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1. WO2012171922 - DIAGNOSTIC DEVICE

Note: Text based on automatic Optical Character Recognition processes. Please use the PDF version for legal matters

Diagnostic Device

The present invention provides an indicator for the in situ detection and indication of the presence of a substance or a microbe at a location, and methods for its use and construction.

It is important to be able to detect, indicate and draw attention to the presence of microbes at a given location, for example in applications to determine whether a wound has become infected or whether foodstuffs are free from chemical and/or microbial contamination and are safe for consumption. There are currently several methods for the detection and indication of the presence or absence of chemical and/or microbial contamination.

However, these methods for microbial contamination generally require a multi-step assay to be performed in a laboratory or other clinical environment and not directly at the location where microbial contamination may exist.

During the process of wound healing there is often a serious danger of microbes entering a wound site, multiplying and resulting in the wound becoming infected. There are currently only indirect clinical methods of determining this condition. This is usually at a point at which infection has taken hold and is considered to be seriously detrimental to the patient. In extreme cases septicaemia will result. One aspect of treatment is to change the dressing to remove some of the infection. It would be desirable to have a convenient and simple early warning of wound infection, and preferably an indication of the extent of any infection.

Several systems have been proposed which are intended to give nursing staff a prior warning of when a dressing should be replaced. Many of these however only provide an indication of the need to change the dressing because the absorptive capacity is nearly or fully exhausted and/or there is the danger of exudate striking through the surface of a dressing, for example a wound dressing including an indicator layer which contains a dye that changes colour on contact with aqueous wound exudate.

Other such systems that have been proposed include a wound dressing including a substrate that allows the extent of wound exudate absorption to be viewed therethrough.

Further such systems include a dressing which has a transparent cover sheet such that the spreading of strike-through exudate from the wound towards a circular line on the cover sheet indicates that the dressing should be changed.

However, if a wound is infected, it is likely that a dressing should be replaced before its absorptive capacity is reached or exceeded. On the other hand it is well understood that over-frequent changing of a patient's dressing leads to sub-optimal wound healing.

The systems proposed above do not provide an indication of the wound condition or of the presence or extent of microbial contamination at a wound site.

There is a clear need therefore for a method of providing an early and/or continuing check on the level of microbial bioburden in a wound or in consumer products that are susceptible to microbial contamination, and on the presence or level of any chemical contamination in consumer products. .

Thus, according to a first aspect of the invention there is provided an indicator suitable for detecting and indicating the presence of a substance or a microbe at a location which indicator comprises:

(a) a sacrificial layer which is susceptible to degradation by the substance or microbe; and

(b) a signalling layer which is adapted to produce a detectable signal which indicates the presence of the substance or microbe;

wherein, in use,

at least part of the sacrificial layer (a) initially

(i) lies on the other side of the signalling layer (b) from the substance or microbe; and

(ii) does not transmit the detectable signal; and

the signalling layer (b)

(i) is initially shielded from means for detecting the detectable signal by the sacrificial layer (a), and

(ii) is at least partially permeable to the substance or microbe.

It will be understood that, in use, over a period of time, the substance or microbe will act on the sacrificial layer on the other side of the indicator signalling layer from the location to reduce its thickness and/or to change its chemical composition until it allows transmission of the detectable signal.

The indicating signal may optionally be visually, electrically or audibly detectable.

Thus, the reduction in thickness or removal of at least part and/or change in chemical composition of the sacrificial layer on the other side of the signalling layer from the location between the signalling layer and the detection means may be enough to transmit the signal, for example it has become transparent or translucent for a visual signal, thus allowing the signalling layer to become visible.

The indicating signal may be electrically detectable. In such case, a pair of electrodes with a low electrical potential difference will be disposed on opposite sides of the combination of the sacrificial layer on the other side of the signalling layer from the location and the signalling layer. The reduction in thickness or removal of at least part and/or change in chemical composition of the sacrificial layer on the other side of the signalling layer from the location may allow the transmission of an indicating signal electric current between the electrodes.

An electrical visual detection and indicator means, such as a meter or a light, may be included in series in the electrical circuit, so that the detectable indicating signal may become visible, or an electric sonic detection and indicator means, such as a buzzer, bleeper or bell may be included in series in the electrical circuit, so that the indicating signal may become audible.

The pair of electrodes with a low electrical potential difference (disposed on opposite sides of the combination of the sacrificial layer on the other side of the signalling layer from the location and the signalling layer) should of course be made of a solid, water-insoluble biocompatible material that is inert to the substance or microbe to be detected at the location may be used for the signalling layer, including, but not limited to such alloys and metals, for example stainless steel or gold, or conductive thermoplastic materials.

Alternatively, the substance to be detected is associated with a microbe, and the indicator may be for example in an antimicrobial wound dressing and be used to monitor the treatment by monitoring the presence of the substance.

It thus monitors the presence of the microbe to give an indication of when the concentration of a substance or microbe at a particular location reaches a predetermined concentration, for example a dangerous level. .

Either or both electrodes, preferably that adjacent to the location may then comprise an antimicrobial substance, including, but not limited to such alloys and metals, for example silver or gold oxides.

To enable the substance or microbe to act on the sacrificial layer on the other side of the signalling layer from the location, the signalling layer (b) must be at least partially permeated by the substance or microbe, so that the latter may access the sacrificial layer.

The sacrificial layer is often all on the other side of the signalling layer from the location and its thickness and/or change in its chemical composition determines the time until it allows transmission of the detectable signal ('the signalling time'). However, in some embodiments, the sacrificial layer may be in a form which extends on both sides of the signalling layer, so that the latter is embedded, encapsulated or enclosed in, or sandwiched by an integral layer (a). The signalling layer may alternatively be sandwiched by two parts of the layer (a).

The substance may be for example a chemical substance in a personal care product or a foodstuff, which could be present at a concentration at which the personal care product or foodstuff is dangerous to apply to the body or to ingest, as appropriate.

Alternatively or additionally, the substance may be a substance associated with a microbe, and degradation of the sacrificial layer (a) by the substance and the production of a detectable signal by the signalling layer indicate the presence of the substance and hence the presence of the microbe. The substance may be, for example, a microbial product, a part of the microbial cell contents, or a substance associated with the location's response to a microbe where the location is a living human or animal body.

One advantage of the invention is that it can give an indication of when the concentration of a substance or microbe at a particular location has reached a predetermined concentration, for example a dangerous level.

In the case of a chemical substance where the location is a personal care product or a foodstuff, the concentration of the substance could be a concentration at which the personal care product or foodstuff is dangerous to apply to the body or to ingest, as appropriate.

In the case of a microbe where the location is a wound in a human or animal, this may be a level at which infection, disease or other pathological condition might develop; after antimicrobial treatment of the location the indicator can give a continuing indication of whether the microbial bioburden has stayed below or again reached an unacceptable level.

Where the location is a foodstuff, the concentration of the microbe to be detected could be a concentration at which the foodstuff is dangerous to eat.

Another advantage of the invention is that it can measure the body's response to the presence of a microbe by way of a measuring a substance associated with the microbe (as described to above) in that it is produced by the body in response to the microbe rather than detecting the microbe directly. The invention therefore provides a speedier and more relevant indication than hitherto of a developing clinical problem.

Whilst traditional methods of microbiological detection and indication tend to be selective to specific species of microbe, the present invention has the advantage over these traditional methods in that by selection of an appropriate sacrificial layer it can give a measure of general, rather than specific, microbial contamination.

A yet further advantage of the indicator according to the invention is that it will monitor microbial activity throughout the entire required time period. As described further hereinafter, the time may be, in a human or animal location, particularly on or in a living human or animal body, for example a wound, the time to when the concentration of the microbe at the location reaches a dangerous and/or infectious level. After antimicrobial treatment of the wound, the time over which it is necessary to monitor the wound for remission or

recurrence of infection, giving a continuing indication of whether the microbial bioburden within the body has stayed below or again reached an unacceptable level, and is therefore a measure of the treatment's continuing efficacy or suitability.

The indicator according to the invention in a dressing for a wound is that it may be indicative of when a dressing needs to be changed and/or when treatment needs to be started, intensified or restarted. This is because the indicator will reveal when infection in the wound has reached a predetermined level. Generally this level is chosen such that there is substantially no risk of the patient becoming diseased, failing to recover or relapsing. On the other hand the level is sufficiently high that the dressing is not replaced too often.

In the case of a foodstuff or personal care product, the required time period may be the usable life of the product, giving a continuing indication of whether the contamination or microbial bioburden within the product is at or has reached an unacceptable level and is therefore a measure of the product's continuing efficacy or suitability.

The sacrificial layer may be adapted to allow the production and detection of the detectable signal after a time ('the signalling time') that is determined by a) the molar concentration of the substance or microbe and the molar rate of degradation of the sacrificial layer by the substance or microbe , and b) the transmissibility of the sacrificial layer of the detectable signal, the strength of the signal, and the level at which it can be detected.

In use, the sacrificial layer (a) will be degraded by the substance or microbe until that part of it which initially lies on the other side of the signalling layer (b) from the substance or microbe, does not transmit the detectable signal; and initially shields the signalling layer (b) from means for detecting the detectable signal is fully eroded or is thin enough or its chemical composition has changed enough to transmit the signal, for example it has become transparent or translucent for a visual signal, or lese resistive for an electrical signal.

The sensitivity of the indicator according to the invention is determined by the nature of layer (a). It can be seen that the thicker layer (a) and/or the lower its molar rate of degradation and/or its transmissibility of the detectable signal is, i) the higher the concentration of the substance or microbe, or

ii) the longer the period of time that the sacrificial layer (a) is exposed to the substance or microbe,

has to be before the sacrificial layer (a) is degraded sufficiently to allow the detectable signal to be transmitted at a level at which it can be detected.

If the layer (a) is in a form which extends on both sides of the signalling layer, so that the latter is embedded, encapsulated, enclosed or sandwiched in an integral layer (a) or is sandwiched by two parts of the layer (a), a relevant parameter that determines the signalling time is the total thickness of the layer (a).

Thus, if only a small concentration of the substance or microbe is present at the location and/or the molar rate of degradation by the substance or microbe present, the signalling time, i.e. the time for a transmitted detectable signal to reach a level at which it can be detected will is be long in comparison to the time where the concentration is larger or the molar rate is greater. As the concentration of a substance or microbe at a particular location increases, the rate of degradation of the sacrificial layer will increase.

In the case of a microbe where the location is a wound in a human or animal, so that accelerating degradation, and hence a decreasing warning time, occurs, for example, at a level which may be pre-set at that at which infection might develop. Where the location is a foodstuff, the concentration of the microbe to be detected at which this is pre-set to occur could be a concentration at which the foodstuff is dangerous to eat.

The structure and thickness of layer (a) and the material used to make it will be chosen according to the location at which the indicator is to be used. For example, if the indicator is designed to detect and indicate the presence of a microbe, the sacrificial layer (a) is structured such that the sacrificial layer allows the transmission of a detectable warning signal before the concentration of the microbe reaches a dangerous level.

On the other hand, the sacrificial layer (a) should be sufficiently robust that the time required before it is degraded by a background level of a microbe or of a substance is long enough such that the indicator has a useful working life.

The relevant structure and thickness of layer (a) and the material used to make it can easily be determined by a person of skill in the art when the location at

which the indicator is to be used and the type of substance or microbe which is likely to be present are known. However, examples of suitable structures and thicknesses for use in layer (a) are given hereinafter.

The location at which the concentration of a substance or microbe is to be detected may be a human or animal location, particularly on or in a living human or animal body, for example a wound; a foodstuff or a personal care product; a domestic location, e.g. a kitchen or bathroom; or an industrial location, e.g. a laboratory location, such as a steriliser or machinery or a surface involved in the production of pharmaceutical products, personal care or food products.

In the case of a microbe, for example where the location is a wound in a human or animal, and in particular where the indication is of a level at which infection might develop, the sacrificial layer (a) preferably comprises a solid, water-insoluble, water-impervious biopolymer which is degradable by the microbe or a substance associated with the microbe.

Examples of suitable solid, water-insoluble, water-impervious biopolymers for use in layer (a) include chitin, chitosan and derivatives thereof, such as amine salts thereof and alkalised and/or optionally salified organic and inorganic acyloxyalkylchitosans, keratan sulphate, hyaluronic acid, chondroitin, polyhydroxybutyrate, polyester amides, polytrimethylene succinate, albumin crosslinked polyvinylpyrrolidone and dextran.

The biopolymer material may be comprised in a sacrificial layer in the form of one or more sheets, films, layers or membranes.

It may also be comprised in a sacrificial layer in the form of one or more woven or non-woven textile fabric cloths, pads, cushions or wadding having one or more plies, and formed from fibres, strands, threads or yarns, which consist essentially of the solid, water-insoluble, water-impervious biopolymer(s), provided that each is spinnable.

Spinnable biopolymers include chitin, chitosan and derivatives thereof, such as amine salts thereof and alkalised and/or optionally salified organic and inorganic acyloxyalkylchitosans, keratan sulphate, hyaluronic acid, chondroitin, polyhydroxybutyrate, polyester amides, polytrimethylene succinate, albumin crosslinked polyvinylpyrrolidone and dextran.

The layer (a) is often in the form of a (preferably continuous) coating, covering or film on one side of the signalling layer, or a (preferably continuous) membrane on the signalling layer, encapsulating or enclosing the signalling layer in an integral layer (a), or sandwiching the signalling layer between two parts of the layer (a), more preferably in the form of a (preferably continuous) coating, covering or film on one side of the signalling layer.

As noted above, over a period of time, the substance or microbe will act on the sacrificial layer on the other side of the signalling layer from the location to reduce the thickness and/or change the chemical composition of the sacrificial layer until it allows transmission of the detectable signal. It will be understood that the structure and physical dimensions of the sacrificial layer (a) of a chosen material, should be chosen with regard to the desired signalling time in which a desired concentration of a substance or microbe is to be detected.

The signalling time is the time at which degradation of the sacrificial layer by a microbe or substance becomes enough for the indicator to produce a detectable signal which is a sign of the presence of the substance or microbe, which in turn depends on

the location at which the indicator will be used, and

the concentration of the substance or microbe present at the location,

the total thickness of the sacrificial layer (a) where it is exposed to degradation by the substance or microbe present, and

the nature of the layer (a) and the substance or microbe, which determine the rate at which the sacrificial layer (a) is degraded until it allows the detectable signal to be transmitted at a level at which it can be detected.

The relevant display time may of course be adjusted inter alia by adjusting the total thickness of the sacrificial layer (a). Subject to the above considerations, where the sacrificial layer is in the form of a membrane, its total thickness may be 0.1 mm - 2mm, for example 0.3 to 1 .5 mm, such as 0.5 to 1 mm in thickness, depending on the material used to make it, and the substance or microbe which degrades it.

Any microbe to be detected may be, for example, a micro-organism, e.g. a bacterium, virus, mould, yeast or fungus, including pathogenic and nonpathogenic micro-organisms.

As noted above, the substance to be detected may be for example a substance or material associated with a microbe, and degradation of the sacrificial layer (a) by the substance and the production of a detectable alerting signal by the signalling layer indicate the presence of the substance and hence the presence of the microbe.

The substance may be, for example, a microbial product, a part of the microbial cell contents, or a substance associated with the location's response to a microbe where the location is a living human or animal body.

Suitable examples of a microbial product as a detectable substance include an enzyme, particularly, an oxidase, lipase, tryptophanase, beta-lactamase, beta-lactamase inhibitor, esterase, dehydrogenase, kinase, hydrolase, protease, nuclease, phosphatase, decarboxylase, and/or carboxylase. The microbial product may also be a naturally occurring organic phosphate such as adenosine triphosphate (ATP), a pyridine nucleotide such as nicotinamide adenine dinucleotide (NADH) or a flavin such as flavin adenine dinucleotide (FADH).

Examples of a substance associated with the location's response to a microbe where the location is in a living human or animal body, e.g. a wound, include an immune cell product, or an enzyme such as lysozyme or a protease.

In particular, dependent upon the wound application for which the indicator is intended, the substance associated with the microbe may be one or more of the following substances, and suitable corresponding materials comprised in the layer (a) may be one or more of the following solid, water-insoluble water-impervious biopolymers:

Substance Biopolymer

lysozyme chitosan and derivatives thereof, such

as amine salts thereof and alkalised

and/or optionally salified organic and

inorganic acyloxyalkylchitosans

protease polyester amide

Otherwise, the substance associated with the microbe may be one or more of the following substances, and suitable corresponding materials comprised in the layer (a) may be one or more of the following solid, water-insoluble, water-impervious biopolymers:

Substance Biopolymer

lipase polytrimethylene succinate

pepsin albumin crosslinked polyvinylpyrrolidone dextranase. dextran,

As noted above, time at which degradation of the sacrificial layer by a microbe or substance becomes enough for the indicator to produce a detectable signal which is a sign of the presence of the substance or microbe, depends inter alia on the nature of the layer (a) and the substance or microbe, which determine the rate at which the sacrificial layer (a) is degraded until it allows the detectable signal to be transmitted at a level at which it can be detected. The relevant signalling time may of course be adjusted for a given substance or microbe inter alia by adjusting the material of the sacrificial layer (a).

Thus, for example, the substance associated with the microbe may be one or more of the following substances, and suitable corresponding materials comprised in the layer (a) may be one or more of the following solid, water-insoluble water-impervious biopolymers:

Substance Biopolymer

lysozyme chitosan and derivatives thereof, such

as amine salts thereof and alkalised

and/or optionally salified organic and

inorganic acyloxyalkylchitosans

pepsin albumin crosslinked polyvinylpyrrolidone

Chitosan is derived from the deacetylation of chitin, the derived product used generally being chitosan which is >50% deacetylated chitin. 50% and higher levels of de-acetylation produce a material having the required mechanical and biochemical characteristics, namely a material which is strong enough to handle but which will degrade in the presence of lysozyme.

We have found that the higher the degree of deacetylation of the chitosan, the more slowly the product is dissolved by lysozyme.

The indicator may be for example in an antimicrobial wound dressing being used to monitor the treatment by monitoring the presence of lysozyme and hence the presence of the microbe to give an indication of when the concentration of a substance or microbe at a particular location reaches a predetermined concentration, for example a dangerous level. In such case, the more infected the treated wound, the greater the concentration of enzyme present in the wound, and therefore the shorter the time in which the lysozyme will break down a given chitosan layer (a), and the higher the degree of deacetylation of a chitosan layer (a) may be for the lysozyme to degrade it in the same time.

Similar considerations apply to derivatives of chitosan, such as amine salts thereof and alkalised and/or optionally salified organic and inorganic acyloxyalkylchitosans.

Similarly, microbial pepsin degrades albumin crosslinked polyvinylpyrrolidone, and the higher the degree of cross-linking of the polyvinylpyrrolidone, the more slowly the product is dissolved by pepsin. The indicator may be for example be used to monitor the presence of pepsin at a particular location and hence the presence of a microbe to give an indication of when the concentration of a microbe at the location reaches a predetermined concentration, for example a dangerous level. In such case, the greater the concentration of microbe present, the greater the concentration of enzyme present, and therefore the shorter the time in which the pepsin will break down a cross-linked PVP layer (a), and the higher the degree of cross-linking of a PVP layer (a) may be for the pepsin to degrade it in the same time.

In summary, it can be seen that the thicker layer (a) and/or the lower its molar rate of degradation and/or its transmissibility of the detectable signal is, the longer the signalling time is until a given substance or microbe at a particular location reaches a predetermined concentration, for example a dangerous level.

The relevant signalling time may thus of course be adjusted for a given substance or microbe inter alia by adjusting the nature of the material of the sacrificial layer (a) and/or the thickness of a layer (a) of a given material, to produce an indicator or an assembly of indicators with a spectrum of signalling times.

Two or more sacrificial layers (a) differing in composition and/or thickness may all be present in a single indicator, or each may be present in a single indicator in an indicator assembly of two or more indicators.

Accordingly, one embodiment of its first aspect the present invention provides an indicator of the present invention which comprises two or more sacrificial layers (a) which differ in composition and/or thickness.

The two or more sacrificial layers will often have a single common signalling layer and will both be on the other side of the signalling layer from the location and the substance or microbe to be detected at the location, and mutually abut in the common plane of the layers. However, in some versions of this embodiment, the sacrificial layers may each be in a form which extends on both sides of the signalling layer, so that the latter is embedded, encapsulated or enclosed in, or sandwiched by integral layers (a). The signalling layer may alternatively be sandwiched by two parts of the layers (a).

Where the sacrificial layers (a) differ in thickness, they will usually be made of the same sacrificial material, for example, the same chitosan or derivative thereof, such as an amine salt thereof or alkalised and/or optionally salified organic or inorganic acyloxyalkylchitosans.

Where the sacrificial layers (a) differ in composition, they will usually be made of different modifications of the same sacrificial material, for example, a chitosan or derivative thereof, such as an amine salt thereof or alkalised and/or optionally salified organic or inorganic acyloxyalkylchitosans, where the chitosan is derived from the deacetylation of chitin with differing levels of deacetylation. However, in some versions of this embodiment, the sacrificial layers (a) may each be made of essentially different materials. The sacrificial materials comprised in each layer (a) may be specific to different substances and/or microbes, thus providing a single indicator for more than one substance and/or microbe.

In a second aspect of the invention there is provided an indicator assembly comprising two or more indicators of the first aspect the present invention and which comprises sacrificial layers (a) in each indicator which differ in composition and/or thickness.

Each indicator in the assembly will have a discrete signalling layer and a discrete sacrificial layer. The indicators in the assembly will usually mutually abut in the common planes of their layers.

The comments above about the position, composition and thickness of the mutually differing sacrificial layers in a single indicator apply equally to the discrete sacrificial layers of the each indicator in the assembly.

The sacrificial materials comprised in each layer (a) of the assembly may be specific to different substances and/or microbes, thus providing an indicator assembly for more than one substance and/or microbe.

The detectable indicating signal is optionally detectable visually, audibly or electrically; preferably it is a visually detectable signal. Often, for example, in use in the latter case, the signalling layer (b) of the indicator is in fluidic communication or in contact with the location. Preferably the sacrificial layer (a) is positioned and adapted such that, in use, the signalling layer (b), at least initially lies between the substance or microbe and at least part of the sacrificial layer (a) at the location, and in use is at least partially permeated by the substance or microbe, so that the latter may access the sacrificial layer. In the case of an indicator in a wound dressing, the sacrificial layer will usually be a layer on that surface of the signalling layer which is distal of the wound.

The sacrificial layer (a) has a thickness and/or chemical composition that, for example, renders it at least initially opaque to a visually detectable signal, often the sight of the signalling layer (b) of the indicator, or renders it initially resistive to an electrically detectable signal. It preferably comprises a solid, water-insoluble, water-impervious biopolymer which is degradable by the microbe or a substance associated with the microbe.

As noted above, over a period of time, the substance or microbe will act on the sacrificial layer on the other side of the signalling layer from the location to reduce the thickness and/or change the chemical composition of the sacrificial layer until it allows transmission of the detectable signal.

This may be because it has disappeared from the between the signalling layer and the detection means or it is thin enough to transmit the signal, for example it has become transparent or translucent, with the result that a visual signal is detected visually, i.e. some or all of the signalling layer becomes visible

Where the signal from the signalling layer is detectable visually, that part of the signalling layer (b) of the indicator providing the signal, for example it an area, graphic, logo or text, should be of a different contrasting colour to that of the sacrificial layer (a) before it is exposed to erosion by any relevant substance and/or microbe, and the sacrificial layer (a) is opaque to the visually detectable signal from the signalling layer (b).

Many of the biocompatible polymer materials that are inert to the substance or microbe to be detected at the location, and are compatible with the material of the sacrificial layer (a). and which may thus be of use in the signalling layer (b) (as described further below) tend to be colourless to white.

Some such materials may thus tend to be transparent or translucent, but opaque when in a thick enough signalling layer, and may be used 'as is'.

Other such materials, however, may never be opaque when in a layer that is a thin enough layer to be used in the indicator of the first aspect the present invention. It is preferred that the latter type of layer is rendered opaque by including at least one suitable biocompatible dye, stain or pigment ('colorant') in the signalling layer (b), for example to make it white, red, orange, green or blue.

Examples of suitable water-insoluble colorants for the signalling layer (b) which are biocompatible materials that are inert to the substance or microbe to be detected at the location) will be known to the skilled person, but depending on the application for which the indicator is intended and the material(s) of the sacrificial layer, these include inorganic pigments, such as titanium pigments: titanium yellow, titanium beige, titanium white and titanium black; carbon black; ultramarine pigments: ultramarine and ultramarine green shade; and zinc pigments: zinc white and zinc ferrite. They also include water-insoluble organic pigments such as alizarin, alizarin crimson, quinacridone, magenta, phthalo green, phthalo blue and pigment red 170

The signalling layer may of course be made of a solid, water-insoluble biocompatible material that is inherently coloured or opaque. Depending on the application for which the indicator is intended and the material(s) of the sacrificial layer, such materials will not require the inclusion of a suitable colorant in the signalling layer (b).

In use, the sacrificial layer (a) needs to be initially opaque to a visually detectable signal from the signalling layer (b).

Many sacrificial layer (a) materials also tend to be colourless to white. Some such materials may thus tend to be transparent or translucent, but opaque when in a thick enough sacrificial layer, and may be used 'as is'.

Other such materials, however, may never be opaque when in a layer that is a thin enough layer to be used in the indicator of the first aspect the present invention, and are transparent or translucent to the extent that in practice the signalling layer (b) can always be viewed therethrough, even before the sacrificial layer (a) is exposed to erosion or change in composition. Examples include a chitosan or derivative thereof, such as an amine salt thereof or alkalised and/or optionally salified organic or inorganic acyloxyalkylchitosans, which may be derived from the deacetylated chitin with differing levels of deacetylation.

It is preferred that the latter type of layer is rendered opaque by including at least one suitable biocompatible dye, stain or pigment ('colorant') in the sacrificial layer (a), for example to make it white, red, orange, green or blue.

Examples of suitable colorants for the sacrificial layer (a) which are biocompatible materials that are inert to the substance or microbe to be detected at the location) will be known to the skilled person, but depending on the application for which the indicator is intended and the material(s) of the sacrificial layer, these include inorganic pigments, such as titanium pigments, for example titanium yellow, titanium beige and titanium white.

Examples of suitable colorants for the sacrificial layer (a) also include natural and synthetic organic dyes, preferably a water-soluble material which is not degradable by the microbe or substance.

Suitable dyes are preferably substantive to the sacrificial material(s) comprised in the layer (a), but not to those in the signalling layer (b).

Suitable dyes may include food dyes, which are favoured because they are biocompatible material that are inert to the substance or microbe to be detected at the location, and are compatible with the material of the sacrificial layer (a). Such materials include naturally-occurring dyes, azo dyes, and anthraquinone and triphenylmethane compounds.

The sacrificial layer may of course be made of a solid, water-insoluble biocompatible material that is inherently coloured or opaque to a visually detectable signal from the signalling layer (b). Depending on the application for which the indicator is intended and the material(s) of the sacrificial layer, such materials will not require the inclusion of a suitable colorant in the sacrificial layer (a) needs to be initially opaque to a visually detectable signal from the signalling layer (b).

Whatever may be the nature of the sacrificial layer (a) or the signalling layer (b), it will be clear to the skilled person, that, as noted above, the colours of the part of the signalling layer (b) providing the signal, for example an area, graphic, logo or text, should be of a different contrasting colour to that of the sacrificial layer (a) before it is exposed to erosion by any relevant substance and/or microbe, and the sacrificial layer (a) is opaque to the visually detectable signal from the signalling layer (b).

Thus, where the sacrificial layer (a) comprises (a), for example, a chitosan or a derivative thereof, such as an amine salt thereof or an alkalised and/or optionally salified organic or inorganic acyloxyalkylchitosans, which, as is well-known to the skilled person, tend to be colourless to white, it may be coloured white with inorganic pigments, such as titanium white or red or orange with a synthetic organic dye, preferably a water-soluble material, such as a food dye which is not degradable by the microbe or substance, and which is substantive to the material) comprised in the sacrificial layer (a), but not to those in the signalling layer (b).

The signalling layer (b) or an area, graphic, logo or text in or on it, should then be of a different contrasting pigment colour to that of the sacrificial layer (a), such as black from titanium or carbon black; or green from ultramarine green shade or blue from ultramarine.

As noted hereinafter, the sacrificial or (preferably) signalling layer may comprise a reagent that changes colour in the presence of a certain microbe or type of microbe, for example a dye, stain, indicator substance or substrate. Current staining techniques in the laboratory enable the observation of microbes (particularly bacteria) directly. Examples of a suitable dye, stain, indicator substance or substrate include methylene blue, Meldola's blue, phenol red, bromo-chloro-indolyl phosphate, alanine amidoacridone, fluorescein diacetate and/or a tetrazolium salt.

The signalling layer may provide an indication of a level at which infection might develop in a wound, or that a microbe is present at a concentration at which a personal care product or foodstuff is dangerous to apply to the body or to ingest, as appropriate.

The location at which the indicator will be used, and where the substance or microbe to be detected is present location is often for example a bodily tissue, foodstuff or personal care product, so that the indicator is often for use in an environment which is a fluid, more often an aqueous fluid.

The signalling layer will be at least partially in fluidic communication or in contact with the environment and should be sufficiently robust chemically and physically to withstand the relevant environment (often a fluid, more often an aqueous fluid) and the substance or microbe for the required time.

The signalling layer must also be at least partially permeable to the substance or microbe, so that the latter may access the sacrificial layer on the other side of the signalling layer from the location. That is, it must allow the diffusion of the substance or microbe, so that the latter may access the sacrificial layer and act on the sacrificial layer to reduce its thickness and/or chemical composition.

The signalling layer may thus suitably be a solid, water-insoluble polymer with channels leading from the location to the sacrificial layer that provide pathways that facilitate diffusion of the substance or microbe through the polymer layer.

The polymer material may be comprised in a signalling layer in the form of one or more sheets, films, layers or membranes, or woven or non-woven pads, cushions or wadding formed from fibres, strands, threads or yarns, and having one or more plies. It is preferably in the form of a membrane in fluidic communication or in contact with the sacrificial layer.

Any solid, water-insoluble biocompatible material that is inert to the substance or microbe to be detected at the location, and is compatible with the material of the sacrificial layer (a) may be used for the signalling layer.

Such materials include, but are not limited to thermoplastic materials such as polypropylene and polyethylene (including high-density polypropylene and polyethylene, ABS, polystyrene, polycarbonate, Nylon, PVC, thermoplastic elastomers, polyester, PVDF, nitrocellulose, polysulphone, cellulose acetate, nylon, a polymer with a hydrophilic component, and/or thermoset plastics, as appropriate to the application for which the indicator is intended and the substance and/or the microbe.

The above materials are compatible with water-insoluble biopolymers which may be used in layer (a) such as chitosan and derivatives thereof, such as amine salts thereof and alkalised and/or optionally salified organic and inorganic acyloxyalkylchitosans and polyester amide. For example, chitosan and such derivatives thereof are compatible with polypropylene and polyethylene (including high-density polypropylene and polyethylene), ABS, polystyrene, polycarbonate, PVC, thermoplastic elastomers, polyester, PVDF, nitrocellulose, polysulphone, cellulose acetate and polyamides. Other such materials compatible with layer (a) materials will be well-known to the skilled person.

Where the indicator is comprised, e.g. in a wound dressing, in use, the signalling layer is usually on a wound-facing face of the dressing, and at least initially lies between the wound and the sacrificial layer (a) and separates the substance or microbe from the sacrificial layer (a) at the wound.

The material of the signalling layer (b) should be compatible with the material of any conventional dressing component layer that may lie between it and the wound, such as absorbent woven or non-woven textile fabric cloths, pads, cushions or wadding having one or more plies, for example a gauze pad having one or more plies, a non-adhesive gel pad, such as a hydrogel pad, or a fluid interactive hydrocolloid layer.

The above signalling layer materials are compatible with materials which may be used in conventional absorbent dressing layers. Otherwise, compatible materials will be well-known to the skilled person.

The signalling layer is preferably attached to the component layer, for example by moulding, welding or bonding.

Signalling layer materials which may be attached to conventional absorbent dressing layers include, but are not limited to thermoplastic materials such as polypropylene and polyethylene (including high-density polypropylene and polyethylene).

It will be understood that the physical dimensions of the signalling layer (b) of a chosen material, including its thickness and the cross-sectional area of the channels through the layer, should be so chosen with regard to the desired time in which a desired concentration of a substance or microbe is to be detected. They should also be chosen such that the diffusion of the substance or microbe through the polymer layer does not become a rate-limiting step.

As noted above, the time is the time at which degradation of the sacrificial layer by a microbe or substance becomes enough for the indicator to produce a detectable signal which is a sign of the presence of the substance or microbe, which in turn depends on

the location at which the indicator will be used,

the concentration of the substance or microbe present at the location,

the total thickness of the sacrificial layer (a) where it is exposed to degradation by the substance or microbe present,

the thickness of the signalling layer (b) if it significantly restricts diffusion of the substance or microbe through it to the sacrificial layer (a),

the nature of the layer (a) and the substance or microbe, and

the rate at which the sacrificial layer (a) is degraded until it allows the detectable signal to be transmitted at a level at which it can be detected.

If the indicator is comprised, e.g. in a wound dressing, then the diffusion rate through layer (b) should (for example) not be less than the rate of degradation of chitosan and derivatives thereof, such as amine salts thereof and alkalised and/or optionally salified organic and inorganic acyloxyalkylchitosans in the presence of diffused lysozyme.

On the other hand, the signalling layer (b) should be sufficiently robust that it can maintain its structural integrity in use, and layer has sufficient structural rigidity to support the indicator alone or with an optional backing layer that is sufficiently stiff to support the indicator and to give it structural rigidity.

As noted above, in use, the signalling layer (b) at least initially lies between the substance or microbe and at least part of the sacrificial layer (a) at the location at which the indicator will be used.

It provides an indication that a substance or microbe to be detected is present the location (often for example a bodily tissue, foodstuff or personal care product) of a level at which infection might develop in a wound, or at a concentration at which a personal care product or foodstuff is dangerous to apply to the body or to ingest, as appropriate.

It will be understood that to do so, the signalling layer must have channels leading from the location to the sacrificial layer, or that part of the sacrificial layer that lies on the other side of the signalling layer from the location, that provide pathways that facilitate diffusion of the substance or microbe, and optionally the components of the location environment which are fluid, more often an aqueous fluid, through the signalling layer (b). The cross-sectional area of the channels through the layer (b) should be so chosen with regard to the desired time in which a desired concentration of a substance or microbe is to be detected. They should also be chosen such that the diffusion of the substance or microbe through the polymer layer does not become a rate-limiting step.

Where the signalling layer (b) is in the form of one or more sheets, films, layers or membranes, preferably formed as a membrane in contact with the sacrificial layer, the signalling layer (b) may be in the form of a net or netting, mesh, web, grid or lattice, or other arrangement or pattern of apertures, holes, openings, perforations, slits or slots, that provide pathways that facilitate diffusion of the substance or microbe, and optionally the components of the location environment which are fluid, more often an aqueous fluid, through the signalling layer.

Where the layer (b) is embedded, encapsulated or enclosed in, or sandwiched by, an integral layer (a), it can be seen that the apertures, holes, openings, perforations, slits or slots, that provide pathways that facilitate diffusion of the substance or microbe through the signalling layer may contain part of the encapsulating, enclosing or sandwiching layer (a).

Such a signalling layer may be formed, as appropriate to the application for which the indicator is intended, by casting, or jet, mist or spray coating a relevant polymer or compatible polymers to a suitable thickness onto the surface of a correspondingly embossed former of e.g. stainless steel, as appropriate.

Alternatively, slits or slots that provide pathways that facilitate diffusion of the substance or microbe, and optionally the components of the location environment which are fluid, through the signalling layer may be formed by slitting or slicing, or similarly cutting or incising a precursor of the signalling layer.

The slit layer, or a correspondingly scored layer, may additionally be stretched conventionally to form a net or netting, mesh, web or other like arrangement or pattern of apertures, holes, openings or perforations.

The cross-sectional area of the channels through the layer which are in the form of apertures, holes, openings, perforations, slits or slots, in such net or netting, mesh, web, grid or lattice, or other arrangement or pattern, may suitably be 30 micron to 5 mm gauge, for example 65 micron to 1 mm, such 100 to 150 micron gauge.

Alternatively, in some applications for which the indicator is intended, the pathways may be provided by interconnecting transmitting channels, which are conduits or pores of smaller cross-sectional area.

These conduits or pores may suitably be 10 micron to 1 .5 mm gauge, for example 30 to 500 micron, such 100 to 150 micron gauge.

These interconnecting transmitting channels of smaller cross-sectional area pass through the layer, which may thus suitably be a solid, water-insoluble, open-cell polymer foam layer.

Such a layer (b) may be sandwiched by the layer (a), but in general the interconnecting channels that provide pathways that facilitate diffusion of the substance or microbe through the signalling layer do not then contain part of the sandwiching layer (a).

Such channels may be created by first mixing a polymer and a channelling agent, heating the mixture above the polymer's melting point and then allowing it to cool, and then removing the channelling agent. The resultant polymer matrix contains a network of interconnecting channels through it. Suitable channelling agents include polyglycol, polyethylene glycol, EVOH, or glycerin. The above materials are in incompatible with the other polymer material(s) comprised in the layer (b).

Such channels through the layer are formed by phase separation of the layer polymer(s) and channelling agent on cooling, and the latter materials may be removed from the matrix of the layer (b) by leaching or by other methods which will be well-known to the skilled person.

The polymer material may be comprised in a signalling layer in the form of one or more woven or non-woven textile fabric cloths, pads, cushions or wadding formed from fibres, strands, threads or yarns, and having one or more plies. It may be in the form of a pad in contact with the sacrificial layer.

The spaces between the fibres, strands, threads or yarns form channels leading from the location to the sacrificial layer that provide pathways that facilitate diffusion of the substance or microbe through the polymer layer.

They may suitably be of a cross-dimension equivalent to that noted above for a signalling layer which is a membrane in the form of a net or netting, mesh, web or other like arrangement or pattern of apertures, holes, openings or perforations.

In some applications, and with some such textile fabrics, the channels that facilitate diffusion of the substance or microbe through the signalling layer may beneficially act as wicking means that facilitates diffusion of the substance or microbe, and optionally the components of the location environment which are fluid, more often an aqueous fluid, through the signalling layer (b) between the location and the sacrificial layer, particularly if the fabric comprises a polymer with a hydrophilic component.

As noted above, in use, the signalling layer (b) at least initially lies between the substance or microbe and at least part of the sacrificial layer (a) at the location at which the indicator will be used. It is in fluidic communication and/or adjacent to, or preferably in contact with the sacrificial layer.

As noted above, in use, the signalling layer (b) at least initially lies between the substance or microbe and at least that part of the sacrificial layer (a) that is on the other side of the layer (b) from the substance or microbe at the location at which the indicator will be used.

The layer (b) is in fluidic communication with and/or adjacent to, or preferably in contact with the sacrificial layer, and is preferably a membrane in the form of a net or netting, mesh, web or other like arrangement or pattern of apertures, holes, openings or perforations, in contact with the sacrificial layer, inter alia for mutual support and/or structural rigidity. They may optionally be fixed together such that one face or both faces of layer (b) is/are covered by layer (a), or one or both faces of layer (b) is/are partially covered by layer (a) or vice versa. They may be joined to each other by adhering, moulding, welding or bonding.

The sacrificial layer may be formed in situ on the signalling layer by casting, or dip, jet, mist or spray coating a relevant polymer or compatible polymers to a suitable thickness onto the surface, and, as appropriate optionally into the apertures, holes, openings, perforations, slits or slots, in the signalling layer.

Where

a) the sacrificial layer is in the form of one or more woven or non-woven textile fabric cloths, pads, cushions or wadding having one or more plies, and formed from fibres, strands, threads or yarns, which consist essentially of the solid, water-insoluble, water-impervious biopolymer(s), and

b) the signalling layer is in the form of a net or netting, mesh, web or other like arrangement or pattern of apertures, holes, openings or perforations, or one or more woven or non-woven textile fabric cloths, pads, cushions or wadding formed from fibres, strands, threads or yarns, and having one or more plies, the two layers may be interwoven, interlaced or knitted, or bound, sewn, stitched or tied together.

As noted above, usually, the signalling layer (b) should be sufficiently robust that it can maintain its structural integrity in use.

The layer should have sufficient structural rigidity to support the indicator alone or with an optional backing layer that is sufficiently stiff to support the indicator and to give it structural rigidity. When it is in the form of a net or netting, mesh, web or other like arrangement or pattern of apertures, holes, openings or perforations, in contact with the sacrificial layer, the strands of such a structure are integral or may optionally be fixed together, for example by adhering, moulding, welding or bonding, such they form an integral layer (b).

However, in some embodiments, the sacrificial layer may be in the form of a one or more membranes or woven or non-woven textile fabric cloths, pads, cushions or wadding formed from fibres, strands, threads or yarns, and having one or more plies. It may be in a form which is sufficiently robust that it can maintain its structural integrity in use, and has sufficient structural rigidity to support the indicator alone or with an optional backing layer that is sufficiently stiff to support the indicator and to give it structural rigidity.

In such embodiments, the signalling layer may be in the form of a net or netting, mesh, web or other like arrangement or pattern of apertures, holes, openings or perforations, which are not integral or fixed together, for example by adhering, moulding, welding or bonding, such they form an integral layer (b). The signalling layer may be in the form of fibres, strands, threads or yarns, which are interwoven, interlaced, embroidered or knitted into, or bound, sewn, stitched or

tied onto and/or into the robust sacrificial layer (a). As the sacrificial layer is eroded in use, the signalling layer in such embodiments usually loses its structural integrity, thus producing an additional signal.

Alternatively, but less preferably, the layers of the indicator may form separate integers, demountably attached to each other within an indicator device according to the invention (as described further hereinafter) by, for example, appropriate fastening means, such as press-clips or similar means.

As noted above, the substance to be detected may be for example a substance or material associated with a microbe, and degradation of the sacrificial layer (a) by the substance and the production of a detectable alerting signal by the signalling layer indicate the presence of the substance and hence the presence of the microbe.

The location at which the indicator will be used, and where the substance or microbe to be detected is present is often for example a bodily tissue, foodstuff or personal care product, so that in such case, the signalling layer may provide an indication of a level at which infection might develop in a wound, or that a microbe is present at a concentration at which a personal care product or foodstuff is dangerous to apply to the body or to ingest, as appropriate.

Suitable examples of a microbial product as a detectable substance include an enzyme, particularly, an oxidase, lipase, tryptophanase, beta-lactamase, beta-lactamase inhibitor, esterase, dehydrogenase, kinase, hydrolase, protease, nuclease, phosphatase, decarboxylase, and/or carboxylase. The microbial product may also be a naturally occurring organic phosphate.

It will be understood that traditional methods of microbiological detection and indication tend to be selective to specific species of microbe. The present invention has the advantage over these traditional methods in that by selection of an appropriate sacrificial layer it can give a measure of general, rather than specific, microbial contamination.

However, an advantage of the indicator according to the invention is that it may be adapted to monitor specific microbial activity throughout the entire required time period, once attention has been drawn to general, rather than specific,

microbial contamination. Thus, the sacrificial or (preferably) signalling layer may comprise a reagent that changes colour in the presence of a certain microbe or type of microbe, for example a dye, stain, indicator substance or substrate. Current staining techniques in the laboratory enable the observation of microbes (particularly bacteria) directly. Examples of a suitable dye, stain, indicator substance or substrate include methylene blue, Meldola's blue, phenol red, bromo-chloro-indolyl phosphate, alanine amidoacridone, fluorescein diacetate and/or a tetrazolium salt.

It is also possible to probe for bacteria using antigen specific antibodies, conjugated with an appropriate label for indirectly visualising the bacteria, in the sacrificial or (preferably) signalling layer.

The time to the point at which degradation of the sacrificial layer by a microbe or substance becomes enough for the indicator to produce a detectable signal which is a sign of the presence of the specific microbe may be used to quantify concentration at which the microbe is present at a given location at which the indicator is used, for example in contact with a wound, or a personal care product or foodstuff.

The indicator according to the invention may be used in a dressing for a wound, and may be used to be indicative of when a dressing needs to be changed and/or when treatment needs to be started, intensified or restarted. This is because the indicator will reveal when infection in the wound has reached a predetermined level. Generally this level is chosen such that there is substantially no risk of the patient becoming diseased, failing to recover or relapsing. On the other hand the level is sufficiently high that the dressing is not replaced too often.

Thus, according to a third aspect of the invention there is provided a dressing for a wound, suitable for detecting and indicating the presence of a substance or a microbe at the wound, which comprises a dressing layer and an indicator according to the first aspect of the invention, which indicator comprises:

(a) a sacrificial layer which is susceptible to degradation by the substance or microbe, and

(b) a signalling layer which is adapted to produce a detectable signal which indicates the presence of the substance or microbe;

wherein, in use, the signalling layer is on a wound-facing face of the dressing.

As used herein, the term "dressing" includes wound coverings, such as adhesive dressings, e.g. plasters, non-adhesive dressings, bandages, and jackets, sleeves and splints for wounded limbs, i.e. where a wound-contacting integer is part of a larger product. For example the dressing could be a non-adhesive gel type dressing such as a hydrogel, an adhesive gel-type dressing, a fluid interactive hydrocolloid dressing capable of adhering to both dry and moist skin surfaces or a hydrocolloid dressing including a polymeric foam layer.

Often one face or both faces of layer (b) is/are covered by layer (a), or one or both faces of layer (b) is/are partially covered by layer (a) or vice versa. Usually, the signalling layer at least initially lies between the wound and the sacrificial layer (a) and separates the substance or microbe from the sacrificial layer (a) at the wound.

The layers may be joined to each other by adhering, moulding, welding or bonding.

One advantage of including an indicator according to the invention in a dressing for a wound is that it may provide an indication of the wound condition or of the presence or extent of microbial contamination at a wound site, and may be indicative of when a dressing needs to be changed and/or when treatment needs to be started, intensified or restarted. This is because the indicator will reveal when infection in the wound has reached a predetermined level.

Generally this level is chosen such that there is substantially no risk of the patient becoming diseased, failing to recover or relapsing. On the other hand the level is sufficiently high that the dressing is not replaced too often.

Suitable examples of a microbial product as a detectable substance include an enzyme, particularly, a lipase, pepsin or dextranase. Examples of a substance associated with the location's response to a microbe where the location is a living human or animal body include an immune cell product, or an enzyme such as lysozyme or a protease.

The sacrificial layer preferably comprises a biopolymer such as polytrimethylene succinate, albumin crosslinked polyvinylpyrrolidone, dextran, chitosan and derivatives thereof, such as amine salts thereof and alkalised and/or optionally salified organic and inorganic acyloxyalkylchitosans or polyester amide, more preferably chitosan and derivatives thereof, such as amine salts thereof and alkalised and/or optionally salified organic and inorganic acyloxyalkylchitosans.

The indicator is provided with a conventional dressing backing layer which usually covers the indicator on its side away from the wound, and protects it from degradation by an external substance or microbe, and, dependent upon the wound application for which the indicator is to be used, may provide means to secure the dressing to the body, such as an adhesive layer.

The backing layer may optionally take any form generally known in the art. Optionally the backing layer is sufficiently stiff to support the indicator and to give it structural rigidity. The backing layer may alternatively not be sufficiently stiff to support the indicator and to give it structural rigidity. The indicator according to the invention in a dressing for a wound is often one in which the signal is detected visually, i.e. some or all of the signalling layer becomes visible. In such case, the backing layer is at least partially transparent or translucent, thus allowing the signalling layer to become visible through the backing layer, and may be a thin polyurethane layer overlying the indicator according to the invention.

Such a backing layer may be provided with an application layer, for example of cardboard, that is sufficiently stiff to support the indicator and to give it structural rigidity. The latter layer may be removable after application of the dressing, or if it is not transparent and not removable, it should have a window above the indicator signalling layer.

The indicating signal may be electrically detectable. In such case, a pair of electrodes with a low electrical potential difference will be disposed on opposite sides of the combination of the sacrificial layer on the other side of the signalling layer from the location and the signalling layer. The former may be in the form of electrically conductive portions of the backing layer.

The dressing according to the invention may also be provided with a moisture sensitive indicator adapted to make known to the medical practitioner and/or patient when the dressing is saturated with moisture. A suitable moisture sensitive indicator is, for example, cobalt chloride which changes from blue to pink in the presence of moisture.

The dressing according to the invention may optionally include further layers, such as a wound-contacting layer.

To enable the substance or microbe to be detected at the location to act on the sacrificial layer on the other side of the signalling layer from the location, the substance or microbe must be able to access that part of the sacrificial layer.

In some embodiments, the sacrificial layer may be in a form which extends on both sides of the signalling layer, so that the latter is embedded, encapsulated or enclosed in, or sandwiched by an integral layer (a). However, the sacrificial layer is often all on the other side of the signalling layer from the wound and its thickness and/or change in its chemical composition determines the time until it allows transmission of the detectable signal.

The signalling layer and/or any part of the sacrificial layer that is on the same side of the signalling layer should be at least partially in fluidic communication or in contact with the location, and thus any wound-facing layer must be at least partially permeable by the substance or microbe.

Subject to that, any wound-facing layer may optionally take any form generally known in the art. For example the layer could be a gauze pad having one or more plies, a non-adhesive gel pad, such as a hydrogel pad, or a fluid interactive hydrocolloid layer.

The dressing according to the invention may optionally further comprise, e.g. in any wound-facing layer, a conventional component such as an antiseptic agent, an anti-bacterial agent, and/or an emollient.

As noted above, the material of the signalling layer (b) should be compatible with the material of any such wound-facing layer. The signalling layer is preferably attached to the wound-facing layer, for example by moulding, welding or bonding.

Such compatible materials include, but are not limited to thermoplastic materials such as polypropylene and polyethylene (including high-density polypropylene and polyethylene, ABS, polystyrene, polycarbonate, Nylon, PVC, thermoplastic elastomers, polyester, PVDF, nitrocellulose, polysulphone, cellulose acetate, nylon, a polymer with a hydrophilic component, and/or thermoset plastics, as appropriate to the application for which the indicator is intended and the substance and/or the microbe.

Signalling layer materials which may be attached to conventional absorbent dressing layers include, but are not limited to thermoplastic materials such as polypropylene and polyethylene (including high-density polypropylene and polyethylene).

The indicator is preferably included within the dressing according to the invention by attaching it, for example by moulding, welding or bonding to the backing layer.

Where either or both layers of the indicator comprise a plurality of threads, and the backing layer is a woven or non-woven textile fabric cloth, pad or cushion formed from fibres, strands, threads or yarns, and having one or more plies, and is sufficiently robust that it can maintain its structural integrity in use, either or both of the two indicator layers may be interwoven, interlaced or knitted, or bound, sewn, stitched or tied into or onto the backing layer.

Aspects of the invention are illustrated by reference to the following drawings. It is to be understood that the figures are included by way of example only and that the invention includes any variants of the embodiments portrayed.

In the drawings:

Figure 1 is an exploded schematic perspective view of an indicator according to the invention;

Figure 2 is a schematic cross-sectional view of an island type dressing incorporating the indicator according to the invention which is shown in Figure 1 ;

Figure 3 is a schematic perspective view of the island type dressing shown in Figure 2;

Figure 4 is a schematic perspective view of the island type dressing shown in Figure 2 where the signalling layer of the indicator is producing a detectable visual signal;

Figure 5 is a cross-sectional view of a polyurethane type dressing comprising the indicator according to the invention which is shown in Figure 1 ;

Figure 6 is a perspective view of the polyurethane type dressing shown in Figure 5.

Figure 1 shows a first embodiment of an indicator according to the invention. The indicator 6 comprises a signalling layer 8 in the form of a disc of coloured planar inert polymer net 8, here a red high-density polyethylene net (Smith & Nephew Extruded Films, 21 g/m2, gauge 120 microns) under a coterminous sacrificial layer 7 in the form of a planar biopolymer membrane 7, here an opaque chitosan layer. Chitosan which is >50% deacetylated chitin is used. This and higher levels of de-acetylation produce a material having the required mechanical and biochemical characteristics, namely a material which is strong enough to handle but which will degrade in the presence of lysozyme.

The upper face of the signalling layer net disc 8 is covered by the biopolymer layer 7. In use, the net 8 is placed in contact with microbially infected wound fluid containing lysozyme, with the opaque chitosan sacrificial layer 7 uppermost.

The lysozyme in the wound dissolves the chitosan sacrificial layer 7, exposing the red colour of the net signalling layer 8 when viewed from distally of the wound, thereby detecting and revealing the presence of lysozyme, and hence microbial infection in the wound. .

Figures 2 to 4 show a dressing 9 comprising an indicator 6 according to the invention, which has been applied to a wound. It is an island wound dressing 9 which comprises a transparent backing layer top sheet 10, here a thin polyurethane layer overlying the indicator 6 according to the invention.

The indicator 6 comprises a signalling layer 8, here an orange high-density polypropylene mesh below a biopolymer, here chitosan, sheet sacrificial layer 7.

The backing layer 10 is provided with an application layer 16 (not shown) of cardboard, that is sufficiently stiff to support the dressing indicator 6 and to give it structural rigidity during application of the dressing 9, but is removed after application of the dressing 9.

A conventional wound-facing layer 1 1 , here a gauze pad having one or more plies and which is permeable by the substance or microbe that is to be detected encloses the indicator 6 on the underside of the backing layer 10, to which the layer 1 1 is adhered.

The signalling layer 8 of the indicator 6 is visible through the transparent backing sheet 10 of the dressing 9 when the substance or microbe has permeated through the signalling layer 8 and has eroded enough of the chitosan sacrificial layer 7 for the signalling layer to be visible.

The dressing 9 may take other forms known in the art. For example, the backing layer 10 could be a woven or textile fabric cloth, having one or more plies formed from yarns, and having a transparent window 14 (not shown) over the indicator 6, and any or all of the indicator 6 layers 7, 8 and the dressing pad 1 1 may be a textile fabric that may be interwoven, interlaced or knitted, or bound, sewn, or stitched into or onto the backing layer 1 1 . .

Figures 5 and 6 show a polyurethane dressing 12 which comprises a backing layer top sheet 13, here a polyurethane layer enclosing an indicator 6 according to the invention. The indicator 6 comprises a signalling layer 8, here a green high-density polyethylene permeable web below a biopolymer, here chitosan, sheet sacrificial layer 7. The backing layer 1 1 is provided with an application layer 16 (not shown) of cardboard, that is sufficiently stiff to support the dressing indicator 6 and to give it structural rigidity during application of the dressing 9, but is removed after application of the dressing 9. The backing layer 13 has a window 14 underneath which is placed the indicator 1 which comprises signalling layer 2 and biopolymer layer 3. Again, in use, the signalling layer 8 is visually exposed by erosion or change in composition of the sacrificial layer 7.

The present invention is further illustrated by the following Examples, which are not intended to limit the scope of the present invention.

Examples 1 - Preparation of sacrificial layers (a)

Example 1 a - Formation of a sacrificial layer (a): A chitosan acetate membrane (poly (D-glucosamine acetate, 60% deacetylated chitin)

A homogenous slurry of 1 % (w/w) purified 60% chitosan was prepared in 25% acetic acid by continuous stirring overnight at 20°C. 25g of slurry was laid out in a Petri dish and dried in an oven a 30°C for 3 days. The dried sheet of chitosan acetate was washed in absolute ethanol for one hour, washed in distilled water for 1 hr, and then dried.

Example 1 b - Formation of a sacrificial layer (a): A chitosan membrane (poly (D-glucosamine, 60% deacetylated chitin)

A homogenous slurry of 1 % (w/w) purified 60% chitosan was prepared in 25% acetic acid by continuous stirring overnight at 20°C. 25g of slurry was laid out in a Petri dish and dried in an oven a 30°C for 3 days. The dried sheet was washed in absolute ethanol for one hour, washed in sodium hydroxide 2% v/w, washed in distilled water for 1 hr, and then dried.

Example 1 c - Formation of a sacrificial layer (a): Chitosan acetate membranes (poly (D-glucosamine acetate, >60% deacetylated chitin)

Several homogenous slurries of 1 % (w/w) purified chitosan, each a deacetylated chitin (degree of deacylation > 60%) were prepared in acetic acid (at several concentrations between 1 and 95% w/w) by continuous stirring overnight at 20°C. 25g of each slurry was laid out in a Petri dish and dried in an oven a 30°C for 3 days. The dried sheet of chitosan acetate was washed in absolute ethanol for one hour, washed in distilled water for 1 hr, and then dried.

Example 1 d - Formation of a sacrificial layer (a): Chitosan membranes (poly (D-glucosamine, >60% deacetylated chitin)

Several homogenous slurries of 1 % (w/w) purified chitosan, each a deacetylated chitin (degree of deacylation > 60%) were prepared in 25% acetic acid by continuous stirring overnight at 20°C. 25g of each slurry was laid out in a Petri dish and dried in an oven a 30°C for 3 days. The dried sheet was washed in

absolute ethanol for one hour, washed in sodium hydroxide 2% v/w, washed in distilled water for 1 hr, and then dried.

Example 1 e - Formation of a coloured sacrificial layer (a): A chitosan acetate membrane (poly (D-glucosamine acetate, 60% deacetylated chitin)

A 1 % (w/v) solution of chitosan in 1 % (v/v) acetic acid was prepared and left stirring for 24 hours using a magnetic stirrer at room temperature. 5% (v/v) red food dye was added to the chitosan slurry with stirring. Perspex sheets (230 x 300mm, 150 x 150mm and 250 x 250mm) were cleaned using hexane, IPA wipes and rinsing with distilled water. Mastic was used to line the edges of each Perspex sheet to form a peripheral dam to contain, and ensure no leaking of, the chitosan slurry when it was poured onto each sheet. The slurry was poured onto each Perspex sheet at a loading of 2318.84g/m2 of chitosan slurry, left to settle for 5 min, and then each coated sheet was placed in a level oven for 24 hours at 50°C. The films were carefully peeled away from the Perspex sheets and stored at room temperature.

Example 1f - Formation of a coloured sacrificial layer (a): A chitosan membrane (poly (D-glucosamine, 60% deacetylated chitin)

A 1 % (w/v) solution of chitosan in 1 % (v/v) acetic acid was prepared and left stirring for 24 hours using a magnetic stirrer at room temperature. 5% (v/v) orange food dye was added to the chitosan slurry with stirring. Perspex sheets (230 x 300mm, 150 x 150mm and 250 x 250mm) were cleaned using hexane, IPA wipes and rinsing with distilled water. Mastic was used to line the edges of each Perspex sheet to form a peripheral dam to contain, and ensure no leaking of, the chitosan slurry when it was poured onto each sheet. The slurry was poured onto each Perspex sheet at a loading of 2318.84g/m2 of chitosan slurry, left to settle for 5 min, and then each coated sheet was placed in a level oven for 24 hours at 50°C.

A 2% (w/v) NaOH solution was poured onto each dried chitosan film, and each Perspex sheet bearing a film was gently shaken for 1 hour at room temperature, and then washed several times with distilled water. The chitosan films on the Perspex sheets were placed back in the oven for 24 hours at 50°C to dry. The films were carefully peeled away from the Perspex sheets and stored at room temperature.

Example 1 g - Formation of a coloured sacrificial layer (a): Chitosan acetate membranes (poly (D-glucosamine acetate, >60% deacetylated chitin)

A 1 % (w/v) solution of chitosan in 1 % (v/v) acetic acid was prepared and left stirring for 24 hours using a magnetic stirrer at room temperature. 5% (v/v) green food dye was added to the chitosan slurry with stirring. Perspex sheets (230 x 300mm, 150 x 150mm and 250 x 250mm) were cleaned using hexane, IPA wipes and rinsing with distilled water. Mastic was used to line the edges of each Perspex sheet to form a peripheral dam to contain, and ensure no leaking of, the chitosan slurry when it was poured onto each sheet. The slurry was poured onto each Perspex sheet at a loading of 2318.84g/m2 of chitosan slurry, left to settle for 5 min, and then each coated sheet was placed in a level oven for 24 hours at 50°C. The films were carefully peeled away from the Perspex sheets and stored at room temperature.

Example 1 h - Formation of a coloured sacrificial layer (a): Chitosan membranes (poly (D-glucosamine, >60% deacetylated chitin)

A 1 % (w/v) solution of chitosan in 1 % (v/v) acetic acid was prepared and left stirring for 24 hours using a magnetic stirrer at room temperature. 5% (v/v) blue food dye was added to the chitosan slurry with stirring. Perspex sheets (230 x 300mm, 150 x 150mm and 250 x 250mm) were cleaned using hexane, IPA wipes and rinsing with distilled water. Mastic was used to line the edges of each Perspex sheet to form a peripheral dam to contain, and ensure no leaking of, the chitosan slurry when it was poured onto each sheet. The slurry was poured onto each Perspex sheet at a loading of 2318.84g/m2 of chitosan slurry, left to settle for 5 min, and then each coated sheet was placed in a level oven for 24 hours at 50°C.

A 2% (w/v) NaOH solution was poured onto each dried chitosan film, and each Perspex sheet bearing a film was gently shaken for 1 hour at room temperature, and then washed several times with distilled water. The chitosan films on the Perspex sheets were placed back in the oven for 24 hours at 50°C to dry. The films were carefully peeled away from the Perspex sheets and stored at room temperature.

Example 1 i - Formation of a coloured sacrificial layer (a): Chitosan acetate membranes (poly (D-glucosamine acetate, 60% deacetylated chitin)

A 1 % (w/v) solution of chitosan in 1 % (v/v) acetic acid was prepared and left stirring for 24 hours using a magnetic stirrer at room temperature. 5% (v/v) titanium white powder was added to the chitosan slurry with stirring. Perspex sheets (230 x 300mm, 150 x 150mm and 250 x 250mm) were cleaned using hexane, IPA wipes and rinsing with distilled water. Mastic was used to line the edges of each Perspex sheet to form a peripheral dam to contain, and ensure no leaking of, the chitosan slurry when it was poured onto each sheet. The slurry was poured onto each Perspex sheet at a loading of 2318.84g/m2 of chitosan slurry, left to settle for 5 min, and then each coated sheet was placed in a level oven for 24 hours at 50°C. The films were carefully peeled away from the Perspex sheets and stored at room temperature.

Example 1 j - Formation of a coloured sacrificial layer (a): Chitosan membranes (poly (D-glucosamine, >60% deacetylated chitin)

A 1 % (w/v) solution of chitosan in 1 % (v/v) acetic acid was prepared and left stirring for 24 hours using a magnetic stirrer at room temperature. 5% (v/v) titanium yellow powder was added to the chitosan slurry with stirring. Perspex sheets (230 x 300mm, 150 x 150mm and 250 x 250mm) were cleaned using hexane, IPA wipes and rinsing with distilled water. Mastic was used to line the edges of each Perspex sheet to form a peripheral dam to contain, and ensure no leaking of, the chitosan slurry when it was poured onto each sheet. The slurry was poured onto each Perspex sheet at a loading of 2318.84g/m2 of chitosan slurry, left to settle for 5 min, and then each coated sheet was placed in a level oven for 24 hours at 50°C.

A 2% (w/v) NaOH solution was poured onto each dried chitosan film, and each Perspex sheet bearing a film was gently shaken for 1 hour at room temperature, and then washed several times with distilled water. The chitosan films on the Perspex sheets were placed back in the oven for 24 hours at 50°C to dry. The films were carefully peeled away from the Perspex sheets and stored at room temperature.

Examples 2 - Preparation of indicators

Example 2a - Formation of an indicator comprising a layer (a) on layer (b): A chitosan acetate membrane (poly (D-glucosamine acetate, 60% deacetylated chitin) on a high-density polyethylene net

The sheet of Example 1 a was placed over a red-coloured high-density polyethylene net (Smith & Nephew Extruded Films, 21 g/m2 gauge 120 micron) to form an indicator.

Example 2b - Formation of an indicator comprising a layer (a) on layer (b): A chitosan membrane (poly (D-glucosamine, 60% deacetylated chitin) on a high-density polyethylene net

The sheet of Example 1 b was placed over a red-coloured high-density polyethylene net (Smith & Nephew Extruded Films, 21 g/m2 gauge 120 microns) to form an indicator.

Example 2c - Formation of indicators comprising a layer (a) on layer (b): Chitosan acetate membranes (poly (D-glucosamine acetate, >60% deacetylated chitin) on high-density polyethylene net

The sheets of Example 1 c were each placed over a red-coloured high-density polyethylene net (Smith & Nephew Extruded Films, 21 g/m2 gauge 120 microns) to form an indicator.

Example 2d - Formation of indicators comprising a layer (a) on layer (b): Chitosan membranes (poly (D-glucosamine, >60% deacetylated chitin) on high-density polyethylene net

The sheets of Example 1 d were each placed over a red-coloured high-density polyethylene net (Smith & Nephew Extruded Films, 21 g/m2 gauge 120 microns) to form an indicator.

Example 2e - Formation of an indicator comprising a layer (a) on layer (b): A chitosan acetate membrane (poly (D-glucosamine acetate, 60% deacetylated chitin) on a high-density polyethylene net

25g of the slurry of Example 1 a is laid out in a Petri dish over a red-coloured high-density polyethylene net (Smith & Nephew Extruded Films, 21 g/m2 gauge 120 microns) and dried in an oven a 30°C for 3 days to form an indicator.

The dried indicator is washed in absolute ethanol for one hour, washed in distilled water for 1 hr, and then dried.

Example 2f - Formation of an indicator comprising a layer (a) on layer (b): A chitosan membrane (poly (D-glucosamine, 60% deacetylated chitin) on a high-density polyethylene net

25g of the slurry of Example 1 b is laid out in a Petri dish over a red-coloured high-density polyethylene net (Smith & Nephew Extruded Films, 21 g/m2 gauge 120 microns) and dried in an oven a 30°C for 3 days to form an indicator. The dried indicator is washed in absolute ethanol for one hour, washed in sodium hydroxide 2% v/w, washed in distilled water for 1 hr, and then dried.

Example 2g - Formation of indicators comprising a layer (a) on layer (b): Chitosan acetate membranes (poly (D-glucosamine, >60% deacetylated chitin) on high-density polyethylene net

25g of each slurry of Example 1 c is laid out in a Petri dish over a red-coloured high-density polyethylene net (Smith & Nephew Extruded Films, 21 g/m2 gauge 120 microns) and dried in an oven a 30°C for 3 days to form an indicator. The dried indicator is washed in absolute ethanol for one hour, washed in distilled water for 1 hr, and then dried.

Example 2h - Formation of indicators comprising a layer (a) on layer (b): Chitosan membranes (poly (D-glucosamine, >60% deacetylated chitin) on high-density polyethylene net

25g of each slurry of Example 1 d is laid out in a Petri dish over a red-coloured high-density polyethylene net (Smith & Nephew Extruded Films, 21 g/m2 gauge 120 microns) and dried in an oven a 30°C for 3 days to form an indicator. The dried indicator is washed in absolute ethanol for one hour, washed in sodium hydroxide 2% v/w, washed in distilled water for 1 hr, and then dried.

Example 2i - Formation of an indicator comprising a coloured layer (a) on layer (b): A chitosan acetate membrane (poly (D-glucosamine acetate, 60% deacetylated chitin) on a high-density polyethylene net

The red films of Example 1 e are each placed over a green-coloured high-density polyethylene net (21 g/m2 gauge 120 micron) to form an indicator.

Example 2j - Formation of an indicator comprising a coloured layer (a) on layer (b): A chitosan membrane (poly (D-glucosamine, 60% deacetylated chitin) on a high-density polyethylene net

The orange films of Example 1f are each placed over a blue-coloured high-density polyethylene net (21 g/m2 gauge 120 microns) to form an indicator.

Example 2k - Formation of indicators comprising a coloured layer (a) on layer (b): Chitosan acetate membranes (poly (D-glucosamine acetate, >60% deacetylated chitin) on high-density polyethylene net

The green films of Example 1 g are each placed over a red-coloured high-density polyethylene net (Smith & Nephew Extruded Films, 21 g/m2 gauge 120 microns) to form an indicator.

Example 2I - Formation of indicators comprising a coloured layer (a) on layer (b): Chitosan membranes (poly (D-glucosamine, >60% deacetylated chitin) on high-density polyethylene net

The blue films of Example 1 h are each placed over an orange-coloured high-density polyethylene net (21 g/m2 gauge 120 microns) to form an indicator.

Example 2m - Formation of indicators comprising a coloured layer (a) on layer (b): Chitosan acetate membranes (poly (D-glucosamine acetate, >60% deacetylated chitin) on high-density polyethylene net

The white films of Example 1 g are each placed over a red-coloured high-density polyethylene net (Smith & Nephew Extruded Films, 21 g/m2 gauge 120 microns) to form an indicator.

Example 2n - Formation of indicators comprising a coloured layer (a) on layer (b): Chitosan membranes (poly (D-glucosamine, >60% deacetylated chitin) on high-density polyethylene net

The yellow films of Example 1 g are each placed over a purple-coloured high-density polyethylene net (21 g/m2 gauge 120 microns) to form an indicator.

Example 2o - Formation of an indicator comprising a coloured layer (a) on layer (b): A chitosan acetate membrane (poly (D-glucosamine acetate, 60% deacetylated chitin) on a high-density polyethylene net

25g of the red slurry of Example 1 e is laid out in a Petri dish over a green-coloured high-density polyethylene net (21 g/m2 gauge 120 microns) and dried in an oven a 30°C for 3 days to form an indicator. The dried indicator is washed in absolute ethanol for one hour, washed in distilled water for 1 hr, and then dried.

Example 2p - Formation of an indicator comprising a layer (a) on layer (b): A chitosan membrane (poly (D-glucosamine, 60% deacetylated chitin) on a high-density polyethylene net

25g of the orange slurry of Example 1f is laid out in a Petri dish over a blue-coloured high-density polyethylene net (21 g/m2 gauge 120 microns) and dried in an oven a 30°C for 3 days to form an indicator. The dried indicator is washed in absolute ethanol for one hour, washed in sodium hydroxide 2% v/w, washed in distilled water for 1 hr, and then dried.

Example 2q - Formation of indicators comprising a layer (a) on layer (b): Chitosan acetate membranes (poly (D-glucosamine acetate, >60% deacetylated chitin) on high-density polyethylene net

25g of the green slurry of Example 1 g is laid out in a Petri dish over a red-coloured high-density polyethylene net (Smith & Nephew Extruded Films, 21 g/m2 gauge 120 microns) and dried in an oven a 30°C for 3 days to form an indicator. The dried indicator is washed in absolute ethanol for one hour, washed in distilled water for 1 hr, and then dried.

Example 2r - Formation of indicators comprising a layer (a) on layer (b): Chitosan membranes (poly (D-glucosamine, >60% deacetylated chitin) on high-density polyethylene net

25g of the blue slurry of Example 1 h is laid out in a Petri dish over an orange-coloured high-density polyethylene net (21 g/m2 gauge 120 microns) and dried in an oven a 30°C for 3 days to form an indicator. The dried indicator is washed in absolute ethanol for one hour, washed in sodium hydroxide 2% v/w, washed in distilled water for 1 hr, and then dried.

Example 2s - Formation of indicators comprising a layer (a) on layer (b): Chitosan acetate membranes (poly (D-glucosamine acetate, >60% deacetylated chitin) on high-density polyethylene net

25g of the white slurry of Example 1 g is laid out in a Petri dish over a red-coloured high-density polyethylene net (Smith & Nephew Extruded Films, 21 g/m2 gauge 120 microns) and dried in an oven a 30°C for 3 days to form an indicator. The dried indicator is washed in absolute ethanol for one hour, washed in distilled water for 1 hr, and then dried.

Example 2r - Formation of indicators comprising a layer (a) on layer (b): Chitosan membranes (poly (D-glucosamine, <60% deacetylated chitin) on high-density polyethylene net

25g of the yellow slurry of Example 1 h is laid out in a Petri dish over a purple-coloured high-density polyethylene net (21 g/m2 gauge 120 microns) and dried in an oven a 30°C for 3 days to form an indicator. The dried indicator is washed in absolute ethanol for one hour, washed in sodium hydroxide 2% v/w, washed in distilled water for 1 hr, and then dried.

Example 2u - Formation of indicators comprising a layer (a) on layer (b): Chitosan and derivatives thereof on high-density polyethylene net

Correspondingly coloured high-density polypropylene, ABS, polystyrene, polycarbonate, PVC, thermoplastic elastomers, and polyesters are used instead of high-density polyethylene for the net in the indicators of Examples 2a to 2t.

Example 3 - Visual indication of the presence of lysozyme.

Some control uninfected wound fluid was placed in a Petri dish, and in another was placed infected wound fluid containing lysozyme, and a sample of the indicator of Example 2a was placed on top of the fluid in each dish with the chitosan acetate sacrificial layer (a) uppermost.

The control indicator showed no change. However, the lysozyme in the other sample dissolved the chitosan acetate over 1 hour, exposing the red colour of the net signalling layer (b), thereby detecting and revealing the presence of lysozyme, and hence microbial infection in the second sample.

The test is repeated with the indicators of Examples 2b to 2t, each being placed on top of the fluid in each dish with the sacrificial layer (a) uppermost, with similar results.