elements each comprising such an electrode surface.

28. A method of fabrication of an electrode for a sensor comprises the steps of preparing a nanoporous membrane by any suitable method and disposing an electrode element comprising a thin conductive layer integrally on the surface of the nanoporous membrane.

29. A method of fabrication of a conductivity cell incorporating an electrode of the method of claim 28 comprising the steps of: preparing a nanoporous membrane by any suitable method, disposing a first electrode and a second electrode together thereupon by the method of claim 28 to form a conductivity cell such that the nanoporous membrane serves to separate the two electrodes that form the conductivity cell.

30. The method of one of claims 28 to 29 wherein nanoporous membrane is prepared by a particle track etching method, wherein the membrane comprises a polymeric sheet material which has been first damaged through particle beam bombardment and subsequently made porous by selective of the damaged areas of the material to open nanopores to the desired extent.

31. A method of manufacture of a nanowire electrode surface comprises the steps of: preparation of a suitable flexible or rigid supporting substrate onto the surface of which has been defined a pattern of bottom metal electrodes; optionally, depositing a thin (<100 nm) cross-linked polymer resin layer in order to protect the substrate material; depositing thereon a layer of a track-etchable polymer to a thickness in the range 0.1 to 50 micron; creating a nanoporous structure within the layer by a particle track etching method; growing nanowires into the nanoporous regions of the membrane by electroplating from a suitable solution; totally or partially removing the polymer layer to leave the nanowires standing on the surface of the bottom electrodes or protruding above the surface of the partially removed NPM or embedded within the NPM.

32. A method of manufacture of a nanowire electrode surface comprises the steps of: coating one side of a free standing nanoporous membrane with a thin metal film. optionally adhering the nanoporous membrane to a suitable supporting substrate; growing nanowires into the nanoporous regions of the membrane by electroplating from a suitable solution; totally or partially removing the polymer layer to leave the nanowires standing on the surface of the bottom electrodes or protruding above the surface of the partially removed NPM or embedded within the NPM.