Abstract
The present invention relates to a wound dressing comprising an electrochemical sensor configured to detect the level of an electroactive species within a wound environment during use. The present invention also relates to a method of detecting the level of an electroactive species within a wound environment by applying a wound dressing according to the present invention onto a wound and operating an electrochemical sensor to detect an electroactive species within the wound environment.
Claims
1. A wound dressing comprising: an electrochemical sensor configured to detect the level of an electroactive species within a wound environment during use, the electrochemical sensor comprising a working electrode; and wherein the working electrode is a micro electrode or nano electrode.
2. The wound dressing of claim 1, wherein the electrochemical sensor detects the electroactive species by anodic stripping voltammetry, amperometry, potentiometry or electrochemical impedance analysis, preferably, wherein the electrochemical sensor detects the electroactive species by anodic stripping voltammetry.
3. The wound dressing of claim 1, wherein the electroactive species is an anti-microbial agent and/or anti-biofilm agent.
4. The wound dressing of claim 1, wherein: (i) the electroactive species is a metal ion selected from the group consisting of Ag, Al, Au, Ba, Bi, TI, Ce, Co, Cr, Cu, Fe, Ga, Ge, Ir, Mn, Mo, Ni, Pd, Pt, Rb, Re, Rh, Ru, Sb, Se, Sn, Sr, Ti, W, Zn and Zr ions; or (ii) the electroactive species is a non-metal species.
5. The wound dressing of claim 1, wherein the surface area of the working electrode that is exposed to the wound environment during use (exposed surface area of the working electrode) is from about 0.0005 mm.sup.2 to about 10 mm.sup.2.
6. The wound dressing of claim 1, wherein (solution volume probed)/(exposed surface area of the working electrode) is from about 0.5 mm to about 300 mm.
7. The wound dressing of claim 1, wherein the working electrode is elongate, optionally wherein the working electrode is a wire.
8. The wound dressing of claim 7, wherein the wire has a thickness of from about 0.1 ?m to about 150 ?m.
9. The wound dressing of claim 7, wherein (solution volume probed)/(exposed surface area of the working electrode) is from about 0.5 mm to about 100 mm.
10. The wound dressing of claim 1, wherein the working electrode is planar.
11. The wound dressing of claim 10, wherein: the working electrode is a planar laminate structure comprising a working electrode layer, insulating layer and substrate layer, wherein the working electrode layer is sandwiched between the substrate layer and insulating layer to form the laminate structure; the laminate structure includes at least one aperture extending through the insulating layer and the working electrode layer to leave a surface of the working electrode layer exposed to the wound environment during use in order to provide a sensing surface suitable for detecting the electroactive species within the wound environment; and the surface of the working electrode layer exposed comprises exposed edges which are formed at the periphery of the at least one aperture and the exposed edges have a thickness of from about 25 nm to about 75 nm.
12. The wound dressing of claim 10, wherein (solution volume probed)/(exposed surface area of the working electrode) is from about 100 mm to about 200 mm.
13. The wound dressing of claim 1, further comprising a counter and/or reference electrode.
14. The wound dressing of claim 1, further comprising a support layer for the working electrode.
15. A method of detecting the level of an electroactive species within a wound environment comprising: applying the wound dressing of claim 1 onto a wound; and operating the electrochemical sensor to detect the electroactive species within the wound environment.
16. The method of claim 15, wherein the operating the electrochemical sensor comprises detecting the electroactive species by anodic stripping voltammetry, amperometry, potentiometry or electrochemical impedance analysis, preferably by anodic stripping voltammetry.
17. The method of claim 15, further comprising administering an anti-microbial agent and/or anti-biofilm agent into the wound in response to the level of electroactive species detected in the wound environment; optionally wherein the electroactive species detected is identical to the administered anti-microbial agent and/or anti-biofilm agent.
18. The method of claim 15, wherein: (i) the electroactive species is a metal ion selected from the group consisting of Ag, Al, Au, Ba, Bi, TI, Ce, Co, Cr, Cu, Fe, Ga, Ge, Ir, Mn, Mo, Ni, Pd, Pt, Rb, Re, Rh, Ru, Sb, Se, Sn, Sr, Ti, W, Zn and Zr ions; or (ii) the electroactive species is a non-metal species.
19. (canceled)
Description
DESCRIPTION OF FIGURES
[0131] The invention will be further described, by way of example only, with reference to the accompanying Figures.
[0132] FIG. 1 is a schematic representation of an electrochemical sensor arrangement according to the present invention.
[0133] FIG. 2 is a block diagram illustrating the major functional units of a sensor and reporting arrangement according to the present invention.
[0134] FIG. 3 is a cross sectional schematic of an electrode arrangement according to the present invention.
[0135] FIG. 4 is a plan view of an electrode arrangement according to the present invention.
[0136] FIG. 5 is a schematic representation of a wound dressing arrangement including an electrochemical sensor according to the present invention.
[0137] FIG. 6 is a schematic representation of a wound dressing arrangement including an electrochemical sensor according to the present invention.
[0138] FIG. 7 is a schematic representation of an alternative wound dressing arrangement including an electrochemical sensor according to the present invention wherein the counter/reference electrode is electrically isolated from the working electrode.
[0139] FIG. 8 is a schematic representation of a wound dressing arrangement including an electrochemical sensor according to the present invention further including an active release electrode.
[0140] FIG. 9 is a schematic representation of a wound dressing arrangement including an electrochemical sensor according to the present invention further including an active release electrode and wound sensing electrode.
[0141] FIG. 10 shows a graph plotting the results of cyclic voltammetry experiments conducted using a platinum macro electrode, a 100 ?m platinum wire electrode and a 25 ?m platinum wire electrode as described in the experimental data section provided herein.
[0142] FIG. 11 shows a graph plotting the results of anodic stripping voltammetry experiments conducted in seven Mueller-Hinton Broth II (MHB2) solutions containing differing concentrations of added Ag.sup.+ ions using a Pt Nanoband Electrode as described in the experimental data section provided herein.
[0143] FIG. 12 shows an expanded version of FIG. 11 focusing on the section of the graph which illustrates the stripping step of the anodic stripping voltammetry experiments conducted.
[0144] FIG. 13 shows the oxidation charge during the anodic stripping voltammetry experiments of FIGS. 11 & 12 plotted as a function of the Ag.sup.+ ion concentration.
[0145] FIG. 14 shows the results of bacterial proliferation assays in Phosphate buffered saline (PBS), Silver Nitrate (NaNO3), Simulated wound Fluid (SWF) and Mueller Hinton Broth II (MHB2) solutions as a function of the amount of added Ag.sup.+ ions as described in the experimental data section provided herein.
[0146] FIG. 15 shows the results of optical density measurements in Phosphate buffered saline (PBS), Silver Nitrate (NaNO3), Simulated wound Fluid (SWF) and Mueller Hinton Broth II (MHB2) solutions as a function of the amount of added Ag.sup.+ ions as described in the experimental data section provided herein.
[0147] FIG. 16 shows the oxidation charge during anodic stripping voltammetry experiments conducted in Phosphate buffered saline (PBS), Silver Nitrate (NaNO3) and Simulated wound Fluid (SWF) solutions as a function of the Ag.sup.+ ion concentration using a Pt Nanoband Electrode as described in the experimental data section provided herein.
[0148] FIG. 17 shows the same results of FIG. 16 on a logarithmic scale.
[0149] FIG. 18 shows the oxidation charge during anodic stripping voltammetry experiments conducted in simulated wound fluid (SWF) plotted as a function of the Ag.sup.+ ion concentration using a 25 ?m platinum wire electrode as described in the experimental data section provided herein.
[0150] FIG. 19 shows the results of anodic stripping voltammetry experiments involving multiple anodic steps using a nanoband electrode. The experiments were conducted in both simulated wound fluid (SWF) and simulated wound fluid with added silver ions as described in the experimental data section provided herein.
[0151] FIG. 20 shows an experiment using a silver release electrode and a Pt nanoband sensor. The experiment is designed to both generate and detect Ag.sup.+ ions as described in the experimental data section provided herein.
[0152] FIG. 21 shows measurement of iodine in phosphate buffer saline using a Macro electrode versus a platinum based nanoband electrode.
[0153] FIG. 22 is a schematic representation of a micro-wire/micro-strip electrochemical sensor arrangement according to the present invention.
[0154] FIG. 23 shows a graph plotting the results of cyclic voltammetry experiments conducted using a carbon micro-wire electrode with ferrocene carboxylic acid versus a carbon macro electrode as described herein.
[0155] FIG. 24 shows measurement of iodine in phosphate buffer saline using a macro printed carbon electrode (left hand side) as described herein versus a carbon micro-wire based electrode (right hand side).
[0156] FIG. 25 shows the calibration curve for a carbon micro-wire sensor with iodine.
[0157] FIG. 26 shows a schematic of a nanoband electrode sensor incorporated within a wound dressing arrangement.
[0158] FIG. 27 shows a photograph of the wound dressing sensor illustrated in FIG. 26 mounted within a flow cell.
[0159] FIG. 28 shows a plot of an anodic stripping charge for a nanoband electrode sensor incorporated within a wound dressing arrangement subjected to simulated wound fluid flow.
DETAILED DESCRIPTION OF THE INVENTION
[0160] Referring to FIG. 1, there is depicted an electrochemical sensor 10 (comprising components 14, 32 and 34) according to a preferred embodiment of the present invention. The electrochemical sensor 10 forms part of a wound dressing 12 and is configured to monitor concentration of an anti-microbial or anti-biofilm agent in or on the wound dressing 12 or in its surrounding environment (i.e. within the wound environment) while the wound dressing is in use and applied to the body of a patient. The electrochemical sensor 10 is self-contained and is configured to transmit measurement data wirelessly to a separate processing device 13.
[0161] The electrochemical sensor 10 of this embodiment is operable to those skilled in the art to perform anodic stripping voltammetry. It will be appreciated that anodic stripping voltammetry is not the only technique that could be employed by electrochemical sensor 10. In this embodiment, the electrochemical sensor 10 is usable to monitor the concentration of electroactive metal ions, and more specifically of silver ions.
[0162] The electrochemical sensor 10 uses a micro- or nano-electrode arrangement as described herein. These have advantages over larger electrodes in that their small size leads to high current density even at low power. Depletion of the electroactive species in the region of the electrode can also be low, and mass transport effects which typically limit the performance of macro electrodes, are avoided.
[0163] As illustrated in FIGS. 1, 3 and 4 the electrochemical sensor contains an electrode component 14, which in preferred embodiments has a planar laminate structure incorporating a thin electrically conductive layer 16 (i.e. working electrode layer) whose upper surface and lower surface are covered with an insulating layer 22, and a substrate layer 20, respectively. The working electrode layer 16 includes exposed edges 18 which form the exposed sensing surfaces of the electrode itself. In the present embodiment the working electrode layer 16 is platinum and is disposed upon a substrate 20, which in the present embodiment is flexible and formed from a polymer, more specifically polyester.
[0164] Other substrate materials, flexible or rigid, may be adopted in other embodiments. The face of the working electrode layer 16 directed away from the substrate layer 20 carries the insulating layer 22. The insulating layer may be formed of a dielectric material. The insulating layer is advantageously able to perform two functions; it electrically insulates the face of the working electrode layer 16 and also has the ability to serve during manufacture as a resist, defining suitable areas to be etched. The material used for the insulating layer (resist) used in the present embodiment may be epoxy based, e.g. it may be what is known in the art as SU-8. Other suitable materials may be substituted, such as, PE773. The functions of etch resist and electrical insulation may in other embodiments be performed by two separate layers. In the example illustrated in FIGS. 1, 3 and 4, the dielectric insulating layer 22 is absent during manufacture from selected regions which form a grid of square shaped apertures 24. Other grid patterns and aperture shapes may be adopted. After deposition of the layers making up the structure of the electrode component 14, which may for example be by casting, spinning, sputtering, growth or deposition, the apertures 24 are then formed by etching to form voids in the selected regions. Suitable etching processes are well known in the art. The etching process removes the insulating layer 22 and the working electrode layer 16 to form apertures 24 have exposed edges 18 at the periphery of each aperture 24. The exposed edges provide the sensing surface for the working electrode layer 16.
[0165] The thickness of the working electrode layer 16, and hence the thickness (labelled as D) or height of the exposed edges 18, is in the order of 50 nm. The insulating layer 22 may have a thickness of around 3 ?m. The square apertures 24 have side or edge length dimensions of 30 ?m and are in embodiments arranged at intervals of 40 ?m over a 2 mm square area.
[0166] In order to perform anodic stripping voltammetry, the electrochemical sensor 10 may additionally comprise a counter/reference electrode (not shown). The counter electrode and the reference electrode are formed by the same component but they may in other embodiments be separately formed. The counter/reference electrode comprises silver and silver chloride, in this example. Other suitable materials may be used.
[0167] The illustrated electrode component 14 is suitable for performance of anodic stripping voltammetry measurements without need of hydrodynamic control. The electrochemical sensor 10 may be configured to measure anti-microbial or anti-biofilm levels down to 0.01 parts per billion.
[0168] As illustrated in FIG. 1, the electrochemical sensor 10 includes two units 14, 30 which are electrically connected, in this embodiment by electrical wiring. Structure 14 is an electrode component containing a working electrode 16, 18 according to the invention (see FIG. 3) and is coupled to or incorporated within the wound dressing 12. Electrode component 14 itself is small enough to be accommodated or incorporated in a wound dressing 12. The second unit 30 houses associated driver electronics 32 and a power source in the form of a battery 34 and requires no physical connection to a further system, so that the patient is not discomforted by trailing wires leading to an external fixed unit.
[0169] The unit 30 is configured to (i) drive the working electrode 16, 18 of the electrode component 14 to obtain measurements of electroactive species within the wound dressing environment, (ii) receive an output signal from the working electrode and (iii) transmit the resultant measurement data to a separate processing device 13.
[0170] In the depicted embodiment, the driver electronics 32 used may be partly analogue and partly digital, comprising at least one microprocessor. The analogue signal from the working electrode may be applied to a suitable analogue front end which provides and controls the drive signals applied to the electrodes (a suitable commercial example is the LMP91000 from Texas Instruments) and which interfaces with a microcontroller. The driver electronicsand the second unit 30 as a wholecan be small enough and light enough to be suitable for affixing to the exterior of the wound dressing itself, to a wound dressing retainer system, or to the patient 10 or their clothes, without undue stress or inconvenience to the patient.
[0171] Wring from the electrode component 14 to the second unit 30 incorporates a releasable connector comprising a first connector part (plug) wired to the working electrode 16,18 and a second connector part (socket) wired to the electronics of the second unit, so that (a) if the electrode component 14 is disposed of after use, the second unit 30 can be connected to a fresh electrode component and re-used and (b) the wound dressing 12 can be applied without the encumbrance of the second unit 30, which is then connected and secured in place after application of the dressing. A simple push-fit connector is used. The wiring from the electrode component 14 needs in practice to be led out of any wound dressings so that the first connector part is accessible.
[0172] In the present embodiment the electrode component 14 is disposed inside the wound dressing 12 with the exposed working electrode edges 18 facing away from the wound.
[0173] As shown in FIG. 1, the wound dressing 12 comprises a dressing layer 36 with a wound contact surface and a backing layer 38. The electrode component 14 is shown in FIG. 1 between the dressing layer 36 and the backing layer 38 with its apertures (as shown in FIGS. 3 and 4) facing toward the backing layer 38. The insulating layer 22 is shown in FIG. 1 on the uppermost surface of the electrode component 14. The electrode arrangement may be adhered in place, or it may be fixed in place using some suitable mechanical arrangement, or it may be incorporated into the structure of the wound dressing. Other arrangements of the electrode component 14 are possible within the scope of the present invention. For example, electrode component 14 may be located at or integrated with the adhesive layers, facing layer, film layer, located at or integrated within the backing layer 38, or located at or integrated within an absorbent core of the wound dressing 12.
[0174] The dressing layer 36 comprises one or more active agents. In the present embodiment these include an anti-microbial or anti-biofilm agent. They may also include a surfactant which could be for example, neutral, anionic, cationic or combinations of these.
[0175] As is well known, the technique of anodic stripping voltammetry comprises electrolytic deposition of the relevant metal (silver in the present example) from solution onto the electrode 18, after which the deposited metal is stripped off to produce a signal proportional to the silver concentration in the solution. An alternative to the anodic stripping process is to make continuous measurements. This is appropriate at higher silver concentrations. In either case the required drive signals are applied to the working electrode 14 under control of software/firmware of the microcontroller, and the result is an output signal which provides an indication of metal ion level in the wound dressing 12, which can thus be used to establish whether the dressing remains fit for purpose, or whether it needs to be changed.
[0176] Measurements may be taken at chosen time intervals. In the present example the anodic stripping sequence takes about three minutes to complete, and an optional electrode conditioning sequence takes a further 1.5 minutes. So the present embodiment is capable of updating the measure of silver ion concentration at five minute intervals (or continuously, if continuous measurements are being made). The reporting frequency chosen in practice may be lower, however, in order to conserve battery lifetime, e.g. once an hour. The driver electronics 32 buffer the measurement data and upload it to a network interface, which in the present embodiment is in a personal area network (PAN).
[0177] The electrode component 14 of the electrochemical sensor 10 needs to be wetted out in order to function. In the present embodiment the dressing layer 36 is moist and the liquid it carries serves to wet out the electrodes. In other embodiments the electrodes may be wholly or partly wetted out by fluid from the wound itself. Wound fluid typically contains 0.9% sodium chloride making it conductive. The electrochemical sensor 10 may incorporate a sensor configured to detect when the system has been bathed in fluid (i.e. when it is operational). This sensor may measure resistance. A reduction in resistance between two electrodes when the wound fluid forms a conductive path between them may be detected as an indication that wound fluid has wetted out the sensor. Multiple electrodes may be provided at spatial intervals across the dressing or through its thickness to establish the degree of spread of exudate.
[0178] The wireless interface used to transmit the measurement data to the processing device 13 may take any suitable form. A range of suitable communications protocols is known to the skilled person. In the present embodiment it uses the Bluetooth (RTM) Low Energy standard and the processing device 13 takes the form of a Bluetooth equipped tablet computer or mobile phone running an app for display of the measurement data to the user. A user, which may or may not be a clinician, can thus pair the tablet computer or mobile phone 13 periodically to the electrochemical sensor 10 to obtain the stored data and assess whether the dressing needs to be changed. Alternatively, or additionally the electrochemical sensor 10 may be configured to push a warning signal to the processing device 13 in response to measurement data indicative of a need to change the dressing.
[0179] The information provided to the user includes an indication of whether the electrochemical sensor 10 is operational, and can include some form of easily comprehensible indication of the current condition of the wound dressing 12. This may for example be single number, or a colour coded signal, e.g. red for concern, amber for deteriorating condition, and green to indicate good anti-microbial or anti-biofilm level.
[0180] The overall configuration of a system embodying the invention is represented in FIG. 2, showing the electrode component 14 whose output is received by the driver electronics 32 and transmitted by network interface 50 of the driver electronics 32 to the processing device 13. The data is additionally or alternatively transmitted, directly by the network interface 50 or via the processing device 13, to a remote host 52. The remote host 52 may collate and analyse data from multiple sources, e.g. in order to facilitate assessment and development of the technology, and to monitor its efficacy.
[0181] It will be appreciated that the structure of a wound dressing according to the present invention may vary, as required, depending on its use. FIGS. 5-9 illustrate wound dressing structures forming various embodiments of the present invention.
[0182] Referring to FIG. 5, there is a depicted a wound dressing 501 according to the present invention configured to monitor concentration of an electroactive species (e.g. anti-microbial or anti-biofilm agent) in or on the wound dressing or in its surrounding environment (e.g skin) while the wound dressing is in use and applied to the body of a patient. The wound dressing 501 has a laminate structure including a backing layer 502, dressing layer 503, a support layer 504 and a wound contact layer 505. The dressing layer 503 and wound contact layer 505 may optionally include an active ingredient (e.g. an anti-microbial or anti-biofilm agent). Fabricated to the wound facing surface of the support layer 504 is an electrode component 506 comprising or consisting of a working electrode configured for sensing electroactive species within the wound environment and a counter (or reference) electrode 507. As depicted by the curved arrow in FIG. 5, it will be appreciated that the electrode component 506 and counter/reference electrode 507 may alternatively be fabricated to the non-wound facing surface of the support later 504.
[0183] FIG. 6 also depicts a wound dressing 601 according to the present invention configured to monitor concentration of an electroactive species (e.g. anti-microbial or anti-biofilm agent) in or on the wound dressing or in its surrounding environment while the wound dressing is in use and applied to the body of a patient. Like FIG. 5, FIG. 6 possess a laminate structure including a backing layer 602, dressing layer 603, a support layer 604 and a wound contact layer 605. The dressing layer 603 and wound contact layer 605 may optionally include an active ingredient (e.g. an anti-microbial or anti-biofilm agent). In this embodiment, an electrode component 606 comprising or consisting of a working electrode configured for sensing electroactive species within the wound environment is fabricated to the non-wound facing surface of the support layer 604 whilst the counter/reference electrode 607 is fabricated to the wound facing surface of the support layer 604. Alternatively and illustrated by the curved arrow in FIG. 6 it will be appreciated that electrode component 606 may fabricated to the wound facing surface of the support layer 604 whilst the counter/reference electrode 607 is fabricated to the non-wound facing surface of the support layer 604. Electrode orientations like these, where electrodes are located on opposing surfaces of the support layer 604 can reduce the overall sensor footprint on the wound facing surface on the support layer 604. This, advantageously, decreases the impact that the sensor has on the flow of fluid (e.g. wound exudate) within the wound dressing or in its surrounding environment while the wound dressing is in use and applied to the body of a patient.
[0184] FIG. 7 illustrates a wound dressing 701 according to the present invention configured to monitor concentration of an electroactive species (e.g. anti-microbial or anti-biofilm agent) in or on the wound dressing or in its surrounding environment while the wound dressing is in use and applied to the body of a patient. The wound dressing 701 includes a backing layer 702, counter/reference electrode 704, spacing layer 703, electrode component 706 (comprising or consisting of a working electrode configured for sensing electroactive species within the wound environment wherein the working electrode is in the form of a wire electrode) and wound contact layer 705. As shown in the embodiment of FIG. 7, the electrode component 706 and counter/reference electrode 704 are positioned within the dressing 701 separate from one another and are not fabricated to a common support layer likes FIGS. 5 and 6. The electrode component 706 and counter/reference electrode 704 are configured to be electrically isolated from one another which can be achieved by positioning the electrode on different layers of the dressing structure, or by coating one or both of the electrode with an insulating but porous material. In the embodiment illustrated in FIG. 7, the spacing layer 704 provides an insulating barrier between the electrode component 706 and counter/reference electrode 704 thus electrically isolated them from one another. The wound contact layer 705 may optionally include an active ingredient (e.g. an anti-microbial or anti-biofilm agent). This type of wound dressing arrangement has the advantage that the working wire forming part of the electrode component 706 has minimal impact on fluid flow within the dressing 701. This also reduces the costs associated with the manufacture of the wound dressing since the use of separate electrode components may be less than the cost of manufacturing an integrated sensor.
[0185] FIG. 8 depicts a wound dressing 801 according to the present invention configured to release and monitor concentration of an electroactive (e.g. anti-microbial or anti-biofilm agent) in or on the wound or in its surrounding environment while the wound dressing is in use and applied to the body of a patient. The wound dressing includes a backing layer 802, counter/reference electrode 803 and spacing layer 804. The spacing layer 804 electrically isolates the counter/reference electrode 803 from the other conductive components within the wound dressing any may be in the form of an insulating coating formed on the counter/reference electrode 803. The dressing also includes an active release electrode 807, active release layer 808 and another spacing layer 809. The active release electrode 807 and active release layer 808 electrically isolated from other components by virtue of the spacing layer 804 and 809. The purpose of the active release electrode 807 and active release layer 808 is to control release of a species forming part of the active release layer 808 by controlling an electrochemical reaction on the active release electrode which cause release of the electroactive species. The electrode component 806 (comprising or consisting of a working electrode configured for sensing electroactive species within the wound environment wherein) is configured to sense the concentration of the electroactive species being released within the wound dressing environment. Wound dressing 801 also includes a wound contact layer 805.
[0186] Like FIG. 8, FIG. 9 depicts a wound dressing 901 according to the present invention configured to release and monitor concentration of an electroactive (e.g. anti-microbial or anti-biofilm agent) in or on the wound dressing or in its surrounding environment while the wound dressing is in use and applied to the body of a patient. The wound dressing includes a backing layer 902, counter/reference electrode 903, spacing layer 904, an active release electrode 907, active release layer 908 and another spacing layer 909 as described for wound dressing 801 above. In addition, wound dressing 901 also includes an electrode component 906 (comprising or consisting of a working electrode configured for sensing electroactive species within the wound environment) configured to sense the concentration of the electroactive species being released within the wound dressing environment from the active release electrode 907 and active release layer 908 as described for the embodiment illustrated in FIG. 8. The wound dressing 901 however also includes a wound status sensor 910 configured to monitor a suitable wound status parameter (e.g. temperature, pH, moisture content, toxin levels, signalling compounds, enzyme and/or growth factors being released by the wound). The active release electrode 907, active release layer 908 and wound status sensor 910 are in communication such that an electroactive species can be released from the release layer 908 via an electrochemical reaction on the active release electrode 907 in response to a change in the wound status parameter monitored by wound status electrode 910. The level of the electroactive species is then detected by the electrode component 906.
[0187] Wound dressing 901 also includes a wound contact layer 905.
Experimental Data
Electrode Types
[0188] Tables 1 and 2 describe various parameters for a planar macro electrode, a Nanoband electrode, a number of micro-wire electrodes, a circular planar macro electrode. The Nanoband electrode is flat planar platinum electrode, as depicted in FIGS. 3 and 4. The electrode has a non-conductive substrate layer 20, a dielectric (insulating) layer 22 and a platinum coating 16 (50 nm thickness) on the non-conductive substrate layer, located between the non-conductive substrate layer and the dielectric layer. Square apertures 24 are formed in the electrode by etching down through the dielectric layer and platinum coating to form square apertures having edge dimensions of 30 ?m (length)?30 ?m (width) with platinum edge (50 nm thickness) exposed as the sensing surface, depicted in FIG. 3 as end faces 18. The apertures have an interval (spacing distance) of 30 ?m. This type of Nanoband electrode structure is described in more detail with reference to FIG. 3 and Falk M, Sultana R, Swann M J, Mount A R, Freeman N J. Nanoband array electrode as a platform for high sensitivity enzyme-based glucose biosensing. Bioelectrochemistry. 2016 December; 112:100-5. doi: 10.1016/j.bioelechem.2016.04.002. Epub 2016 Apr. 14. PMID: 27118384.
[0189] To provide a meaningful comparison between the electrodes, the dimensions of each electrode (i.e. the electrode length and width show in Table 1) have been chosen such that the surface area of the dressing that is capable of being probed by the electrode, herein referred to as the footprint area probed, is approximately the same for all electrodes, namely approximately 4 mm.sup.2.
[0190] In Tables 1 and 2 below, the occluded footprint describes the 2D surface area of the electrode structure. The values calculated for the occluded footprint area are based on the maximum physical dimensions of the electrodes in the plane of the electrode. The footprint area probed is the same, but with the addition of the maximum diffusion length (200 ?m) extending beyond the edge of the electrode. When used in a dressing, this surface area corresponds to the area of probe that is adjacent the surface of the wound that may affect or block fluid flowing away from a wound.
[0191] For the Nanoband electrodes, the footprint calculations are the same as for the macro electrode. As the array spacing is smaller than the maximum diffusion length, meaning the diffusion fields from adjacent apertures overlap, the volume probed is also approximated to the same as the macro electrode. As the planar electrodes are coated onto a support film this volume only extends on one side of the electrode.
[0192] The solution volume probed describes the maximum volume of solution that could be probed for a given working electrode during use. It will be appreciated that when the working electrode forms part of a wound dressing the solution volume probed is equivalent to the volume of wound fluid (e.g. wound exudate) within the wound environment that could be probed for a given working electrode. The solution volume probed is calculated based on a probing distance of 200 ?m which is measured in a perpendicular direction from the surface of the working electrode. In Tables 1 and 2, the solution volume probed is calculated assuming a maximum diffusion length of analyte species to each electrode surface of 200 ?m which corresponds to the probing distance mentioned above. Using the Nanoband electrode in Table 2 as an example, the planar dimensions of the electrode are 7 mm?3 mm (equal to the occluded footprint). Assuming a maximum 200 ?m diffusion length for an analyte within the solution, this extends the planar dimension of the solution probed to approximately 3.2 mm?7.2 mm. The footprint area probed is therefore 3.2?7.2=23.04 mm.sup.2. The thickness of solution probed is the diffusion length 200 ?m which gives a solution volume probed of 3.2 mm?7.2 mm?0.2 mm=4.6 mm.sup.3.
[0193] The exposed surface area of the working electrode describes the surface area of the working electrode which is exposed to the solution which is being probed and available for sensing (i.e. the area that is capable of receiving ions from the solution). It will be appreciated that when the electrode forms part of a wound dressing the exposed surface area of the working electrode is equivalent to the surface area of the working electrode that is exposed to the wound environment during use. That is, the surface area of the working electrode that is available for detecting an electroactive species during use (e.g. the surface area of the electrode which is available for sensing Ag ions within a wound exudate). The solution volume probed/exposed surface area of the working electrode describes the maximum volume of solution that the working electrode is capable of probing for a given surface area of exposed working electrode. This provides a guide as to the sensitivity of the working electrode. Taking the example of the embodiment described in FIG. 3, the exposed electrode surface area would correspond to the area of exposed end faces 18.
TABLE-US-00001 TABLE 1 Solution Exposed volume probed/ surface Exposed Footprint Occluded Solution area of surface area of area footprint volume working working Electrode probed Length Width area probed electrode electrode Type (mm.sup.2) (mm) (mm) (mm.sup.2) (mm.sup.3) (mm.sup.2) (mm) Planar Macro 4.08 3.2 1 3.2 0.816 3.2 0.26 Pt Micro-wire 4.1 8 0.1 0.8 1.541 2.513 0.61 (100 ?m) Pt Micro-wire 4.34 10 0.025 0.25 1.434 0.785 1.83 (25 ?m) Pt Micro-wire 4.13 10 0.005 0.05 1.305 0.157 8.31 (5 ?m) Pt Micro-wire 4.09 10 0.001 0.01 1.280 0.031 40.74 (1 ?m) Nanoband 4.08 3.2 1 3.2 0.816 0.0053 153.0 electrode Circular 4.52 2 2 3.14 0.905 3.14 0.288 Planar Macro (Carbon) Micro-wire 4 10 0.005 15 0.942 0.05 18.8 (5 ?m, Carbon)
[0194] It is desirable for the solution volume probed/exposed surface area of working electrode ratio to be as high as possible. This is attributed to a high degree of sensitivity (signal to noise) and reduced tendency for the signal to deteriorate due to mass transport hindrances.
[0195] As can be seen in Table 1, micro-wires have the smallest occluded footprint and therefore have the smallest impact on the flow of fluid away from the wound and into the dressing. The solution volume probed is also increased when using wire electrodes over planar electrodes. Without wishing to be bound by theory, this is because diffusion is available from all sides of the wire (i.e. from an area radially surrounding the wire).
[0196] However, the solution volume probed per electrode area, and therefore the sensitivity (i.e. signal to noise) of the working electrode, is greatest for the planar Nanoband electrode. This arises due to the highly reduced exposed surface area of the Nanoband electrode.
[0197] The experiments detailed below were performed using the electrodes detailed in Table 2. The structure of the planar macro electrode is as described above for Table 1. The Nanoband electrode structure is as described above and includes edge dimensions of 30 ?m (length)?30 ?m (width) with an exposed platinum edge thickness of 50 nm and aperture spacing intervals of 30 ?m. The Nanoband electrode includes approximately 5830 apertures.
[0198] Table 2 shows the dimensions of the electrodes used in the accompanying experimental studies described below.
TABLE-US-00002 TABLE 2 Solution Exposed volume probed/ surface Exposed Footprint Occluded Solution area of surface area of area footprint volume working working Electrode probed Length Width area probed electrode electrode Type (mm.sup.2) (mm) (mm) (mm.sup.2) (mm.sup.3) (mm.sup.2) (mm) Planar 8.6 7 1 7 1.728 7 0.247 Macro (Pt) Micro-wire 12.6 25 0.1 2.5 4.745 7.85 0.604 (100 ?m, Pt) Micro-wire 10.7 25 0.025 0.625 3.554 1.96 1.81 (25 ?m, Pt) Nanoband 23.0 7 3 21 4.608 0.035 131.7 electrode
[0199] Mass transport to micro electrodes vs mass transport to macro electrodes FIG. 10 shows the results of three cyclic voltammetry experiments, where the potential (E) of different electrodes were swept between ?0.1 and 0.5 V with respect to a Ag/AgCl electrode in 1 mM ferrocene carboxylic acid prepared in phosphate buffered saline (PBS) solution and platinum counter electrode. The planar electrodes were coated onto a polyethylene terephthalate (PET) film. The electrodes assessed as shown in FIG. 10 were a macro platinum (Pt) planar electrode 14 mm?1 mm (shown as green), a 100 ?m platinum wire electrode (shown as blue) and a 25 ?m platinum wire electrode (shown in black) (as described in Table 2 above). The data for the 25 ?m platinum wire electrode shows a classic sigmoidal shape with no drop in current above a potential of 0.3V. The 100 ?m platinum wire electrode shows some drop in current above 0.3V and the macro electrode shows the largest drop in current at this potential. Without wishing to be bound by theory, this effect occurs due to a mass transport limitation. Importantly, it has been observed that for measuring low concentrations of Ag ions using macro electrodes in the system illustrated in FIG. 10, it is usually necessary to rotate the electrode rapidly to provide sufficient mass transport to the electrode surface. This requirement to agitate the electrode is of course impractical when the electrode is intended to be housed within a wound dressing. Advantageously, this requirement is eliminated when using micro or nano electrodes according to the invention, rendering the working electrodes of the present invention eminently suitable for use in wound care applications.
Use of Anodic Stripping Voltammetry to Measure Silver Ions
[0200] To detect low concentration of metal ions, such as silver, electrochemical methods such as anodic stripping voltammetry can be used. Anodic stripping voltammetry reduces the metal ions onto the electrode surface over a certain period of time and then re-oxidises them in one step to produce a clearly measurable signal.
[0201] FIG. 11 shows the measured current over time from seven anodic stripping voltammetry experiments conducted in Mueller-Hinton Broth II (MHB2) solutions that contained differing concentrations of added Ag.sup.+ ions. The concentrations ranged between 0 ppm and 100 ppm and the electrode was a platinum Nanoband electrode supplied by NanoFlex Ltd as described above in connection with Table 2. In each experiment, the potential of the electrode was initially held at ?0.2V with respect to a standard/reference Ag/AgCl electrode for 100 seconds. The potential was then increased to ?0.05V and held for 1 second, and then further increased to +0.2V and held for another 100 seconds. The initial 100 s is the reductive collection stage and the second 100 s is the anodic re-oxidation stage.
[0202] FIG. 12 shows an expanded section of FIG. 11 that focuses on the oxidation current observed for each concentration of Ag.sup.+ ions during the beginning of the anodic re-oxidation stage (the stripping step). The concentration values given in FIGS. 11 and 12 are nominal values.
[0203] The data shown in FIGS. 11 and 12 can be used calculate the oxidation charge during the anodic stripping step as a function of the Ag.sup.+ ion concentration added to the MHB2.
[0204] This is displayed in FIG. 13 plotted using the precise values for concentrations instead of the nominal concentrations. The results show that a significant response is observed for concentrations above 1 ppm. Without wishing to be bound by theory, the slope is not linear because at higher concentrations AgCl is precipitated from the solution. The response observed in FIG. 13 is surprising, as the amount of chloride in the solution should limit the Ag.sup.+ concentration above ?1 ppm. MHB2 Contains 17.5 g/L Casein hydrolysate and 3 g/L beef extract and so is unlikely to contain the higher affinity metal ion binding sites that are found in serum albumins, but may contribute to stabilizing Ag.sup.+ or nanoscale Ag/CI aggregates in solution.
[0205] FIG. 16 plots the oxidation charge against Ag.sup.+ ion concentration obtained from similar anodic striping experiments using the Pt Nanoband electrode but in PBS, Silver Nitrate (NaNO3) and simulated wound fluid (SWF) solutions. Simulated wound fluid was made up of 33 g/L BSA, 100 mM NaCl, 40 mM NaHCO.sub.3, 4 mM KCl and 2.5 mM CaCl.sub.2. (see Bradford C, Freeman R, Percival SL. In vitro study of sustained anti-microbial activity of a new silver alginate dressing. J Am Col Certif Wound Spec. 2009; 1(4):117-120. Published 2009 Oct. 6. doi:10.1016/j.jcws.2009.09.001). In these experiments, Ag.sup.+ ions were added at concentrations between 0 ppm and 300 ?m. The data in FIG. 16 is presented after a blank background subtraction. FIG. 17 plots the same data as FIG. 16 but on a log scale. The data for the simulated wound fluid in FIGS. 16 and 17 are particularly relevant, showing a strong response at and above 30 ppm.
[0206] Similar anodic striping experiments were also conducted using microelectrodes. FIG. 18 displays the oxidation charge as a function of Ag.sup.+ concentration using a 25 ?m Pt wire electrode. The Ag.sup.+ ions were added to a simulated wound fluid (SWF) containing 33 g/L bovine serum albumin (BSA). Each point displayed is the average of a duplicate sequential measurements, and associated error bars have been calculated. As seen for the Pt nanoband electrode, the sensor response is evident at 30 ppm and above. Without wishing to be bound by theory, this may be because the serum albumin, which contains higher affinity metal ion binding sites reduces the effective Ag.sup.+ concentration until the added Ag.sup.+ concentration approaches that of the BSA. The molar concentration of 33 g/L BSA is 0.5 mM and 30 ppm Ag.sup.+ corresponds to 0.28 mM.
Bacterial Proliferation Assays
[0207] To assess the antibacterial efficacy of the silver, and to correlate this with the sensor response, bacterial proliferation assays were performed on MHB2, PBS, NaNO3 and SWF solutions with added silver at concentrations between 0.3 to 300 ppm. FIG. 14 shows the number of Colony Forming Units of Pseudomonas that were measured after a bacterial proliferation assay in the different solutions as a function of the amount of added Ag.sup.+.
[0208] The bacterial proliferation assay data shows that in NaNO.sub.3 and PBS solutions the efficacy of the Ag.sup.+ is already apparent at the lowest concentration measured (0.3 ppm), however in SWF solutions the efficacy of Ag.sup.+ is seen only for 30 ppm added Ag.sup.+ and above. This mirrors the electrochemical sensor data which shows a poor response of the sensor to Ag.sup.+ below 30 ppm, presumably due to preferential binding to the BSA in SWF.
Optical Density Measurements
[0209] Optical density measurements can be used to indicate the presence of AgCl precipitates/aggregates in solution. FIG. 15 shows Optical density data measurements for solutions of PBS, SWF and NaNO.sub.3 as a function of concentration of added Ag.sup.+ ions. No bacteria where present for the measurements. This OD data shows the impact of AgCl precipitation in SWF above 30 ppm and in PBS above 1 ppm, which result in the nonlinear sensor measurements.
Using Multiple Anodic steps
[0210] The effect of the anodic stripping technique is to concentrate the reduced metal ion (Ag) onto the sensing electrode and to re-oxidise it in an anodic step. Due to the poor solubility of Ag.sup.+ in SWF and other chloride containing media, this process, which generates high concentrations of Ag.sup.+ at the electrode surface, can have the effect of precipitating insoluble silver salts. These salts are then present and can be re-reduced at the next measurement increasing the subsequent anodic stripping signal and enhancing the size of the silver signal. When silver levels in the SWF reduce again, this additional silver is solubilized and the signal reduces. This effect can be ameliorated by using a short initial reduction step and progressively increasing the reduction time until a signal is detected, which limits the amount of Ag deposited on the electrode. Further, instead of a single anodic stripping potential step, either a slow voltage scan or multiple smaller potential steps can be used, such that the silver ions are released at a reduced rate back into the solution. This is relevant for high concentrations of Ag.sup.+. Another advantage of performing multiple small potential steps is that once above the maximum silver oxidation potential, the charge passed can be used as a measure of the size of the background charging current present during the Ag oxidation steps.
[0211] FIG. 19 shows the current passed during multiple anodic steps for two nanoband electrodes after 50 s at a reducing potential (?0.2V) in simulated wound fluid, one in the presence of added silver (30 ppm) and one without any added silver. The steps observed in the current after 55 s correspond to incremental increases of the potential by 0.05V, starting at ?0.05V. The step at 60 s shows anodic stripping of the deposited Ag, whereas the steps before and after are just the background charging capacitance of the electrode.
Electrochemical Ag.SUP.+ Generation
[0212] FIG. 20 shows the results of an experiment designed to generate and detect Ag.sup.+ ions in a gauze dressing. The experiment involved a Ag/AgCl counter/reference electrode, a Ag/AgCl silver release electrode and a Pt nanoband sensor (as described in Table 2). During the experiment, the potential of the Ag/AgCl release electrode (as shown in blue) is scanned (0V to ?0.3V to +0.3V to 0V) to reduce and then oxidize silver. Meanwhile the Ag sensing electrode (orange) is held at ?0.2V. At this potential Ag.sup.+ ions in solution can be reduced on the sensing electrode surface producing a negative sensor collector current. The sensor collector current is shown on the right hand Y-axis, while the current of the release electrode is shown on the central axis. Note the two axes do not share the same origin. The x-axis on the plot meanwhile shows the potential of the Ag release electrode being scanned. Initially when the experiment starts at 0V and scans to negative potentials, there is a large reduction in the magnitude of the negative sensor collector current on the sensing electrode. This current reaches zero as the Ag electrode scans to ?0.3V to reduce silver. As the scan then increases the potential of the release electrode and goes above zero, the Ag release electrode passes positive current, oxidizing Ag to Ag.sup.+ which is in part released into the solution. The magnitude of the negative reduction current on the Ag sensing electrode therefore increases again as it reduces the Ag.sup.+ ions in solution that were produced by the generator (the Ag release electrode). Finally, as the release electrode potential is reduced back to zero and less silver is generated, the reduction current of the sensor electrode also returns to zero.
Detection of Non-Metallic Anti-Microbial/Anti-Biofilm Agent (Iodine)
[0213] In this example, the measurement of a non-metallic anti-microbial/anti-biofilm agent using a nano-electrode is performed. The data shown in FIG. 21 illustrates the measurement of iodine in phosphate buffered saline (PBS). Iodine can be measured either at oxidative or at reductive potentials. The plot on the left hand side of FIG. 21 shows the measurement of iodine using a platinum based macro electrode (as described in Table 1 above under Planar Macro) at iodine concentrations of 500 ?M and 1.5 mM.
[0214] The plot on the right hand side of FIG. 21 shows the measurement of iodine using a Pt nanoband array sensor with printed Ag/AgCl reference electrode and Platinum counter electrode (as described in Table 1 above under Nanoband electrode) at iodine concentrations of 500 ?M and 1.5 mM The data in left hand side plot of FIG. 21 shows the reduction of iodine as the curve moves towards a potential of 0V (vs. Ag/AgCl) as function of time. The response shown in the right hand side plot for the nanoband electrode shows significantly less decay with time due to improved diffusion and analyte depletion characteristics as compared to the macro electrode. This results in a more stable measurement being achieved by the nanoband electrode. This example demonstrates that the use of micro or nano electrodes over an extended area of a wound dressing for the measurement of anti-microbial/anti-biofilm agents will result in less depletion of the anti-microbial/anti-biofilm agent levels as a result of the measurement process itself. This overcomes the analyte depletion issues associated with macro electrodes where agent is significantly consumed as part of the measurement mechanism which can lead to an unstable analyte measurement.
[0215] FIG. 22 is a schematic representation of a micro-wire or micro-strip electrochemical sensor arrangement according to the present invention. The schematic shows a cross section of the arrangement with the electrode labelled as 16, the insulating layer labelled as 22 and substrate layer labelled as 20.
Cyclic Voltammetry Experiments Conducted Using a Carbon Micro-Wire Electrode
[0216] FIG. 23 shows a normalised cyclic voltammogram of a carbon micro-wire electrode in 1 mM Ferrocene Carboxylic Acid in PBS. The plot displays a sigmoidal shape which is characteristic of micro-electrodes and in contrast to a macro electrode also plotted. The current is normalised to the maximum current for each electrode. The macro electrode is a 3.3 mm diameter circular printed carbon electrode on a PET substrate, which is part of a 3-electrode electrochemical sensor with carbon counter electrode and Ag/AgCl reference electrode. The circular surface and edges of the marco carbon electrode are exposed for analyte detection. The micro-wire electrode in this example has a width (or thickness) of approximately 5 ?m and a length of approximately 3.3 mm.
[0217] FIG. 24 shows the measurement of iodine (at varying concentrations) with a carbon micro-wire electrode as compared to a conventional macroscopic printed carbon electrode. Both are utilised with a Ag/AgCl reference electrode and carbon counter electrodes. The macro electrode is a 3.3 mm diameter circular printed carbon electrode on a PET substrate, which is part of a 3-electrode electrochemical sensor with carbon counter electrode and Ag/AgCl reference electrode. The circular surface and edges of the marco carbon electrode are exposed for analyte detection. The micro-wire electrode in this example has a width (or thickness) of approximately 5 ?m and a length of approximately 3.3 mm. As can be seen from the plot on the right hand side, the micro-wire sensor rapidly reaches a steady state measurement.
[0218] The dimensions of the carbon macro electrode and carbon micro-wire utilisted for the experiments illustrated in FIGS. 23 and 24 are provided in Table 3 below.
TABLE-US-00003 TABLE 3 Solution Exposed volume probed/ surface Exposed Footprint Occluded Solution area of surface area of area footprint volume working working Electrode probed Length Width area probed electrode electrode Type (mm.sup.2) (mm) (mm) (mm.sup.2) (mm.sup.3) (mm.sup.2) (mm) Circular 10.75 3.3 3.3 8.55 2.15 8.553 0.251 Planar Macro (C) Micro-wire 1.32 3.3 0.005 4.95 0.311 0.0165 18.8 (5 ?m, C)
[0219] FIG. 25 shows a graph plotting the iodine reduction charge versus concentration for a printed carbon micro-wire electrode having a width (or thickness) of approximately 5 ?m and a length of approximately 3.3 mm.
Simulated Wound Fluid Flow Test
[0220] FIG. 26 shows a schematic of a nanoband electrode sensor (as described in Table 1 above) incorporated within a wound dressing arrangement. The wound dressing arrangement includes a silver loaded carboxymethyl cellulose dressing material as a lower layer and a polypropylene (nonwoven) upper layer. The sensor component is sandwiched between the carboxymethyl cellulose dressing layer and polypropylene layer. The sensor dressing is mounted into a flow cell for measurement under simulated wound fluid (SWF). The blue arrows represents the direction of flow of the simulated wound fluid whereas the red arrow represents the direction that the working electrode is facing in this setup.
[0221] FIG. 27 provides a photograph of the type of wound dressing sensor represented in FIG. 26 mounted within a flow cell.
[0222] FIG. 28 shows the results from an experiment performed with a nanoband electrode sensor (as described in Table 1 above) incorporated within a wound dressing arrangement as shown in FIGS. 26-27 which is subjected to flow of simulated wound fluid over the course of 24 hours at a flow rate of 2 mL/hour. The decaying signal for silver, measured by the sensor, can be clearly seen as time progresses and can be used as an indication for when the levels of active silver within the wound fluid drop below therapeutic levels and the wound dressing needs to be changed.
[0223] It will be appreciated that numerous modifications to the above described sensor may be made without departing from the spirit and scope of the invention, for instance, the scope of the invention as defined in the appended claims. Moreover, any one or more of the above aspects/embodiments could be combined with one or more feature of the other aspects/embodiments and all such combinations are intended with the present disclosure.
[0224] Optional and/or preferred features may be used in other combinations beyond those explicitly described herein and optional and/or preferred features described in relation to one aspect of the invention may also be present in another aspect of the invention, where appropriate. The described and illustrated embodiments are to be considered as illustrative and not restrictive in character, it being understood that only the preferred embodiments have been shown and described and that all change and modifications that come within the scope of the invention as defined in the claims are desired to be protected.
[0225] It should be understood that while the use of words such as preferable, preferably, preferred, or more preferred in the description suggest that a feature so described may be desirable, it may nevertheless not be necessary and embodiments lacking such a feature may be contemplated as within the scope of the invention as defined in the appended claims. In relation to the claims, it is intended that when words such as a, an or at least one are used to preface a feature there is no intention to limit the claim to only one such feature unless specifically stated to the contrary.
