WO2005122296A2

WO2005122296A2 - An electrode device, process for making an electrode device and a method of electrochemical detection using an electrode device

Info

Publication number: WO2005122296A2
Application number: PCT/EP2005/006109
Authority: WO
Inventors: James Davis; Laura Yonge
Original assignee: University Of Surrey
Priority date: 2004-06-07
Filing date: 2005-06-07
Publication date: 2005-12-22
Also published as: WO2005122296A3; GB0412670D0

Abstract

An electrode device comprising a laminate assembly of alternating insulating layers and electrode layers is disclosed. The laminate comprises at least one channel for carrying a fluid, which extends through the insulating and electrode layers. The electrode device has applications as an electrochemical detector, particularly for use with liquid chromatographic detection where multiple electrode devices can be used in series to form a detector array, sample-sensing structures and multi -titre plates for high throughput screening 3D multi-parametric detection. A process for producing the electrode device is also disclosed, together with methods of using the electrochemical device in electrochemical detection.

Description

An Electrode Device, Process for Making an Electrode Device and a Method of Electrochemical Detection using an Electrode Device

Scope of the Invention

The present invention relates to an electrode device and in particular an electrode device comprising a laminate assembly of alternating insulating layers and electrode layers, said device having at least one channel for receiving a fluid extending through the layers of the laminate. The electrode device is useful as an electrochemical detector for analytes such as chemical species carried in a fluid. More particularly, the electrode device can be used with liquid or ion chromatographic techniques, as a generic sensor plate for reagentless 3D multi parametric electrochemical detection and as a disposable sampling-sensing structure.

Discussion of Prior Art

Analytical detection and determination of an analyte in a fluid can be carried out by a number of spectroscopic and non-spectroscopic techniques such as UV, infrared and mass spectroscopy, nuclear magnetic resonance and electrochemical detection. Electrochemical detection is proving to be a facile and versatile analytical method. This analytical technique relies upon the detection of an electrochemical response from an analyte at the working or indicating electrode.

Electrochemical detection typically utilises two electrodes, a working and a reference/counter (or auxiliary) electrode. The latter electrode is normally of large area when compared to that of the working electrode. The detector can be used in either potentiometric or amperometric mode. In potentiometric mode the analytical signal is derived from the potential difference that develops between the two electrodes under zero current flow. In amperometric operation, the working electrode is maintained at a particular potential with respect to the counter electrode by a potentiostat with the current from the induced electrode process serving as the analytical signal. In amperometric or coulometric electrochemical detection, a third electrode called a reference electrode can also be present. When all three electrodes are connected to a potentiostat, the potential of the working electrode can be held at a particular potential with respect to the reference electrode. The cuirent flow can then be monitored.

Conventionally, the electrodes for electrochemical detection are formed from metallic or carbon plates, films or meshes, or conducting foams. Different electrode substrates and the use of different operating potentials can elicit significantly different responses from a given analyte. It is usual for only a single electrode substrate to be present in a given electrode. A number of separate electrodes are therefore required to analyse the response of particular analyte to a variety of electrode substrates. This requirement for different electrodes is therefore costly. The present invention provides an electrode device formed of a laminate of insulating and conducting layers wherein each conducting layer can be formed of a different electrode substrate. The electrode device can be simply and economically manufactured using known lamination techniques.

Liquid chromatographic techniques can separate a complex matrix of analytes. These analytes are then conventionally analysed using UV or mass spectroscopy which provide good resolutive capacity and structure determination. However, the interpretation of complex mass spectra arising from co-eluting analytes can be far from trivial and are generally more suited to investigative studies rather than routine determination. Recently, liquid chromatographic techniques followed by the electrochemical detection of analytes has been shown to be a versatile analytical option when attempting to characterise complex matrices of chemical species. Electrochemical detectors can exhibit sensitivities which are superior to conventional UV spectroscopic techniques and are also significantly less expensive, both to purchase and to operate, than mass spectrometers. The combination of electrochemical detection with liquid chromatographic techniques therefore has great utility in routine determinations without compromising selectivity.

The electrochemical properties of an analyte are governed by a variety of factors such as electrode substrate and potential, and are independent of those factors influencing column retention. These properties therefore allow multi-dimensional resolution. For example, performance can be improved by utilising an array comprising multiple detectors placed in series, each detector formed of a different electrode substrate or operating at a different potential. Consequently, two analytes eluting at the same time can be resolved at the electrochemical detection array as a result of their electrode substrate and potential dependent responses.

Conventional post-column electrochemical detectors are usually employed as a discrete, single channel detector operating under amperometric control. A plurality of detectors can be combined to form electrochemical array systems which currently dominate liquid chromatographic electrochemical detection techniques. These anays invariably employ an unmodified porous graphite working surface of the electrode. Porous graphite working surfaces are common principally because of the ease with which graphite foams can be fabricated. However, graphite foams do not exhibit any particularly advantageous electrode response characteristics when compared to other electrode substrates such as platinum, gold, silver, copper or nickel.

The adaptation of the array approach using amperometric or coulometric methodologies to other substrates would offer an additional level of resolution, where liquid chromatographic column retention, operating potential and substrate reactivity could combine to enhance the analytical profiling of a given sample.

Summary of the Invention

The present invention provides an electrode device which can be used as an electrochemical detector in liquid chromatography and exhibits improved performance. The electrode device can be optimised to particular applications or analytes and can be readily prepared and integrated with conventional liquid chromatographic architectures.

Electrochemical detector an'ays formed from the electrode device of the invention can be used in the liquid chromatographic analysis of nutritional components in physiological fluids such as plasma. Such arrays are applicable to the analysis of complex matrices of biological analytes and allow the physiological influence of nutritional supplements to be assessed.

The electrode device of the invention also has applications as a disposable electro- analytical sensor system for decentralised testing within a variety of agri-food, biomedical and environmental contexts. Electrode passivation occuπing through the progressive accumulation of molecular debris at an electrode substrate is one of the major obstacles that can hamper the application of electro-analytical techniques. The application of photolithographic and screen printing technologies to sensor production allow the mass production of near identical electrodes rendering single shot electrochemical analysis economically viable.

However, current photolithographic and screen printing technologies require expensive equipment and a high degree of user expertise in order to achieve reproducible production of the system. The electrode device of the invention can be provided as a disposable sensor strip which is simple to manufacture in a conventional research setting and is relatively inexpensive.

The electrode device of the invention also has applications in the field of electrochemical high throughput screening systems. Conventionally, most microtitre plates used in such systems are based around single parameter analysis that typically utilises spectroscopic detection. The electrode device of the invention can be provided as a multi-titre plate which enables multi-parametric reagentless detection through the use of a variety of electrode substrates or potentials. In contrast to conventional electrochemical high throughput screening systems, these properties can be achieved within each discrete well in the array. The microtitre plate electrode device of the invention is particularly advantageous in conventional high throughput screening robot systems.

Statement of Invention

According to the invention, there is provided an electrode device comprising a laminate assembly of alternating insulating layers and electrode layers, said device having at least one channel for receiving a fluid extending through the layers of the laminate.

The presence of a channel through the laminate exposes the conductive surface of the electrode layers in the channel which, upon contact with a fluid phase, form electrodes. The electrode device can therefore serve as an electrochemical detector.

In a preferred embodiment of the invention, the elecu'ode device preferably has insulating layers laminated on both sides of an electrode layer to ensure the electrical insulation of each electrode layer from neighbouring layers and from the analyte fluid.

In another preferred arrangement, the electrode device has an insulating foil as the insulating layer. The insulating layer can be made from any non conducting film that can effectively adhere to the conductive substrate through heat treatment or adhesive coating. More preferably, the insulating foil is a polyester foil.

The electrode device can have a single or a plurality of electrode layers. It is preferred that the electrode layer has a thickness of 10-100 μm.

If there is only a single electrode layer, the electrode device can operate as an electrochemical detector in single channel amperometric mode. In this mode, the counter/reference electrode is external to the electrode device. Such a detector configuration can be used in liquid chromatographic detection where the counter electrode is foπned by a metallic chromatographic column, such as conventional steel columns.

A plurality of electrode layers in the electrode device is preferred. When more than one electrode layer is present, at least one electrode layer may form the working electrode and one electrode layer forms an internal counter/reference electrode, allowing the electrode device to operate in either potentiometric (two electrode) or amperometric mode.

Alternatively, the electrode device having more than one electrode layer present can operate in multichannel amperometric or coulometric mode. In this mode, more than one of the electrode layers forms the working electrodes and one electrode layer forms an internal counter/reference electrode, with an external electrode forming the reference/counter electrode.

If there are more than two electrode layers present, the electrode device can operate in amperometric or coulometric mode without the need for an external electrode. In this mode, at least one of the electrode layeis may fonn the working electrode, with at least one other electrode layer forming an internal reference electrode and a further electrode layer forming an internal distict counter electrode. This mode has the advantage that each of the three electrodes necessary for amperometric function are present in the electrode device.

When the electrode device comprises a plurality of electrode layers, these are formed from the same or different conductive members. The conductive member may comprise at least one of carbon, a conductive polymer, metal or alloy and may be present as a film, foil or printed layer. Preferred metals are copper, nickel, chromium or zinc and alloys thereof as well as silver, platinum or gold.

Multiple electrode layers can be held at the same or different potentials and can be formed of the same or different conductors in a single electrode device to form an electrode or detector array. Individual electrode layers may also be formed of more than one conducting material to provide multiple electrode substrates in a single conducting layer. Detector arrays can also be formed by combining multiple electrode devices in series.

In a further preferred arrangement, the electrode layer is adapted to establish an external electrical contact at a location spatially removed from the analyte channel.

In certain devices, all the layers may be connected in parallel, for example with a common contact strip provided along an edge of the device. However, in other embodiments it is desirable to allow each layer to be provided with its own electrical connection. This configuration allows the independent control of the potential of the electrode layers.

The electrode device of the invention allows redox recycling and signal amplification where different anode/cathode material combinations can be harnessed to counter problems associated with single electrode substrate arrays. Such single electrode substrate arrays can exhibit an inability to reversibly reconvert an electrode product of an analyte which leads to breaks in the redox cycle.

For instance, the electrode layers in the laminate of the electrode device can not only alternate between oxidising and reducing potentials but also in terms of substrate material. For example, in the case of aminothiol analysis the cathode can be optimised to facilitate the specific reduction of the disulphide.

The electrode layer may be formed of at least one conductor selected from: carbon fibre, carbon cloth, carbon (or "Toray") paper, screen printed carbon or screen printed carbon composite film, inkjet printed carbon or inkjet printed carbon composite film, platinum foil, silver foil, gold foil, copper foil or nickel foil or foils made from alloys of these metals. Such foils are commercially available. It is preferred that the electrode layer is in the form of a strip. Conductive inks which can be applied using an inkjet printer can also be used to form the electrode layer. Screen printed carbon offers a number of advantages as an electrode layer over the pressed fibre matting used in conventional cloth electrodes because catalysts, biological material and/or modifiers can be readily incorporated throughout the bulk of the electrode material through simple mechanical blending

In parti cular, the catalyst may comprise at least one of platinum, palladium or gold particulates or a metallocyanine. Metallophthalocyanine/carbon electrodes can be provided for the detection of reduced aminothiols. Furthermore, the biological material may be an enzyme, such as an oxidase enzyme.

In certain situations, the close proximity of electrode layers within the laminate and the sequential nature of the addressing of the electrodes could result in the problem of crosstalk between the layers loaded with enzyme sensing components in the same channel.

Such a diffusion of electroactive enzymatic by-products to an adjacent sensing electrode layer in the channel could lead to the erroneous amplification of the analytical signal at that layer. This diffusion of eleciroactive enzymatic by-products is exemplified by the measurement of hydrogen peroxide originating from unmediated oxidase systems.

A second problem can arise where the enzyme itself desorbs from its original electrode layer and is then lost within the centre of the channel or relocates through non-specific binding at another electrode layer. This situation will lead to the artificial depression of the signal from one layer and the potential amplification of the signal from another layer. The extent to which these problems could affect the detection method will depend, to a large extent, on the time it takes to sequentially scan each conducting layer.

Both of these crosstalk problems can be minimised by recessing the electrode layer from the adjacent insulating layers and/or optionally entrapping the enzyme within a permselective membrane. The former solution can be achieved through the use of pre -patterned films where the surface area of a specific channel is varied from layer to layer. Recessing the electrode layer presents a diffusional barrier minimising the transport of enzyme and electrodetectable by-product to neighbouring layers. The electroploymerisation of monomers derived from pyrrole, aniline, indole, thiophene or phenol within each channel results in the localised deposition of a permselective barrier entrapping the enzyme and thereby preventing co-connection/crosstalk. The thickness of the barrier is preferably in the range of a few nanometers, for instance 10-100 nm.

It is also possible to reduce the cost of the electrode device by the judicious patterning or placement of the electrode layers. This can be achieved by localising a more expensive conductor in the region of the channel with a different less costly conductor present which is not in the region of said channel e.g. by coating or electroplating.

Consequently, expensive detector materials such as catalysts e.g. gold or platinum, or carbon loaded with biological material, such as an enzyme, can be localised in the region of the fluid channel with an inexpensive copper band facilitating electrical contact to external instrumentation. This provides a cost effective option which will allow the commercial exploitation of application optimised devices.

It is known that different electrode substrates, even when poised at different operating potentials can elicit significantly different responses from different analytes. The versatility of the electrode device of the invention in facilitating the incorporation of different electrode materials within the channels allows the implementation of chemometric methodologies to interpret and quantify multi -electrode responses to improve selectivity.

The fluid channel preferably extends through the entire thickness of the laminate of the electrode device, intersecting every insulating and electrode layer. This provides open channels to allow a fluid to be carried. Open channels can also allow the filling of the channels by capillary action and avoid the problem of air blockages preventing channel filling in a closed-ended system. Consequently, it is preferred that the base of the channel is not enclosed by a substrate present on the base of the laminate. However, a base substrate closing one end of the channel may be attached to the laminate.

The cross-section of the channel determines the shape of the electrode in the electrode device. The channel can have a cross section of any shape. It is preferred that a circular cross section is used which forms a ring electrode where the channel intersects the electrode layer. The surface area of the ring electrode can be simply calculated from the diameter of the channel and the thickness of the electrode layer. An increase in the diameter of the channel increases the size of the ring electrode formed in the electrode layer with a con"esponding increase in the magnitude of the response to an analyte.

The channels can be formed by directly coring the laminate to reveal the internal electrode layers. Channels of circular cross-section can be achieved by drilling, the diameter of the drill bit corresponding to the diameter of the channel formed. It is preferred that the coring is earned out prior to lamination to avoid the problems of inter-layer smearing and shorting which may be associated with post-lamination channel coring.

In another preferred arrangement, the channel is formed by a patterned or random perforation of the electrode layer. For example, patterns can be provided which mimic the structure of conventional carbon foam coulometric detectors by providing non-linear channels which cause turbulent fluid flow.

In a further preferred arrangement, the electrode device has a plurality of channels. In particular, the channels can be arranged randomly or in a geometric pattern such as in a single or multiple rows.

In another preferred arrangement, the cross-section of a channel in an electrode layer can be greater than the cross-section of said channel in the two adjacent insulating layers, such that the channel in the electrode layer is recessed from the channel in said adjacent insulating layers. In an alternative preferred arrangement, the cross-section of a channel in an insulating layer can be greater than the cross-section of said channel in the two adjacent electrode layers, such that the channel in the insulating layer is recessed from the channel in said adjacent electrode layers, increasing the surface area of the adjacent electrode layers.

An extended electrode array comprising a plurality of electrode devices placed in series is another preferred aspect of the invention. Such an array is suitable as a post liquid chromatography electrochemical detector.

A sampling-sensing strip comprising a series of electrode devices arranged in a row wherein the electrode layers are formed by a common conducting member is a further aspect of the invention. The sampling-sensing structure is preferably disposable.

The electrode device can be provided as a strip with a single row of channels forming multiple detection sites. This approach is acceptable when considering the use of the strip as a conventional electrode where analysis is not conducted directly within the bulk sample matrix but rather in the individual channels. The channels are used to extract a defined sample volume with the analysis done in that extract.

Preferably, the mouths of the channels on the surface of the laminate are covered by a removable protective layer. It is also preferred that the channels are circular in cross- section with a diameter of 1 mm or less to maintain film contact across the ring electrode.

Any of the conductors discussed above can be used to form the electrode layer. Strips of metallic films or foils such as those already mentioned are ideally suited, and can optionally be electroplated with other conducting materials as already discussed. The terminating end of the conductive strip can allow the connection of the conducting layer to an external device such as a potentiostat. It is preferred that a plurality of conducting layers are present, with the terminating end of the conductive strip of each allowing the individual connection of each electrode layer to the external device.

A multi-titre screening plate comprising an array of electrode devices in accordance with the invention wherein at least some of the electrode layers are formed by a common conducting member is a further aspect of the invention. The multi -titre plate is suitable for reagentless multi -parametric detection. Such multi -titre plates can be used in high throughput screening robot sensing systems.

The multi-titre screening plate comprises a plurality of channels, preferably in a grid formation. It is preferred that the base of the laminate is not enclosed. Delivery of the sample to the channels can therefore be achieved simply by capillary action. This obviates the problems of air lock that are traditionally associated with sample delivery to small volume closed channels. This provides an additional advantage over conventional plate designs in that because the sensing layers are locked within the walls of the channels in the laminate structure, simple air displacement of the solution will allow the re-use of the piate.

It is possible to seal the base of the laminate using a substrate material, such as that used for the insulating layer of the electrode device. However, this embodiment requires a minimum channel size of 0.5 mm to allow the filling of the channels without the problems associated with air lock.

The multi-titre plate should preferably have at least 3 and more preferably 6 individually addressable electrode layers. Typical plates have 96 channels (each preferably of 0.35 mL) and 384 channels (each preferably of 0.12 mL (normal) or 0.4 mL (deep well)).

The channel volume is defined by the channel cross-section and laminate thickness. The laminate thickness is preferably 1-4 mm. The channels are preferably circular in cross-section. Diameters of 0.1-1 mm can be achieved using conventional mechanical engineering methods. This provides channels with preferred volumes of 0.1 -5 microlitres. Channel formation by laser ablation using an excimer or copper vapour high energy source provides a minimum diameter of 10 micrometers, providing well volumes of a few nL or less.

The present invention also relates to a process for the manufacture of an electrode device comprising the steps of: i) arranging sequential layers of insulating and electrode layers to form a lay- up; and ii) laminating said lay-up.

The integrity of the structure and electrical isolation of the device components are maintained by the bonding of the insulating layer between each conducting layer in the laminate. This can be achieved using commercial lamination techniques. Hot lamination techniques can be used for chemical sensing layers. It is preferred that hot lamination is carried out by baking the device for several hours. This provides a laminate which is resistant to splitting when the analyte fluid vehicle is water. Suitable insulating materials for hot lamination are commercially available polyester laminating pouches, which can be resin-backed and other non conducting plastics, such as polyvinylchloride, polystyrene etc..

However, cold lamination techniques involving adhesive layers should be used for the encapsulation of temperature sensitive agents such as enzymes contained within the electrode layers.

In addition, production of the electrode device by lamination allows large numbers of identical detectors to be fabricated from a single laminate sheet.

The laminate construction of the electrode device of the invention allows the assembly of a number of different electrode devices in series to form the backbone of a larger detection array that is analogous to a commercial multi-channel coulometric array detector. In contrast to conventional arrays, the present invention allows the selection of a variety of pre-packaged electrode devices, each consisting of different electrode substrates, allowing the technician to tailor the structure of the detection array to a given application. Such devices are ideally suited for detector arrays in liquid chromatographic electrochemical detection.

In a preferred arrangement, the channel is formed by coring the lay-up prior to lamination. In an alternative preferred arrangement, the channel is formed by using pre-pattemed insulating and electrode layers which are pre- perforated in the position of the channel.

In another preferred arrangement, the surface of the channel at the electrode layer is coated with a different conductive material by electroplating. In this way an economic advantage can be obtained by plating expensive detector materials such as gold or platinum only at the surface of the conductive material at the channel. Furthermore, internal reference electrodes can be incorporated into the electrode device in this way. For instance, silver can be electroplated onto the surface of the conductive material furthest from that of the working electrode . The electroplated silver can then be electrochemically oxidised to produce a silver oxide pseudo reference electrode or chloridised to form a conventional silver/silver chloride solid reference. Such pseudo reference electrodes obey the Nerast equation for a reversible oxidative process.

In a further preferred embodiment, the surface of the electrode layer at the channel is electropolymerised with a permselective membrane. Suitable membrane materials are electroploymers of monomers derived from pyrrole, aniline, indole, thiophene or phenol.

In another preferred embodiment, the electrode layer of the electrode device of the invention can be manufactured by methods know in silicon technology. In particular, films of electrode layers comprising conductive materials can be formed using vapour deposition, optionally with the use of an appropriate mask, onto a thermally stable insulating layer. Alternatively, the electrode layer can be formed by the solvent casting of conducting films. The conductive, such as metallic, particles forming the electrode layer can be uniformly dispersed in a solvent to provide a homogeneous dispersion, admixed with a fugitive polymer and cast onto an substrate, dried and stripped from the substrate to provide a conductive-particle-fugitive polymer film. This can then be applied to the insulating layer preheated above the glass transition temperature of the fugitive polymer to form the electrode layer. This operation is then followed by vapour deposition of further conductive particles forming the electrode layer as discussed above to provide an improved electrode layer.

The present invention also relates to a method of potentiometric electrochemical detection of an analyte wherein the electrode device, array, strip or plate of the invention is connected to a potentiostat such that the electric potential which develops across an electrode layer is received by an external recording device.

The present invention also extends to a method of amperometric electrochemical detection of an analyte wherein the electrode device, array, strip or plate of the invention is connected to a potentiostat such that the electric potential which develops across an electrode layer is controlled by the potentiostat and the current received by an external recording device.

In a preferred amperometric method, a reference electrode is present and the potential of the electrode layer of the device, array, strip or plate is held at a particular potential with respect to the reference electrode.

In a another preferred method of amperometric or potentiometric detection, multiple electrode layers can be connected to the potentiostat in parallel to amplify the magnitude of the response. In another preferred amperometric method, the conducting layers can be individually set to different electrical potentials with the electrical currents from each electrode layer being received by an external recording device.

In a further prefen-ed amperometric method, the conducting layers can be individually set to an alternating pattern of electrical potentials such that the analyte within the channel of the electrochemical module is repeatedly oxidised and reduced with the electrical current from each electrode layer being received by an external recording device. This amplifies the magnitude of the response.

Various recording systems can be used as external recording devices for accepting voltage inputs for potentiometric analysis or voltage/current input for amperometric analysis such as a chart recorder, voltammeter or A/D board. A/D boards can take a number of forms such as usb, serial, PCI or ISA boards. The A/D boards can also be either computer based or remote data logger boards, the latter optionally functioning independently of a PC, with the data being downloaded at a later date.

The electrode device of the invention can be used to detect amino acids, carbohydrates such as monosaccharides, organic acids, purines, oxidised and reduced aminothiols and neurotransmitters.

Brief Description of the Drawings

Some preferred embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings in which:

Figure 1 is a schematic representation of a first embodiment of the invention;

Figure 2 is a schematic representation of a second embodiment of the invention;. Figure 3 is a schematic representation of a third embodiment of the invention;

Figure 4 is a schematic representation of a fourth embodiment of the invention;

Figure 5 is a schematic representation of a fifth embodiment of the invention;

Figure 6 is a schematic representation of a sixth embodiment of the invention;

Figure 7 is a schematic representation of a seventh embodiment of the invention;

Figure 8 shows linear sweep voltammograms of the response for multiple sampling at a single electrode for mM glucose in 0.1M NaOH as the test medium; and

Figure 9 shows the current-time response of a redox system of five working electrodes for 0.5 mM ferrocene carboxylic acid in 0.12 M Britton Robinson buffer.

Detailed Description of the invention

The electrode device of the invention, in its most general mode, is based upon the encapsulation of a electrode layer, which is preferably a thin conductive film such as a metal foil, carbon paper or screen printed carbon, between insulating layers of an insulating polymer, which is typically a polyester or resin composite, to form a multi - layer laminate.

Figure 1 shows an exploded view of a first embodiment of the invention. In this embodiment, an electrode device 2 is formed with a fluid inlet 4 to receive an analyte and a fluid outlet 6. Arranged between the inlet 4 and outlet 6 are alternating layers of insulating layers 8 and electrode layers 10. Each of these layers 8, 10 is formed with a circular opening 12 which openings 12 together define a fluid channel 14 through the device. It will be understood that the figure shows the insulating and electrode layers 8, 10 in an exploded condition, and that it in practice these layers are suitably held together to form a laminated structure. The openings 12 may be preformed in the layers 8, 10 but preferably they are formed after the layers are laminated, for example by drilling.

Electrical connections, not shown, are made to the conductive layers 10.

Two types of simple connection arcangements to achieve this are shown in Figure 2. In this embodiment, electrode layers 16, 18 are formed as rectangular plates. Insulating layers 20, 22, 24 are laminated with the electrode layers 16, 18 so as to leave exposed contact pads 26, 28 on the electrode layers to which leads 30 may be attached, e.g. by bonding (by, for example a conductive epoxy), soldering or through suitable connectors (i.e. zero insertion force sockets), for connection to the external device. This allows the layers 16, 18 to be individually addressed.

Alternatively, a second channel can be formed through the laminate to expose the electrode layers 16, 18 on the channel wall. The second channel does not cany analyte fluid. The electrode layers 16, 18 can then be attached either individually υr in series from the exposed portions of the electrode layers in this second channel as discussed above.

These types of electrode connection therefore not only provide a simple route to the construction of a coulometric array, but can also be used as an interdigitated array for use in redox cycling detection, as will be discussed further below.

In the embodiments of Figures 1 and 2, the fluid channel is formed as a series of aligned holes in the various device layers. The embodiment of Figure 3 is similar in construction to that of Figure 1 except that it provides a more complex fluid channel. In this embodiment, the device 30 comprises a series of insulating layers 32 each of which is in the form of a square ring. Laminated between the insulating layers 32 are electrode layers 34, 36. The first electrode layers 34 each comprise a central circular opening 38 while the electrode layers 36 each comprise four circular openings 40 arranged in a square formation around a central region 42. The arrangement of the electrode layers is such that fluid entering through the inlet 4 passes though the central opening 38 in the uppermost electrode layer 34, and then impinges upon the central region 42 of the underlying electrode 36 and is deflected laterally in order to flow through the openings 40 in the electrode 36.

This arrangement has the advantage of significantly increasing the surface area of the electrodes within the device, the upper and lower surfaces of the electrode layers now forming part of the exposed surface of the electrode layers.

Thus, various patterns can be used for the electrode and insulating layers. For example, patterns can be provided which mimic the structure of conventional carbon foam coulometric detectors by providing non -linear channels which cause turbulent flow.

Figure 4 shows a schematic representation of a device 60 having a recessed electrode layer in a channel containing an adsorbed enzyme 65. Target analyte 67a, 67b is converted to enzyme product 68a, 68b by the adsorbed enzyme 65. In this embodiment, conductive electrode layers 62 alternate with insulating layers 64 and are recessed relative to the insulating layers 64. Recessing the electrode layers presents a diffusional barrier minimising the transport of enzyme and electrodetectable by-product to neighbouring layers.

Localised permselective membrane 66 prevents the desorption of an enzyme from the electrode layer, thus preventing the artificial depression of the signal in one layer and the potential amplification of the signal from another layer.

The electrode device of the invention advantageously provides individually addressable electrode layers which can be used to amplify an analyte signal, for instance the signal of an analyte having thiol functionality. Figure 5 shows such an arrangement.

In this embodiment, alternating electrode layers 72a, are held at an oxidising potential while alternating intermediate electrode layers 72b are held at a reducing potential.

The device would operate in a mode where conducting layers 72a, 72b separated by insulating layers 74 alternate between an oxidising and a reducing potential to form an inter-digitated array 70 for use in redox cycling detection. This could be used for species that possess electrochemical reversibility (such as catecholamines, ferrocenyl or quinoid indicators) but could also be used for systems where the re-conversion of the consequent oxidised or reduced form is irreversible at the generating substrate.

Such a device arrangement highlights the case where different electrode substrates (i.e. alternating carbon and copper/zinc/nickel (or alloys thereof) electrodes) contained within the laminate assembly may be used to effect the determination of species not amenable to redox cycling at conventional single substrate materials. The analytical signal is only extracted from those layers 72a held at the oxidising potential (e.g. carbon poised at +1 V). The oxidised species is then re-reduced at a neighbouring, second substrate electrode (i.e. copper/zinc/nickel or alloys thereof), held at a reducing potential (i.e. -IV). The repeated oxidation and reduction of the same analyte (e.g. 2RS^" ^ R-S-S-R) as it progresses along the detector channel 78 results in a substantial amplification of the signal as increasing numbers of eloctrode layers are added to the assembly.

The use of a strongly alkaline mobile phase (e.g. 0.1 M NaOH) as the fluid in liquid chromatographic-electrochemical detection of thiols has the benefit of minimising hydrogen evolution at the cathodic electrodes whilst favouring oxidation as a consequence of the deprotonation of the RSH group to form RS^" . The influence of dissolved oxygen is minimal if the analytical signal is extracted from the electrode layers 72a forming the anodic electrodes. It is preferred that the mobile phase forming the fluid is degassed to prevent the aerial oxidation of the thiol. Target thiols such as ascysteine, N-acetylcysteine, glutathione and their corresponding oxidised dimer (R-S-S-R) forms can be analysed by this method.

Another example of a redox system is the dopamine/ascorbate system, which is well known for the characterisation of new electrodes. In this case, the particular analyte redox potentials vary depending on electrode substrate, surface treatment and pH. The reduction and oxidation signals from commercial Glassy Carbon electrodes generally overlap causing problems with signal discrimination in this system. However, the electrode device of the present invention can be constructed from different electrode materials in different electrode layers of the laminate, providing a simple means of separating and discriminating the dopamine and ascorbate signals.

Figure 6 is a schematic representation of an integrated strip like sampling-sensing structure 80 as described above. In this representation, three conducting layers 82 are present in the laminate separated by insulating material 84. The structure 80 has four channels or wells 86 formed therein, spaced from each other along the length of the strip 80.

The terminating ends 88 of the electrode layers 82 can be seen to extend beyond the insulating laminate 84 to allow the connection of the electrode layers to an external device. An adhesive cover strip 90 is placed over the upper surface 92 of the device 80 to cover the wells 86 to protect them from contamination and uncontrolled filling. A similar adhesive cover strip (not shown) is also placed on the lower surface of the strip.

A particular sensing well 86 can be exposed simply by removing the strips 90 from either end of the well 86 which can then be dipped into the bulk fluid to be analysed to receive the sample. The sampling-sensing structure is then removed from the bulk solution and the electrochemical analysis carried out.

A fresh detection well 86 can be activated by simply cutting away the used well 86 and exposing the next well 86 in line by removing the protective tape 90 covering the ends of the well 86. It is pointed out that while cutting the laminate 80 to remove a used well 86 will also reveal the edge of the electrode layer in the laminate, the "dip- extract-analyse" sampling methodology means that the sample fluid is effectively confined within the well 86. The exposed /severed edge of the electrode layer is not in contact with the sample fluid after extraction from the bulk solution and so does not participate in the electroanalysis procedure.

The three dimensional nature of the laminate structure of the electrode device allows the volume of the fluid present in the channel to be controlled. This sampling methodology however necessitates a compromise in the design process as the detection channel must retain a thin film of solution after "dip-sampling". Increasing the cross-section of the channel lessens the ability of the thin laminate to hold the film through capillarity. While the optimal configuration will obviously depend upon the thickness of the laminate, diameters of 1 mm or less are preferred to maintain film contact across the ring electrodes and thereby allow electrochemical measurements to be made directly in the extracted fluid.

A multi-titre plate 100 embodying a further aspect of the invention is show in Figure 7. The multi-titre plate 100 comprises a plurality of channels or wells 102 arranged in a grid-like array. As many channels 102 may be provided as are necessary, and this embodiment shows 20 channels 102. The channels 102 are formed by aligned apertures formed in the successive layers, as in the embodiment of Figure 1 above.

In the described embodiment, the electrode layers are formed as parallel strips 104 of conductor. The strips 104 are preferably metal foils or screen printed carbon. The strips can be deposited onto the underlying insulating layer 106 which is preferably a polyester or resin composite.

The strips 104 of each consecutive electrode layer area are preferably oriented perpendicular to those of the adjacent layer, as shown, to facilitate the making of electrical connections thereto. As can be seen from Figure 7, each strip 104 extends beyond the insulating layers 106 of the structure on one side to allow connection to an external device.

Each well 102 can be accessed individually by activating the appropriate strips 104. Assigning one of the electrodes within each well 102 to act as the counter electrode allows the sequential addressing of the remaining three electrodes and thereby facilitates the detection and quantification of three different analytes, depending upon the electrode substrates, catalysts, biological agents and detection mode employed.

Such a multi-titre plate can be used to identify key biomarkers for the broad spectrum electrochemical screening of diabetes, hyperlipidaemias, renal failure, thrombophilic disorders and established coronary or peripheral vascular disease. The multi-titre plates of the invention could be used in high throughput screening in diabetes, lipid and haematology clinics, renal dialysis units and clinical biochemistry laboratories.

The following Examples are non-limiting and illustrate the laminate electrode devices of the invention, their production and their methods of use.

Example 1

This Example illustrates the manufacture of an integrated sampling-sensing strip according to the embodiment of Figure 6 and discloses a method of electrochemical detection using such a sampling-sensing strip.

Fabrication of Laminates: Strips of copper metal (100 micrometer thick) were thermally sandwiched between layers of commercial polyester laminating pouches (75 micrometer). The laminate assembly was built up with alternating layers until three distinct metal layers were encapsulated. The polymer laminate enclosed the entire metal strip except the terminating edge used to connect to the potentiostat. Sensing channels were created periodically along the strip by mechanically drilling (1 mm - 2 mm diameter holes) through the centre - thereby exposing the metal layers within the core. These ring electrodes, when suitability conditioned, would ultimately seive as working, counter and reference electrodes. The drilling process was clean in that there was no evidence of inter-layer smearing. No evidence of short-circuits between individual electrode layers was found with a digital multi-meter.

Electrode Conditioning: Electrodes designated to serve as either working or counter electrodes were repeatedly cycled in 0.1 M NaOH / 2 mM Glucose in deionised water (Elgastat) prior to conducting the electrochemical characterisation. This facilitated the formation of a stable copper oxide layer possessing a low background current. Initial studies were conducted using a conventional macro counter electrode (platinum wire) and reference electrode (Ag / AgCl, 3 M NaCl, BAS Technicol). Subsequent studies utilised an internal silver oxide pseudo reference electrode whereby the layer furthest away from the designated working electrode was plated with silver (-0.4 V, 1

Electrochemical detection: The oxidation of glucose is a model system for the assessment of electrode behaviour within the laminate. Linear sweep voltammograms were obtained with a microAutolab computer controlled potentiostat (Eco-Chemie, Utrecht, the Netherlands). The electrode response oi a 1 mm diameter copper / copper oxide ring electrode to increasing additions of glucose (0.5 mM) in 0.1 M NaOH showed the irreversible oxidation of the monosaccharide at +0.7 V with the oxidation current found to increase linearly with increasing concentrations of glucose.

The individually addressable nature of the laminate structure is a key advantage of the fabrication methodology. The efficacy of the encapsulation procedure was therefore tested through comparing isolated electrode layers within the assembly and through the sequential co-connection of these to form what would essentially become a larger electrode. This behaviour is confirmed when the response to 0.5 mM glucose at 1, 2 and 3 electrodes (sequentially connected in series) was found to lead to a linear increase in current response. The increase in oxidative current shows the independent nature of the electrode in each conducting layer. The magnitude of the response at individual electrodes was improved by increasing the diameter of the detection channel. Diameters of 1 mm or less were necessary to maintain film contact across the ring electrodes and thereby allow electrochemical measurements to be made directly in the extracted fluid.

Figure 8 shows voltammograms highlighting the response of the ring electrodes to glucose (0-2 mM, 0.1 M NaOH) within a 1 mm diameter detection channel using a conventional three electrode system. In this configuration negligible current is allowed to pass through the reference electrode layer. The middle electrode within the three electrode laminate was designated as the counter. It was then possible to plot the obseived oxidation current versus glucose concentration as is shown in the insert to Figure 8.

The reproducibility of the electrode responses was assessed using the detection of 1 mM glucose in 0.1 M NaOH as the test medium. The responses for multiple sampling at a single electrode are shown in the right hand side linear sweep voltammogram, allowing the determination of the analyte concentration in the test medium.

Example 2

This Example illustrates the manufacture of a multi-electrode device according to the embodiment of Figure 1 and discloses a method of electrochemical detection using such a device.

Fabrication of Laminates: Five independently addressable electrodes of carbon fibre matting (E-Tek Inc., USA, B-2 Toray Carbon Papers 0.09-0.35 mm thick, 0.42-0.47 g/cm³) with connecting copper foil contacts were thermally laminated between layers of resin backed polyester laminating pouches (75-125 μm thick) to form an electrode device according to the invention. The separation between the electrode layers was varied by adjusting the number of polyester laminating layers between each conductive carbon layer. A polyester thickness of 225 μm corresponding to three layers of 75 μm laminating pouches was preferred between electrode layers. The carbon fibre microtubes were initially completely encapsulated between the insulating laminating pouches and baked at 100 °C for 1 h. The baking process is important because the insulating layer is composed of a two part system comprising a polyester outer layer backed by a thermally activated resin. Prolonged baking is preferable to ensure the complete permeation of the molten resin through the carbon network and therefore serves to provide a complete seal which is impermeable to water.

A sensing channel was created by mechanically drilling a 0.5 mm diameter hole through the centre of the laminate - thereby exposing the carbon fibre layers within the core. These ring electrodes serve as the five working electrodes of the device. The drilling process was clean in that there was no evidence of inter-layer smearing or short-circuits.

Construction of detector: A separate chloridised silver wire was added to the system to provide a reference electrode. A further separate platinum wire was added as a counter electrode to complete the electrochemical detector. The detector was placed in a flow injection system through which 0.12 M Britton Robinson buffer (pH 7, 0.1 M KCl) composed of acetic, boric and phosphoric acids each at a concentration of 0.04 M was pumped at a rate of 1 mL/min. The sample loop had a volume of 50 μL.

Electrochemical analysis: The detector array was connected to an EcoChemie PGSTAT 12 (Eco-Chemie, Utrecht, The Netherlands) multi-array potentiostat. The potential of the five carbon fibre working electrodes were fixed in series at +0, +0.8, +0, +0.8 and +0 V respectively, as shown in the insert to Figure 9. This choice of potentials facilitated the reduction/ oxidation/ reduction/ oxidation/ reduction cycling of the model ferrocene carboxylic acid redox system to be investigated.

The test medium comprised 0.5 mM ferrocene carboxylic acid solution prepared in

0.12 M Britton Robinson buffer (pH 7, 0.1 M KCl). The test medium was injected into the sample loop upstream of the detector array. The first carbon fibre working electrode in the series was held at a potential of 0 V to serve as a blank. It is apparent from the essentially uniform current-time response for this electrode over time shown in Figure 9 that no reducible components were detected in the test medium.

Upon reaching the second working electrode in the laminate device the ferrocene carboxylic acid analyte was oxidised resulting in a corresponding positive current peak. The electro-generated oxidised ferrocenyl species was then reduced back to ferrocene carboxylic acid upon reaching the third working electrode. This reduction is shown as a negative current peak for the third carbon fibre electrode in Figure 9.

The magnitude of the reduction peak for the third working electrode is smaller than the magnitude of the oxidation peak for the second electrode. The reduction in current in absolute terms is due in part to turbulence through the laminate device and in part to the geometrical design of the channel resulting in a portion of the oxidised species not reaching the surface of the third electrode.

The fourth and fifth working electrodes function in an identical manner to the second and third electrodes respectively. Figure 9 shows the corresponding positive current peak for the oxidation of the ferrocene carboxylic acid at the fourth working electrode and the consequent negative current reduction of the electro-generated oxidised species back to ferrocene carboxylic acid at the fifth working electrode.

In this manner, the electrode device according to the present invention can be used as a probe for the detection of an electrochemically reversibly species such as ferrocene carboxylic acid. The redox recycling in this system performed by the pairs of working electrodes held at oxidising and reducing potentials can he harnessed to provide signal amplification of the oxidisable analyte.

Claims

Claims:

1. An electrode device comprising a laminate assembly of alternating insulating layers and electrode layers, said device having at least one channel for receiving a fluid extending through the layers of the laminate.

2. The electrode device of claim 1 wherein said electrode layer is adapted to establish an external electrical contact at a location spatially removed from said channel.

3. The electrode device of any of the preceding claims wherein said insulating layer is an insulating foil.

4. The electrode device of any of the preceding claims comprising a plurality of electrode layers.

5. The electrode device of claim 4 wherein the electrode layers comprise different conductors.

6. The electrode device of any of the preceding claims wherein said electrode layer is in the form of a strip.

7. The electrode device of any of the preceding claims wherein at least one conductor is localised in the region of the channel in the electrode layer and at least one different conductor is not in the region of said channel.

8. The electrode device of any of the preceding claims wherein the surface of the electrode layer forming the surface of a channel has a coating of a different conductor.

9. The electrode device of any of the preceding claims wherein said electrode layer comprises at least one of carbon, conductive polymer, metal or alloy.

10. The electrode device of any of the preceding claims wherein said electrode layer comprises at least one of carbon fibre, carbon paper, screen printed carbon or screen printed carbon composite film, inkjet printed carbon or inkjet printed carbon composite film, a conductive ink, platinum foil, silver foil, gold foil, copper foil or nickel foil or NiCu or NiCr alloys.

1 1. The electrode device of any of the preceding claims wherein the electrode layer comprises carbon and a catalyst and/or biological material.

12. The electrode device of claim 11 wherein said catalyst comprises at least one of platinum, palladium or gold particulates, metallocyanine or quinone

13. The electrode device of claim 11 wherein said biological material is an enzyme.

14. The electrode device of claim 13 wherein said enzyme is an oxidase enzyme.

15. The electrode device of claim 13 or claim 14 wherein said enzyme is secured by polymer entrapment behind an electropolymerised permselective membrane.

16. The electrode device of any of the preceding claims wherein the surface area of a channel in an electrode layer is greater than the surface area of said channel in the two adjacent insulating layers, such that the channel in the electrode layer is recessed from the channel in said adjacent insulating layers.

17. The electrode device of claims 1 to 15 wherein the surface area of a channel in an insulating layer is greater than the surface area said channel in the two adjacent electrode layers, such that the channel in the insulating layer is recessed from the channel in said adjacent electrode layers.

18. The electrode device of any of the preceding claims wherein the channel is formed by a patterned or random perforation of the electrode layer.

19. The electrode device of any of the preceding claims having a plurality of channels.

20. The electrode device of claim 16 wherein said channels are arranged randomly or in a geometric pattern.

21. The electrode device of any of the preceding claims having a substrate attached to the base of the laminate to seal the ends of the channels.

22. An extended electrode array comprising a plurality of electrode devices according to claims 1 to 20 placed in series.

23. A sampling-sensing strip comprising a series of electrode devices according to claims 1 to 20 arranged in a row wherein the electrode layers are formed by a common conducting member.

24. A sampling-sensing strip according to claim 23 wherein the mouths of the channels on the surface of the laminate are covered by a removable protective layer.

25. A multi-titre screening plate comprising an array of electrode devices according to claims 1 to 21 wherein the electrode layers are formed by a common conducting member.

26. A process for the manufacture of an electrode module according to any one of claims 1 to 20 comprising the steps of: i) arranging sequential layers of insulating and electrode layers to form a lay- up; and ii) laminating said lay-up.

27. The process according to claim 26 wherein the channel is formed by coring the lay-up prior to lamination.

28. The process according to claim 26 wherein the channel is formed by using pre- pattemed insulating and electrode layers which are pre-perf orated in the position of the channel.

29. The process according to any one of claims 26 to 28 wherein the lamination is hot lamination.

30. The process according to any one of claims 26 to 28 wherein the lamination is cold lamination using pressure active adhesive layers.

31. The process of any one of claims 26 to 30 wherein the surface of the channel at the electrode layer is coated with a different conductor by electroplating.

32. The process of any one of claims 26 to 30 wherein a permselective membrane is electropolymerised on the surface of the electrode layer at the channel.

33. A method of electrochemical detection of an analyte wherein the electrode device according to claims 1 to 21, the electrode array according to claim 22, the sampling - sensing strip according to claim 23 or claim 24 or the multi-titre screening plate according to claim 25 is connected to an external recording device such that the electric potential which develops across an electrode layer is measured under zero current flow.

34. A method of electrochemical detection of an analyte wherein the electrode device according to claims 1 to 21, the electrode array according to claim 22, the sampling - sensing strip according to claim 23 or claim 24 or the multi-titre screening plate according to claim 25 is connected to a potentiostat such that the electric potential which develops across an electrode layer is controlled by a potentiostat and the current received by an external recording device.

35. The method of claim 34 wherein the potential of an electrode layer of the device, array, strip or plate is held at a particular potential with respect to the reference electrode.

36. The method of claim 34 or claim 35 wherein a plurality of electrode layers are individually set to different electrical potentials with the electrical currents from each electrode layer being received by an external recording device.

37. The method of claim 34 or claim 35 wherein the electrode layers are individually set to an alternating pattern of electrical potentials such that the analyte within a channel is repeatedly oxidised and reduced with the electrical current from each electrode layer being received by an external recording device.

38. The method of any of claims 33 to 35 wherein the electrode layers are connected in parallel to the potentiostat.

39. The method of any of claims 33 to 38 wherein the external recording device is a chart recorder, voita meter or A/D boatd.

40. A method of electrochemical detection of an analyte wherein a plurality of electrodes are connected to a potentiostat such that the electric potential which develops across an electrode is controlled by the potentiostat and the electrodes are individually set to an alternating pattern of electrical potentials such that the analyte is repeatedly oxidised and reduced with the electrical current from each electrode being received by an external recording device.