WO2014125237A1

WO2014125237A1 - Novel biosensor

Info

Publication number: WO2014125237A1
Application number: PCT/GB2013/050318
Authority: WO
Inventors: Paul Davis; Jan Jezek; Andrew Austin
Original assignee: Microarray Limited
Priority date: 2013-02-12
Filing date: 2013-02-12
Publication date: 2014-08-21

Abstract

This invention relates to a biosensor for determining the presence of an analyte in a wound exudate, the biosensor comprising a sensing means and a hydrogen peroxide indicator means, wherein the sensing means comprises an aqueous composition comprising an oxidase enzyme and two or more protective substances.

Description

Novel Biosensor

This application is a continuation-in-part of international patent application no.

PCT/GB2012/051944 filed on 10 August 2012. International patent application no.

PCT/GB2012/051944 claimed priority from United Kingdom patent application no. 1113880.7 filed on 12 August 2011.

This invention relates to improvements to biosensors containing proteins such as

oxidoreductases, for example oxidase and/or peroxidase enzymes.

Background to the invention

Products in the healthcare market, particularly products involving contact with broken skin such as wound dressings, require sterilisation to avoid being a source of infection to the individual being treated. Other products that fall into this category include biosensors which may be used to provide readings concerning the contents of biological fluids such as wound exudates. In certain instances these biosensors are integrated into composite wound dressings although they may be a separate article of manufacture.

Advanced biosensors typically contain enzymes which catalyse reactions involved in a diagnostic or reporting step. Whereas it would be appropriate to sterilise such biosensors using ionising radiation, unfortunately many proteins in an aqueous environment exhibit a high sensitivity to ionising radiation leading to loss of enzymatic activity. Thus, sterilising protein containing biosensors using ionising radiation is a challenge.

The use of organic additives to stabilise proteins from the adverse effect of ionising radiation in other contexts has been described. For example, WO03/026704 (Clearant) describes the use of a multitude of organic substances to stabilise biological material such as bone products and collagen as well as anti-insulin monoclonal immunoglobulin and Factor VIII. Ascorbate, which is a strong reducing agent, appears to be particularly favoured. In general the issues involved in stabilising biological materials are not the same as those involving stabilising purified proteins. Moreover biosensors typically contain reagents in addition to proteins which have to be compatible with the stabilising additives. For example, components of biosensors commonly rely on redox reactions which rules out the inclusion of strong oxidising or reducing agents as stabilising additives. WO2007/034198 (Insense) discloses the use of substances which are hydroxyl radical quenchers for stabilising proteins in an aqueous environment.

WO2007/003936 (Insense) discloses aqueous compositions comprising a protein and one or more stabilising agents which compositions are relatively stable to storage. This document does not make any reference to stability to ionising radiation

A specific type of biosensor of interest in the art is a lactate biosensor which can report on the level of lactate present in wound exudate. Recent developments have shown that localised lactate production in wounds reflects processes and conditions that affect wound healing. High levels of lactate (e.g. above 18 mM) are known to be deleterious to wound healing whilst levels around 3 mM are helpful.

Lactase oxidase catalyses the reaction of lactate with oxygen to form pyruvate and hydrogen peroxide. A lactate biosensor based on the lactase oxidase catalysed reaction of lactate to form hydrogen peroxide which may be detected using a hydrogen peroxide indicator means is described in WO2012/120293 (Archimed). Hydrogen peroxide may be determined taking advantage of reactions catalysed by peroxidase enzymes which are also sensitive to ionising radiation.

Other oxidase enzymes are known. For example urate oxidase catalyses the reaction of urate with oxygen to form 5-hydroxyisourate and hydrogen peroxide. Glucose oxidase catalyses the reaction of glucose with oxygen to form 5- D-glucono-5-lactone and hydrogen peroxide. The class of amino acid oxidases includes glutamate oxidase which catalyses the reaction of glutamate with oxygen to form 2-oxoglutarate and hydrogen peroxide. Another example of an amino acid oxidase is glycine oxidase, in the class of deaminating oxidases. Glycine oxidase catalyses the reaction of glycine with oxygen and water to form glyoxylate and hydrogen peroxide.

It is useful to determine urate in wound exudate because it has been reported that urate accumulation in the fluid of non-healing wounds correlates with delayed healing. It is well known in the art that urate is elevated in the blood of patients with diseases that involve inflammation and ischaemia (Fernandez et al, Int Wound J. 2012, 9(2): 139-49). It has also been reported that sodium urate crystals can be deposited in wounds which generate an undesirable inflammatory response and inhibit wound healing. It is also useful to determine glucose in wound exudates because it has been reported that high glucose levels in wounds lead to inhibition of focal adhesion kinases which mediate wound healing. Moreover, very low levels of glucose (for example in a wound tending to be relatively dry) can result in a lack of healing activity from the cells responsible for the healing process, due to a deficiency in the energy-generating metabolism depending on glucose.

The present invention takes advantage of the usefulness of such oxidase enzymes in biosensors for determining analytes in wound exudates.

We have now invented an improvement to biosensors thereby to facilitate their stabilisation to ionising radiation with improved efficacy, convenience or compatibility as compared with the prior art. Summary of the invention

According to a first aspect of the invention there is provided a biosensor for determining the presence of an analyte in a wound exudate, the biosensor comprising a sensing means and a hydrogen peroxide indicator means, wherein the sensing means comprises an aqueous composition comprising an oxidase enzyme, which enzyme is capable of catalysing oxidation of the analyte present in the wound exudate to produce hydrogen peroxide, and two or more protective substances, the first protective substance being the anion of an organic carboxylic acid not being an amino acid and having a rate of reaction with hydroxyl radicals of greater than 10⁹ Lmol ^~1s^~1 and the second protective substance, different to the first protective substance, being selected from the list consisting of phenylalanine, tryptophan, tyrosine, nicotinate, purine, methionine and malate, provided that none of the protective substances is the analyte itself, such that upon contact of the sensing means with wound exudate, the analyte if present is oxidised to form hydrogen peroxide by the action of the oxidase enzyme and the hydrogen peroxide so produced triggers the hydrogen peroxide indicator means to indicate the presence of the analyte in the wound exudate.

According to a further aspect of the invention there is provided a process for sterilising a biosensor according to the invention which comprises irradiating a biosensor according to the invention with ionising radiation. There is also provided a sterilised biosensor.

According to a further aspect of the invention there is provided a method of stabilising a biosensor for determining the presence of an analyte in a wound exudate, the biosensor comprising a sensing means and a hydrogen peroxide indicator means, wherein the sensing means comprises an aqueous composition comprising an oxidase enzyme, which enzyme is capable of catalysing oxidation of the analyte present in the wound exudate to produce hydrogen peroxide, such that upon contact of the sensing means with wound exudate, the analyte if present is oxidised to form hydrogen peroxide by the action of the oxidase enzyme and the hydrogen peroxide so produced triggers the hydrogen peroxide indicator means to indicate the presence of the analyte in the wound exudate, said method comprising including said composition two or more protective substances, the first protective substance being the anion of an organic carboxylic acid not being an amino acid and having a rate of reaction with hydroxyl radicals of greater than 10⁹ Lmol ^"1s^"1 and the second protective substance, different to the first protective substance, being selected from the list consisting of phenylalanine, tryptophan, tyrosine, nicotinate, purine, methionine and malate, provided that none of the protective substances is the analyte itself.

The biosensor is protected from experiencing loss in activity on exposure to ionising radiation as compared with a similar biosensor not containing the two or more protective substances.

Other aspects of the invention will be apparent from the following disclosure. Brief description of the Figure

Figure 1 shows an example biosensor according to an aspect of the invention.

Detailed description of the invention

Suitably the wound is a wound on the skin of an animal such as a human or lifestock animal (e.g. a ruminant animal) or horse, suitably a human.

In an embodiment of the invention the biosensor is a lactate biosensor i.e. the analyte is lactate. In this embodiment, the oxidase enzyme is lactate oxidase. None of the protective substances is lactate.

In the case of a lactate biosensor the wound may, for example, be a diabetic foot ulcer.

In an embodiment of the invention the biosensor is a urate biosensor i.e. the analyte is urate. In this embodiment, the oxidase enzyme is urate oxidase. None of the protective substances is urate.

In an embodiment of the invention the biosensor is a glucose biosensor i.e. the analyte is glucose. In this embodiment, the oxidase enzyme is glucose oxidase. None of the protective substances is glucose.

In an embodiment of the invention the biosensor is an amino acid biosensor i.e. the analyte is an amino acid. In this embodiment, the oxidase enzyme is an amino acid oxidase. None of the protective substances is an amino acid.

For example, the biosensor is a glutamate biosensor i.e. the analyte is glutamate. In this embodiment, the oxidase enzyme is glutamate oxidase. None of the protective substances is glutamate.

For example, the biosensor is a glycine biosensor i.e. the analyte is glycine. In this

embodiment, the oxidase enzyme is glycine oxidase. None of the protective substances is glycine.

For example, the biosensor is a D-lactate biosensor i.e. the analyte is D-lactate. Such a biosensor is expected to be especially useful for determining D-lactate in wounds of ruminant animals. In this embodiment, the oxidase enzyme is D-lactate oxidase. None of the protective substances is D-lactate.

Certain oxidase enzymes require cofactors for activity in which case such cofactors will also be present in the composition. Example cofactors include flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide (NAD). For example, glycine oxidase requires FAD as cofactor.

Suitably the oxidase enzyme is present in fully hydrated condition. In one embodiment of the invention, the composition contains the anion of an organic carboxylic acid not being an amino acid having a rate of reaction with hydroxyl radicals of greater than 10⁹ Lmol ^"V¹ as the first protective substance. In such compositions of the invention the organic carboxylic acid as first protective substance may be a monocarboxylic acid. Alternatively it may be a dicarboxylic acid. The organic carboxylic acid may, for example, be selected from the group consisting of lactic acid, nicotinic acid, malic acid, benzoic acid, cinnamic acid, folic acid, salicylic acid and phthalic acid. For use in a lactate biosensor the first carboxylic acid is not lactic acid.

The rate of reaction with hydroxyl radicals of a substance may for example be measured by pulsed radiolysis under the conditions described in Buxton G.V, Greenstock C.L, Helman W.P. and Ross A.B. J. Phys. Chem. Ref. Data. 17: 513-886 (1988).

In one embodiment of the invention, the composition contains lactate as first protective substance. In another embodiment of the invention, the composition contains nicotinate as first protective substance. In another embodiment of the invention, the composition contains malate as first protective substance.

Suitably the first protective substance has a rate of reaction with hydroxyl radicals of greater than 10¹⁰ Lmol ^"V¹.

In one embodiment the first protective substance is methionine. Mixtures of enantiomers including racemic mixtures may be used, or a single enantiomeric form may be used, especially the L enantiomer.

Suitably the first protective substance is not a strong reducing agent such as ascorbate or an organic carboxylic acid with a free thiol group. Suitably the first protective substance is not an oxidising agent, for example suitably it is incapable of oxidising iodide to iodine under the conditions of the particular application.

Suitably the concentration of the first protective compound is 5 to 200 mM and more suitably in the range 10 to 100 mM, e.g. 10 to 50 mM for example 10 mM, 25 mM or 50 mM.

In an embodiment, the second protective substances is selected from the list consisting of phenylalanine, tryptophan, tyrosine, nicotinate, methionine and malate.

In one embodiment of the invention, the composition comprises nicotinate as second protective compound.

In another embodiment of the invention, the second protective compound is selected from phenylalanine, tryptophan and tyrosine. Mixtures of enantiomers including racemic mixtures may be used, or a single enantiomeric form may be used, especially the L enantiomer. In another embodiment of the invention, the composition contains tryptophan as second protective compound. In another embodiment of the invention, the composition contains methionine as second protective compound.

Suitably the concentration of the second protective compound is 5 to 200 mM and more suitably in the range 10 to 100 mM, e.g. 10 to 50 mM for example 10 mM, 25 mM or 50 mM. Thus a composition for use according to the invention may comprise (i) nicotinate and methionine; or (ii) lactate and phenylalanine; or (iii) nicotinate and tryptophan; or (iv) lactate and nicotinate; or (v) nicotinate and purine; or (vi) malate and nicotinate; or (vii) methionine and tryptophan.

In further embodiments, a composition for use according to the invention may comprise (viii) lactate and tryptophan; or (ix) nicotinate and phenylalanine; or (x) lactate and methionine; or (xi) methionine and an aromatic amino acid selected from phenylalanine, tryptophan and tyrosine (e.g. methionine and phenylalanine); or (xii) malate and phenylalanine; or (xiii) malate and tryptophan; or (xiv) lactate and tryptophan.

A composition for use according to the invention may also comprise a third different protective compound selected from the list consisting of phenylalanine tryptophan, tyrosine, nicotinate, purine, methionine and malate.

Thus a composition for use according to the invention may contain (i) lactate, nicotinate and phenylalanine; or (ii) lactate, nicotinate and methionine; or (iii) nicotinate, tryptophan and phenylalanine; or (iv) nicotinate, tryptophan and methionine; or (v) nicotinate, methionine and purine; or (vi) nicotinate, methionine and phenylalanine; or (vii) lactate, methionine and purine or (viii) lactate, nicotinate and tryptophan; or (ix) lactate, tryptophan and purine; or (x) lactate, tryptophan and methionine as first, second and third protective compounds respectively.

Suitably the concentration of the third protective substance is 5 to 200 mM.

In an embodiment, none of the protective substances is an amino acid.

In an embodiment, none of the protective substances is methionine.

Suitably the pH of the composition is 4 to 9 e.g. 4.5 to 8.5. For example, when the composition is a composition containing glucose oxidase the pH is suitably 4.5 to 5.5 e.g. around 5. When the composition is a composition containing lactose oxidase, the pH is suitably around 7 e.g. between pH 6 and 7. When the composition is a composition containing urate oxidase, the pH is suitably around 8 e.g. between pH 7.5 and 8.5. More generally the pH of the composition is within 1 pH unit e.g. within 0.5 pH unit of the pH at which a protein in the composition has maximum stability especially maximal thermal stability during storage. The storage stability is measured with respect to a stability aspect of the protein that is of critical importance for a specific application. For example, enzyme activity, measured by a suitable colorimetric or electrochemical method, may be a critical stability aspect for an enzyme used in a biosensor. In other applications, aggregation of a protein, measured by techniques such as size exclusion chromatography, differential light scattering or light obscuration techniques, may be the critical stability aspect for which optimal conditions are sought. In some cases, more than one stability aspect of a protein is critical for a particular application. The pH at which all critical aspects are best controlled is then sought, which thus becomes the pH of maximal thermal stability.

Ionising radiation that may be used to stabilise a biosensor according to the invention includes gamma radiation, electron beam radiation of X-ray radiation, especially gamma radiation. The total radiation dose may be at least 5 kGy, e.g. at least 15 kGy, e.g. at least 25 kGy e.g. at least 50 kGy. Preferably, the total radiation dose is between 25 to 50 kGy .Stabilisation with ionising radiation may be performed at a temperature of 4 to 40 °C, for example 15 to 30 °C. In an alternative embodiment it may be performed at low temperature e.g. below 4 °C such as below 0 °C such as below -20 °C.

In one embodiment of the invention, the aqueous composition is a hydrated hydrogel.

The oxidase enzyme, the protective substances and the hydrogen peroxide indicator means may, for example, be dissolved in the water together with swelling agent(s) to form the hydrogel. Suitable hydrogels are disclosed in WO03/090800 which is herein incorporated by reference in its entirety. An exemplary hydrogel comprises poly 2-acrylamido-2-methylpropane sulfonic acid or a salt thereof, preferably in an amount of about 20% by weight of the total weight of the gel.

Suitably the hydrogen peroxide indicator means permits the quantitative determination of hydrogen peroxide production. In this way it is possible for the biosensor to determine the concentration of the analyte in the wound exudate.

Suitably the hydrogen peroxide indicator means comprises a chromogenic material, typically together with a peroxidase enzyme. Suitably the chromogenic material comprises iodide. Iodide (e.g. as potassium iodide) is oxidised by hydrogen peroxide to form iodine.

Production of iodine may be determined by the colorimetic change caused on its contact with a complexing agent such as starch or polyvinyl acetate.

In the presence of a complexing agent such as starch or polyvinyl acetate a wide range of bright colours can be generated to provide a visually perceptible indicator. The hydrogen peroxide indicator means may, for example, provide a visual indicator which is in proportion to the concentration of hydrogen peroxide (and thus analyte) in the sensing means.

Accordingly, in an embodiment, the hydrogen peroxide indicator means comprises iodide and starch optionally together with a peroxidase enzyme such as horseradish peroxidase.

When the hydrogen peroxide indicator means comprises iodide then the protective substances must be compatible with iodide and iodine for example they must not be capable of oxidising iodide to iodine or reducing iodine to iodide. In general the protective substances must be compatible with the chromogenic material.

Other chromogenic materials which quantitatively react with hydrogen peroxide will be known to a skilled person. For example, suitable other chromogenic materials which may be employed in the invention are described in EP103288A (herein incorporated by reference in its entirety), see in particular pages 7 and 8.

Suitable peroxidase enzymes may be of plant, animal or bacterial origin and include for example those described in US3983005, US4211845, GB2036963 and JP 57 (1982)-99192 (each of which documents is herein incorporated by reference in its entirety). Peroxidase enzymes are suitably of class E.C.1 1 1.1.7. An example peroxidase enzyme is horseradish peroxidase.

In another embodiment, the hydrogen peroxide indicator means comprises an electrochemical sensor. Electrochemical sensors involve use of an ammeter for determining the current generated in the sensing means by the redox activity of the oxidase enzyme. The biosensor can be provided with electrodes which can be separably attached to an ammeter.

A biosensor according to the invention may be calibrated by reference to samples containing known concentrations of analyte. Thus the biosensor may be packaged together with a colour chart (for use when the indicator means comprises a chromogenic material) or a current chart (for use when the indicator means comprises an electrochemical sensor) which, by

comparison, can allow the concentration of analyte in the wound exudate to be determined. Alternatively a calibrated absorption coefficient may be determined by exposure of the hydrogen peroxide indicator means to light of an appropriate frequency (again for use when the indicator means comprises a chromogenic material). Alternatively the hydrogen peroxide indicator means may comprise one or a plurality of indicator regions each region being responsive to a certain amount or several different certain amounts of hydrogen peroxide. For example each region may comprise a different amount of oxidase enzyme, peroxidase, chromogenic material (e.g. iodide and/or starch) or other reaction condition variable. For example when the sensing means comprises a hydrogel, the indicator regions may comprise a different extent of crosslinking of the hydrogel thus restricting the amount of analyte that may diffuse to each indicator region.

Suitably the biosensor is provided with means for preventing the contact of the sensing means with any catalase in the wound exudate which could interfere with the sensing means by causing the decomposition of hydrogen peroxide. More generally, suitably the biosensor is provided with means for preventing the contact of the sensing means with molecules in the wound exudate which are significantly larger than the analyte. In an embodiment, the biosensor is provided with means for preventing the contact of the sensing means with molecules in the wound exudate of molecular weight greater than 100,000 Da or greater than 50,000 Da or greater than 25,000 Da or greater than 10,000 Da or greater than 1 ,000 Da. Suitably the biosensor is provided with a semi-permeable membrane to protect the sensing means from contact with molecules in the wound exudate which are significantly larger than the analyte. Suitably the semi-permeable membrane has a size cut-off of 100,000 Da or less e.g. 50,000 Da or less e.g. 25,000 Da or less e.g. 10,000 Da or less e.g.1 ,000 Da or less. Suitably the size cut-off is 500 Da or more. Suitably the semi-permeable membrane has a size cut-off in the range 500-25,000 Da e.g. 500-10,000Da e.g. 500-1000 Da i.e. it prevents the passage of molecules having a molecular weight above a weight in these ranges. This prevents the biosensor from giving erroneous readings such as may be caused by other components that may be present in the wound exudate, especially catalase.

Suitably the sensing means comprises a hydrated hydrogel and the means for preventing the ingress of molecules significantly larger than the analyte into the sensing means is provided by the hydrogel.

The hydrogen peroxide indicator means is also desirably protected from catalase in a similar manner. The semi-permeable membrane or the hydrogel referred to above can protect both the sensing means and the hydrogen peroxide indicator means from catalase.

In an embodiment, the biosensor is packaged as (or as a component part of) a wound-dressing suitable for direct application to the wound. According to this embodiment, the wound exudate enters the biosensor directly from the wound. Alternatively the biosensor is not applied to the wound but a sample of wound exudate is extracted from the wound and applied to the biosensor.

The biosensor can be sealed in packaging and comprising a sealed opening which, in use, is exposed and wound exudate introduced to it. When sealed in packaging, the sealed opening is the one and only opening in the packaging. In this embodiment, the biosensor is designed to remain in its packaging during use.

If sealed in packaging, the indicator means will be visible through the packaging.

Suitably the biosensor will be provided with means acting as fluid channel permitting passage of the wound exudate from the sealed opening to the sensing means. Thus, suitably the biosensor according to the invention sealed in packaging comprises a diffusion means situated between the sealed opening and the sensing means.

Suitably the biosensor comprises absorbent wick material which provides a fluid diffusion path from the opening to the sensing means. The absorbent wick material may, for example, comprise a fabric which is dry, partially dry or saturated with water. Suitably the elements of the hydrogen peroxide indicator means are combined with the oxidase enzyme of the sensing means. This means that the hydrogen peroxide produced by the oxidation of the analyte can be determined without need for any significant diffusion of it to any other part of the biosensor.

Since the reaction of the analyte involving the oxidase enzyme requires oxygen, oxygen will be permitted to enter the device from ambient air by diffusion and/or a sufficient concentration of oxygen will be present in the sensing means.

In an aspect of the invention there is provided a biosensor for determining the presence of an analyte in a wound exudate, the biosensor being sealed in packaging and comprising a sealed opening which, in use, is exposed and placed over the wound site, the biosensor further comprising a sensing means and a hydrogen peroxide indicator means, wherein the sensing means comprises an aqueous composition comprising an oxidase enzyme, which enzyme is capable of catalysing oxidation of the analyte present in the wound exudate to produce hydrogen peroxide, and two or more protective substances, the first protective substance being the anion of an organic carboxylic acid not being an amino acid and having a rate of reaction with hydroxyl radicals of greater than 10⁹ Lmol ^"V¹ and the second protective substance, different to the first protective substance, being selected from the list consisting of

phenylalanine, tryptophan, tyrosine, nicotinate, purine, methionine and malate, provided that none of the protective substances is the analyte itself, the biosensor further being provided with means for preventing the contact of the sensing means with molecules in the wound exudate which are significantly larger than the analyte, such that upon contact of the sensing means with wound exudate, the analyte if present is oxidised to form hydrogen peroxide by the action of the oxidase enzyme and the hydrogen peroxide so produced triggers the hydrogen peroxide indicator means to indicate the presence of the analyte in the wound exudate.

In another embodiment the sensing means can further comprise a control region which provides a visual indication of the flow of wound exudate into the sensing means. For example the control region of the sensing means may provide a visual indication of the presence of glucose and may suitably contain glucose oxidase for this purpose. A region upstream of the region containing glucose oxidase is suitably doped with glucose such that ingress of wound exudate into the sensing means causes glucose to come into contact with glucose oxidase leading to production of hydrogen peroxide. Alternatively, the control region of the sensing means may provide a visual indication of the presence of lactate and may suitably contain lactate oxidase for this purpose. A region upstream of the region containing lactate oxidase is suitably doped with lactate such that ingress of wound exudate into the sensing means causes lactate to come into contact with lactate oxidase leading to production of hydrogen peroxide. The hydrogen peroxide produced in these reactions can be detected by means of the hydrogen peroxide indicator mentioned above.

In an embodiment, the sensing means comprises two (or more than two) independent elements, a first for detecting the analyte to be determined and a second (or further) for control or calibration purposes. Each element is accompanied by a hydrogen peroxide indicator means. The second element is adapted to detect the same or a different analyte to that being determined by the first element of the biosensor. The second element is pre-dosed upstream of the region containing the oxidase enzyme with a pre-determined amount of its analyte. The objective is that ingress of wound exudates into the second (or further) element results in a control level indication (e.g. a control depth of colour, or extent of colour change) by the hydrogen peroxide indicator means irrespective of the wound exudate composition. This gives reassurance that the biosensor is working properly. All that is needed is a sufficiency of wound exudate to activate and drive the test process. A positive result in the second (or further) element shows that the biosensor has worked (even if the determination in the first element is negative). The depth of colour or extent of colour change in the second (or further) element can also be used as a comparator against which to judge the concentration of analyte in the wound exudate determined in the first element. When more than one control element is present these can provide a range of comparators to facilitate the determination of the concentration of analyte in the wound exudate determined in the first element.

In an embodiment, the biosensor comprises glucose oxidase. Glucose oxidase may be present because the biosensor contains glucose oxidase as oxidase enzyme in the sensing means of a glucose biosensor or because it is contained within the control region of a biosensor.

According to an embodiment of the invention, the biosensor is a biosensor for more than one analyte (e.g. two or three analytes). This may be achieved by having more than one fluid channel (e.g. more than one diffusion means, such as adsorbent wick material) for wound exudate leading to more than one sensing means suitable for determination of more than one analyte. For example the analytes measured may comprise lactate and urate and two sensing means may be provided which comprise, respectively, lactate oxidase and urate oxidase. For example the analytes measured may be (or include) lactate and glucose and two sensing means may be provided which comprise, respectively, lactate oxidase and glucose oxidase. . For example the analytes measured may be (or include) glucose and urate and two sensing means may be provided which comprise, respectively, glucose oxidase and urate oxidase. . For example the analytes measured may be (or include) lactate, glucose and urate and three sensing means may be provided which comprise, respectively, lactate oxidase, glucose oxidase and urate oxidase. Further details concerning physical features of a biosensor according to the invention may be gleaned from WO2012/120293 (herein incorporated by reference in its entirety) and specifically Figures 1-15 thereof and the associated explanatory text on pages 1 1-16.

Biosensors of the invention may have the advantage that the proteins they contain (or one or more of them) are relatively protected against damage (such as loss of activity, aggregation or chemical modification of the proteins) by ionising radiation used in sterilisation processes as compared with compositions lacking the protective substances. Activity of proteins may be measured using conventional processes. For example enzyme activities may be measured using chromogenic or electrochemical assays. Activity of lactate oxidase may be measured by following the rate of hydrogen peroxide production in a solution of lactate using a chromogenic assay or an electrochemical assay. Activity of glucose oxidase may be measured by following the rate of hydrogen peroxide production in a solution of glucose using a chromogenic assay or an electrochemical assay. Activity of urate oxidase may be measured by following the rate of hydrogen peroxide production in a solution of urate using a chromogenic assay or an electrochemical assay. Activity of amino acid oxidases may be measured by following the rate of hydrogen peroxide production in a solution of the amino acid using a chromogenic assay or an electrochemical assay. Activity of peroxidases e.g. horseradish peroxidase may be measured by measuring the rate of oxidation of a suitable dye (such as tetramethylbenzidine) in the presence of excess hydrogen peroxide using a chromogenic assay.

As used herein, "lactate" means "L-lactate" and lactate oxidase means L-lactate oxidase.

Unless stated otherwise, all amino acids referred to herein are L-amino acids.

The invention will be illustrated by reference to the following non-limiting examples.

Examples

Example 1 - Example lactate biosensor

By reference to Figure 1 , a biosensor 10 is shown sealed in clear transparent packaging 12, and having an opening 14 covered by removable seal 16.

The biosensor 10 comprises a sensing means 18 which comprises a hydrated hydrogel containing lactate oxidase (as oxidase), horseradish peroxidase enzyme (as a component of the hydrogen peroxidase indicator means), two or more protective substances to protect the lactate oxidase and horseradish peroxidase enzymes during sterilisation, iodide as the chromogenic material, and starch.

The biosensor also comprises semi-permeable membrane 20 which allows the free passage of water, lactate and other low molecular weight solutes but prevents passage of high molecular weight solutes such as enzymes e.g. catalase. The biosensor 10 also comprises an absorbent wick material 22 which provides a fluid diffusion path from the opening 14 to the sensing means 18 and comprises a fabric saturated with water, although many other versions are possible.

In use, the seal 16 is removed and the opening 14 is placed over a wound in the skin of a human or animal subject.

Wound exudate then diffuses into the biosensor through opening 14 and diffuses along the absorbent wick 22. Once at the semi-permeable membrane 20 only the lactate and other low molecular weight solutes continue to diffuse into the hydrogel 18.

Once in the hydrogel the lactate oxidase causes oxidation of the lactate to form hydrogen peroxide. The formed hydrogen peroxide then oxidises the iodide with the action of the peroxidase enzyme to form iodine. The iodine then complexes with the starch which forms a distinctive blue colour. This causes a visual indication in a change of colour of the hydrogel, which is visible through the clear transparent packaging.

Example 2 - Example urate biosensor

The biosensor 10 comprises a sensing means 18 which comprises a hydrated hydrogel containing urate oxidase (as oxidase), horseradish peroxidase enzyme (as a component of the hydrogen peroxidase indicator means), two or more protective substances to protect the urate oxidase and horseradish peroxidase enzymes during sterilisation, iodide as the chromogenic material, and starch.

The biosensor also comprises semi-permeable membrane 20 which allows the free passage of water, urate and other low molecular weight solutes but prevents passage of high molecular weight solutes such as enzymes e.g. catalase.

The biosensor 10 also comprises an absorbent wick material 22 which provides a fluid diffusion path from the opening 14 to the sensing means 18 and comprises a fabric saturated with water, although many other versions are possible.

Wound exudate then diffuses into the biosensor through opening 14 and diffuses along the absorbent wick 22. Once at the semi-permeable membrane 20 only the urate and other low molecular weight solutes continue to diffuse into the hydrogel 18.

Once in the hydrogel the urate oxidase causes oxidation of the urate to form hydrogen peroxide. The formed hydrogen peroxide then oxidises the iodide with the action of the peroxidase enzyme to form iodine. The iodine then complexes with the starch which forms a distinctive blue colour. This causes a visual indication in a change of colour of the hydrogel, which is visible through the clear transparent packaging.

Example 3 - Sterilisation protocol

A biosensor device may be sterilised by means of a gamma-radiation service, of which there are many specialist commercial services to choose from (e.g. Isotron Ltd in the UK). The device is typically placed in an aluminium foil pouch and sealed. At the gamma radiation facility the pouch is placed in a radiation chamber for a period of time decided by a trained and qualified operator under conditions that ensure that the device receives a radiation dose of between 25 and 40 kGy.

Example 4 - Effect of selected excipients of the recovery of aqueous enzyme activity of glucose oxidase following sterilisation by gamma radiation (25 kGy)

Glucose oxidase was used as model for determining the effect of various protective substances on oxidoreductase enzymes.

Aqueous solutions (1 ml) of glucose oxidase (350 μg/ml) were prepared with selected additives in 2 ml glass (Type I) vials and sealed with a crimp top. The vials were gamma-irradiated by an industrial sterilisation service (Isotron Ltd, Swindon, Wilts, UK) using a Cobalt 60 gamma source and a 25 kGy dose. The gamma-irradiated solutions were subsequently tested for glucose oxidase activity. This was performed according to the following procedure:_50 μί of the solution was added to 50 mL of deionised water. The following solutions were then added:

• 10 mL of reagent mix (5 parts of 0.1 M sodium phosphate, pH 6 + 4 parts 2% w/w starch + 1 part of 1 mg/mL lactoperoxidase enzyme);

• 5 mL of 100 mM potassium iodide and

• 5 mL of 40% w/w glucose solution.

These were mixed together quickly. Time = 0 was counted from the addition of the glucose. After 5 min, 1 ml of 5 M aq. hydrochloric acid was added to stop the reaction. The absorbance was then read at 630 nm. If the colour intensity was too great to allow an accurate reading, the sample was diluted with a defined volume of deionised water to bring the colour back on scale. The results were expressed as percentage recovery, by reference to the absorbance measured in the pre-gamma irradiation samples.

The effect of a number of excipients in various combinations on the recovery of glucose oxidase activity was assessed. Results are shown in Table 1 below: Table 1. Activity recovery of glucose oxidase in aqueous formulations following gamma irradiation. All formulations were adjusted to pH 5.0 and contained 50 mM NaCI.

Part 1

Part 2

Excipient Lactate + Lactate + Lactate + Nicotinate + Nicotinate +

Nicotinate + Tryptophan + Methionine + Tryptophan + Methionine +

No excipients 69 65 51 76 88

Lactate

(50 mM)

Nicotinate

(50 mM)

Purine

68 96 99 79 101 (50 mM)

Tryptophan

94

(15 mM)

Phenylalanine

102 78 78 99 100 (25 mM)

Methionine

97 93 102 (50 mM)

Malate (50

mM) As can be seen from Table 1 , the compositions containing two protective substances were more effective than the compositions containing a single protective substances (i.e. inclusion of lactate, methionine or nicotinate improved the protective effect of compositions containing another single protective substance). The combinations of three protective substances were yet more effective.

Throughout the specification and the claims which follow, unless the context requires otherwise, the word 'comprise', and variations such as 'comprises' and 'comprising', will be understood to imply the inclusion of a stated integer, step, group of integers or group of steps but not to the exclusion of any other integer, step, group of integers or group of steps. All patents and patent applications mentioned throughout the specification of the present invention are herein incorporated in their entirety by reference.

The invention embraces all combinations of preferred and more preferred groups and suitable and more suitable groups and embodiments of groups recited above.

Claims

1. A biosensor for determining the presence of an analyte in a wound exudate, the biosensor comprising a sensing means and a hydrogen peroxide indicator means, wherein the sensing means comprises an aqueous composition comprising an oxidase enzyme, which enzyme is capable of catalysing oxidation of the analyte present in the wound exudate to produce hydrogen peroxide, and two or more protective substances, the first protective substance being the anion of an organic carboxylic acid not being an amino acid and having a rate of reaction with hydroxyl radicals of greater than 10⁹ Lmol ^"1s^"1 and the second protective substance, different to the first protective substance, being selected from the list consisting of phenylalanine, tryptophan, tyrosine, nicotinate, purine, methionine and malate, provided that none of the protective substances is the analyte itself, such that upon contact of the sensing means with wound exudate, the analyte if present is oxidised to form hydrogen peroxide by the action of the oxidase enzyme and the hydrogen peroxide so produced triggers the hydrogen peroxide indicator means to indicate the presence of the analyte in the wound exudate.

2. A biosensor according to claim 1 wherein the oxidase enzyme is urate oxidase and which is a biosensor for urate in wound exudate.

3. A biosensor according to claim 1 wherein the oxidase enzyme is lactate oxidase and which is a biosensor for lactate in wound exudate.

4. A biosensor according to claim 1 wherein the oxidase enzyme is glucose oxidase and which is a biosensor for glucose in wound exudate.

5. A biosensor according to claim 1 wherein the oxidase enzyme is glutamate oxidase and which is a biosensor for glutamate in wound exudate.

6. A biosensor according to claim 1 wherein the oxidase enzyme is D-lactate oxidase and which is a biosensor for D-lactate in wound exudate.

7. A biosensor according to any one of claims 1 to 6 wherein the first protective

substance is the anion of an organic carboxylic acid not being an amino acid having a rate of reaction with hydroxyl radicals of greater than 10⁹ Lmol ^"V¹.

8. A biosensor according to claim 7 wherein the organic carboxylic acid is a

monocarboxylic acid.

9. A biosensor according to claim 7 wherein the organic carboxylic acid is selected from the group consisting of lactic acid, nicotinic acid, malic acid, benzoic acid, cinnamic acid, folic acid, salicylic acid and phthalic acid.

10. A biosensor according to any one of claims 1 to 9 wherein the concentration of the first protective compound is 5 to 200 mM.

11. A biosensor according to any one of claims 1 to 10 wherein the concentration of the second protective compound is 5 to 200 mM.

12. A biosensor according to any one of claims 1 to 11 which contains lactate as first protective compound.

13. A biosensor according to any one of claims 1 to 11 which contains nicotinate as first protective compound.

14. A biosensor according to any one of claims 1 to 13 wherein the second protective substance is selected from the list consisting of phenylalanine, tryptophan, tyrosine, nicotinate, methionine and malate.

15. A biosensor according to claim 14 which contains nicotinate as second protective compound.

16. A biosensor according to claim 14 which contains tryptophan as second protective compound.

17. A biosensor according to claim 14 which contains methionine as second protective compound.

18. A biosensor according to any one of claims 1 to 11 wherein the composition

comprises (i) nicotinate and methionine; or (ii) lactate and phenylalanine; or (iii) nicotinate and tryptophan; or (iv) lactate and nicotinate; or (v) nicotinate and purine; or (vi) malate and nicotinate; or (vii) methionine and tryptophan.

19. A biosensor according to any one of claims 1 to 11 wherein the composition

comprises (viii) lactate and tryptophan; or (ix) nicotinate and phenylalanine; or (x) lactate and methionine; or (xi) methionine and an aromatic amino acid selected from phenylalanine, tryptophan and tyrosine (e.g. methionine and phenylalanine); or (xii) malate and phenylalanine; or (xiii) malate and tryptophan; or (xiv) lactate and tryptophan.

20. A biosensor according to any one of claims 1 to 19 wherein the composition

comprises a third different protective compound selected from the list consisting of phenylalanine, tryptophan, tyrosine, nicotinate, purine, methionine and malate.

21. A biosensor according to claim 20 wherein the composition contains (i) lactate, nicotinate and phenylalanine; or (ii) lactate, nicotinate and methionine; or (iii) nicotinate, tryptophan and phenylalanine; or (iv) nicotinate, tryptophan and methionine; or (v) nicotinate, methionine and purine; or (vi) nicotinate, methionine and phenylalanine; or (vii) lactate, methionine and purine or (viii) lactate, nicotinate and tryptophan; or (ix) lactate, tryptophan and purine; or (x) lactate, tryptophan and methionine as first, second and third protective compounds respectively.

22. A biosensor according to any one of claims 1 to 21 wherein the pH of the

composition is 4.5 to 8.5.

23. A biosensor according to any one of claims 1 to 22 wherein the hydrogen peroxide indicator means comprises iodide.

24. A biosensor according to any one of claims 1 to 23 wherein the hydrogen peroxide indicator means comprises starch.

25. A biosensor according to any one of claims 1 to 24 wherein the hydrogen peroxide indicator means comprises a peroxidase enzyme such as horseradish peroxidase.

26. A biosensor according to any one of claims 1 to 25 which is packaged as a wound dressing.

27. A biosensor for determining the presence of an analyte in a wound exudate, the biosensor being sealed in packaging and comprising a sealed opening which, in use, is exposed and placed over the wound site, the biosensor further comprising a sensing means and a hydrogen peroxide indicator means, wherein the sensing means comprises an aqueous composition comprising an oxidase enzyme, which enzyme is capable of catalysing oxidation of the analyte present in the wound exudate to produce hydrogen peroxide, and two or more protective substances, the first protective substance being the anion of an organic carboxylic acid not being an amino acid and having a rate of reaction with hydroxyl radicals of greater than 10⁹ Lmol ^"1s^"1 and the second protective substance, different to the first protective substance, being selected from the list consisting of phenylalanine, tryptophan, tyrosine, nicotinate, purine, methionine and malate, provided that none of the protective substances is the analyte itself, the biosensor further being provided with means for preventing the contact of the sensing means with molecules in the wound exudate which are significantly larger than the analyte, such that upon contact of the sensing means with wound exudate, the analyte if present is oxidised to form hydrogen peroxide by the action of the oxidase enzyme and the hydrogen peroxide so produced triggers the hydrogen peroxide indicator means to indicate the presence of the analyte in the wound exudate.

28. A biosensor according to any one of claims 1 to 27 wherein the sensing means

further comprises a control region which provides a visual indication of the flow of wound exudate into the sensing means.

29. A biosensor according to any one of claims 1 to 27 wherein the sensing means

comprises two or more than two independent elements, a first for detecting the analyte to be determined and a second or further for control or calibration purposes, each element being accompanied by a hydrogen peroxide indicator means.

30. A process for sterilising a biosensor according to any one of claims 1 to 29 which comprises irradiating a biosensor with ionising radiation.

31. A method of stabilising a biosensor for determining the presence of an analyte in a wound exudate, the biosensor comprising a sensing means and a hydrogen peroxide indicator means, wherein the sensing means comprises an aqueous composition comprising an oxidase enzyme, which enzyme is capable of catalysing oxidation of the analyte present in the wound exudate to produce hydrogen peroxide, such that upon contact of the sensing means with wound exudate, the analyte if present is oxidised to form hydrogen peroxide by the action of the oxidase enzyme and the hydrogen peroxide so produced triggers the hydrogen peroxide indicator means to indicate the presence of the analyte in the wound exudate, said method comprising including said composition two or more protective substances, the first protective substance being the anion of an organic carboxylic acid not being an amino acid and having a rate of reaction with hydroxyl radicals of greater than 10⁹ Lmol ^"1s^"1 and the second protective substance, different to the first protective substance, being selected from the list consisting of phenylalanine, tryptophan, tyrosine, nicotinate, purine, methionine and malate, provided that none of the protective substances is the analyte itself.