WO2018141017A1

WO2018141017A1 - Absorbent article with indicator

Info

Publication number: WO2018141017A1
Application number: PCT/AU2018/050066
Authority: WO
Inventors: Anushi Rajapaksa; Will Hughes
Original assignee: Murdoch Childrens Research Institute
Priority date: 2017-02-01
Filing date: 2018-02-01
Publication date: 2018-08-09

Abstract

The present disclosure relates to an absorbent article comprising a liquid permeable bodyside liner; an outer cover; and an absorbent body positioned between the bodyside liner and the outer cover for detecting the presence of bilirubin in urine and stabilised enzymes for use in the same. Bilirubin oxidase may be stabilised by covalently binding to one or more polymers such as polyacrylic acid. Such absorbent articles incorporating said enzymes may be used to detect jaundice in a subject.

Description

ABSORBENT ARTICLE WITH INDICATOR

FIELD OF THE INVENTION

The present disclosure relates to an absorbent article for detecting the presence of bilirubin in urine and stabilised enzymes for use in the same. Such articles and enzymes may be used to detect jaundice in a subject.

BACKGROUND OF THE INVENTION

Jaundice is one of the most common neonatal conditions, with approximately 60% of full-term babies and 80% of pre-term babies developing jaundice within the first two weeks of birth. As a result, a high proportion of incidence of jaundice occurs when the infant is at home, meaning there is often a delay or complete failure to detect jaundice at an early stage. In addition, the failure of screening is particularly prevalent in many developing world settings, where morbidity and mortality due to jaundice is disproportionately high (i.e. approximately 30% of newborn deaths). If left untreated, jaundice can result in severe brain injury or death.

Jaundice is characterised by an increased level of bilirubin in tissue and bodily fluids such as blood and urine. However, despite the availability of effective treatment, an effective screening tool for jaundice in infants is yet to be developed. Screening practice for jaundice currently involves a physical examination of the skin and eyes. This physical examination is particularly unreliable in a subject with darker skin tones, and, as a result, the hallmarks of jaundice may not be visible until bilirubin far exceeds a safe level. Following physical examination, a blood test can be performed to measure serum levels of bilirubin. However, blood tests are invasive, expensive and time- consuming. Blood tests also require a clinical setting which may limit the availability of testing.

Dipstick based testing of urine samples are also used for detecting jaundice in a subject, including infants. These generally use a diazonium salt in an acid medium to produce a colorimetric reaction in the presence of bilirubin in a urine sample. Testing requires a urine sample to be obtained from the subject and may also require a medical practitioner. Diazonium salts have also been incorporated into nappies or diapers in an attempt to detect bilirubin in urine. However, the diazonium salts used are irritating to the skin.

Accordingly, methods of detecting symptoms of jaundice at an early stage are required. SUMMARY OF THE INVENTION

The present inventors have identified that bilirubin oxidase can be used at low levels to visually detect the presence of bilirubin in urine. The inventors also identified that crosslinking bilirubin oxidase in a polymer provides stable compositions with improved capacity to detect bilirubin in urine. Important for consumer goods, such compositions can be incorporated into various fabrics and remain stable for long periods of time. These findings indicate that bilirubin oxidase can be incorporated into various absorbent articles to visually detect the presence of bilirubin in urine.

Accordingly, in an example, the present disclosure relates to an absorbent article, comprising:

a liquid permeable bodyside liner;

an outer cover; and

an absorbent body positioned between the bodyside liner and the outer cover;

wherein the absorbent article comprises a bilirubin oxidase that catalyses a colorimetric reaction visible when contacted with urine containing bilirubin so as to detect the presence of bilirubin in the urine.

In an example, the bilirubin oxidase is from Myrothecium sp., Magnaporthe sp., Pleurotus sp., Trachy 'derma sp., Penicillium sp., Bacillus sp., or a combination thereof. In another example, the bilirubin oxidase is from Myrothecium verrucaria, Magnaporthe oryzae, Pleurotus ostreatus, Trachyderma tsunodae, Penicillium janthinellum, Bacillus subtilis, or a combination thereof. In another example, the bilirubin oxidase is selected from the group consisting of Myr. verrucaria MTl, Myr. verrucaria 3.2190, Myr. verrucaria B03, Myr. verrucaria IMER1, Myr. verrucaria FERM-P 5918, Myr. verrucaria IFO 6113, Myr. verrucaria IFO 6133, Myr. verrucaria IFO 6351 and Myr. verrucaria IFO, Magnaporthe oryzae PI 31 or a combination thereof. In another example, the bilirubin oxidase comprises the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2.

In an example, the bilirubin oxidase is encapsulated in a microcapsule. In an example, the microcapsule is a polysaccharide. For example, the microcapsule can be a starch-based material, a cellulose-based material, or a combination thereof.

In another example, the present disclosure relates to a bilirubin oxidase conjugated to a polymer, wherein the bilirubin oxidase has improved stability and capacity to detect bilirubin in urine compared to unconjugated bilirubin oxidase. In an example, the polymer is a polyacid. In an example, the polyacid has a molecular weight between about 1,000 and 100,000. In another example, the poly acid has a molecular weight between about 1,000 and 50,000. In another example, the poly acid has a molecular weight between about 1,000 and 25,000. In another example, the poly acid has a molecular weight between about 1,000 and 10,000. In an example, the polyacid is polyacrylic acid. Accordingly, in an example, the polyacrylic acid can have a molecular weight falling in an above exemplified range. For example, the polyacrylic acid can have a molecular weight between about 1,000 and 10,000. In an example, the bilirubin oxidase conjugated to a polymer is stable for at least 6 months at room temperature. In another example, the bilirubin oxidase is stable for at least 12 months at room temperature. In another example, the bilirubin oxidase can detect bilirubin in urine at 8 mg/dL. In another example, the bilirubin oxidase can detect bilirubin in urine at 6 mg/dL. In another example, the bilirubin oxidase can detect bilirubin in urine at 5 mg/dL. In an example, the bilirubin oxidase is stable for at least 12 months at room temperature and can detect bilirubin in urine at 5 mg/dL.

In another example, the present disclosure relates to a bilirubin oxidase, conjugated to a polyacid, wherein the polyacid has a molecular weight between about 1,000 and 10,000. In this example, the polyacid may be polyacrylic acid.

In another example, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100% of the total surface area of the absorbent article comprises bilirubin oxidase.

In another example, the absorbent article comprises a total amount of bilirubin oxidase less than about 0.4 units/mg of protein. In another example, the absorbent article comprises a total amount of bilirubin oxidase between about 0.001 units/mg of protein and about 0.4 units/mg of protein. In another example, the absorbent article comprises a total amount of bilirubin oxidase between about 0.025 units/mg of protein and about 0.05 units/mg of protein. In another example, the absorbent article comprises a total amount of bilirubin oxidase between about 0.1 and 1.0 μg. In another example, the absorbent article comprises a total amount of bilirubin oxidase between about 0.2 and 0.4 μg.

In an example, the absorbent body comprises the bilirubin oxidase.

In another example, the absorbent body comprises a urine permeable compartment zone. In this example, the compartment zone can comprise the bilirubin oxidase. In an example, the absorbent body comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least 10, at least 25, at least 50, at least 100, at least 1000 urine permeable compartment zones.

In an example, the absorbent body can comprise a control zone. In an example, the absorbent body comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least 10, at least 25, at least 50, at least 100, at least 1000 control zones. In an example, the control zone is compared to a compartment zone comprising bilirubin oxidase, so as to provide a comparative visual colour signal to the user.

In an example, the colorimetric reaction is confined to a compartment zone comprising the bilirubin oxidase.

In an example, the amount of bilirubin oxidase in a compartment zone is between about 0.001 units/mg of protein and about 0.4 units/mg of protein. In another example, the amount of bilirubin oxidase in a compartment zone is between about 0.025 units/mg of protein and about 0.05 units/mg of protein.

In another example, the absorbent body comprises a colourant to enhance the colorimetric reaction visible when contacted with urine containing bilirubin.

In an example, the absorbent article is a nappy or diaper or training pant. In another example, the absorbent article is an absorbent liner. For example, the absorbent liner can be a nappy or diaper liner.

In another example, the present disclosure relates to a method of detecting presence of bilirubin in urine from a subject, the method comprising: fitting a subject with an absorbent article according to the present disclosure and visually assessing the article after the subject has urinated therein for a colorimetric change. In this example, a colorimetric change indicates the presence of bilirubin in the subject's urine. In an example, the colorimetric change is green. In another example, the colorimetric change is purple. In an example, the subject is an infant. In an example, the infant is 0 - 3 weeks old.

In another example, the present disclosure relates to a method of diagnosing jaundice in a subject, the method comprising: fitting a subject with an absorbent article according to the present disclosure and visually assessing the article after the subject has urinated therein for a colorimetric change. In this example, a colorimetric change indicates that the subject has jaundice. In an example, the colorimetric change is green. In another example, the colorimetric change is purple. In an example, the subject is an infant. In an example, the infant is 0 - 3 weeks old.

In another example, the present disclosure relates to a bilirubin oxidase conjugated to a polymer, wherein the bilirubin oxidase has improved stability and capacity to detect bilirubin in urine compared to unconjugated bilirubin oxidase. In an example, the polymer is a polyacid. In an example, the polyacid has a molecular weight between about 1,000 and 100,000. In another example, the poly acid has a molecular weight between about 1,000 and 50,000. In another example, the poly acid has a molecular weight between about 1,000 and 25,000. In another example, the poly acid has a molecular weight between about 1,000 and 10,000. In an example, the polyacid is polyacrylic acid. Accordingly, in an example, the polyacrylic acid can have a molecular weight falling in an above exemplified range. For example, the polyacrylic acid can have a molecular weight between about 1,000 and 10,000. In an example, the bilirubin oxidase conjugated to a polymer is stable for at least 6 months at room temperature. In another example, the bilirubin oxidase is stable for at least 12 months at room temperature. In another example, the bilirubin oxidase can detect bilirubin at 8 mg/dL. In another example, the bilirubin oxidase can detect bilirubin in urine at 6 mg/dL. In another example, the bilirubin oxidase can detect bilirubin in urine at 5 mg/dL. In an example, the bilirubin oxidase is stable for at least 12 months at room temperature and can detect bilirubin in urine at 5 mg/dL.

In another example, the present disclosure relates to a composition comprising a bilirubin oxidase disclosed herein. In an example, the composition is a liquid formulation. Such formulations may be applied to an absorbent article disclosed herein before being fitted to a subject.

Any example herein shall be taken to apply mutatis mutandis to any other example unless specifically stated otherwise.

The present disclosure is not to be limited in scope by the specific examples described herein, which are intended for the purpose of exemplification only.

Functionally-equivalent products, compositions and methods are clearly within the scope of the disclosure, as described herein.

Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.

The disclosure is hereinafter described by way of the following non-limiting

Examples and with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

Figure 1. Bilirubin oxidase catalyses the oxidation of bilirubin to produce a visual colour change. A. In the absence of bilirubin oxidase, bilirubin (0.002% w/v) is coloured yellow. B. A reaction mixture containing 0.002% (w/v) total bilirubin and 0.0025 unit bilirubin oxidase is coloured green.

Figure 2. Colorimetric results at varying concentrations of bilirubin.

Figure 3. Comparison of BOx and BOx/PAA activity over time.

Figure 4. Capacity of BOx and BOx/PAA to detect bilirubin in urine.

DETAILED DESCRIPTION OF THE INVENTION

General Techniques and Selected Definitions

Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., molecular biology, biochemistry, enzymology, pathology, diagnostics, clinical studies, protein chemistry, polymer chemistry and materials chemistry).

As used in this specification and the appended claims, terms in the singular and the singular forms "a," "an" and "the," for example, optionally include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a bilirubin oxidase" optionally includes one or more bilirubin oxidases.

As used herein, the term "about", unless stated to the contrary, refers to +/- 10%, more preferably +/- 5%, more preferably +/- 1%, of the designated value.

The term "and/or", e.g., "X and/or Y" shall be understood to mean either "X and

Y" or "X or Y" and shall be taken to provide explicit support for both meanings or for either meaning.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

The term "subject" is used in the context of the present specification to refer to a human subject. The terms "subject", "wearer" and "user" can be used interchangeably in the context of the present disclosure. In an example, the subject is an infant. The term "infant" is used in the context of the present specification to refer to a human subject aged between about 0 and 5 years. In an example, the subject is 0— 36 months old. In another example, the subject is 0 - 3 weeks old. In an example, the subject is 0 - 2 weeks old. In another example, the subject is 0 - 1 week old. In another example, the subject is 6 - 10 years old. In another example, the subject is an adolescent. For example, the subject can be 10 - 19 years old. In another example, the subject is an adult. For example, the subject can be at least 18 - 21 years old. In another example, the subject is classified as a geriatric person. For example, a geriatric subject may require a part-time or full-time care.

The present disclosure describes the following, various non-limiting embodiments, which relate to the development of an absorbent article that rapidly detects bilirubin in urine.

Bilirubin Oxidase

The enzyme bilirubin oxidase (EC 1.3.3.5) converts bilirubin to biliverdin through an oxidation reaction as follows:

2 bilirubin + O2 2 biliverdin + 2 H2O

If bilirubin is present in urine, it will be oxidised by bilirubin oxidase to biliverdin and H2O. Oxidation of bilirubin to biliverdin is a colorimetric reaction allowing the presence of bilirubin in a reaction medium to be identified visually. The term "colorimetric reaction" is used in the context of the present disclosure to describe the coloured product produced by bilirubin oxidase in the presence of bilirubin.

The bilirubin oxidase incorporated into an absorbent article according to the present disclosure is not particularly limited so long as it can catalyse the above referenced colorimetric reaction. For example, a bilirubin oxidase encompassed by the present disclosure catalyses a colorimetric reaction visible when contacted with urine containing bilirubin. In an example, the bilirubin oxidase catalyses a colorimetric reaction that produces a green product in the presence of bilirubin. In another example, the bilirubin oxidase produces a yellow product in the presence of bilirubin. In another example, the bilirubin oxidase produces a green product in the presence of bilirubin. In another example, the bilirubin oxidase produces a purple product in the presence of bilirubin.

In another example, the bilirubin oxidase detects conjugated bilirubin (e.g. bilirubin conjugated to glucuronic acid). In another example, bilirubin oxidase detects bilirubin IX-alpha or its isoforms Ill-alpha and Xlll-alpha.

In another example, the bilirubin oxidase is from Myrothecium sp., Magnaporthe sp., Pleurotus sp., Trachyderma sp., Penicillium sp., Bacillus sp., or a combination thereof. In another example, the bilirubin oxidase is from Myrothecium sp.. In another example, the bilirubin oxidase is from Magnaporthe sp.. In another example, the bilirubin oxidase is from Pleurotus sp.. In another example, the bilirubin oxidase is from Trachyderma sp.. In another example, the bilirubin oxidase is from Penicillium sp.. In another example, the bilirubin oxidase is from Myrothecium sp. and Magnaporthe sp..

In another example, the bilirubin oxidase is from Myrothecium verrucaria, Magnaporthe oryzae, Pleurotus ostreatus, Trachyderma tsunodae, Penicillium janthinellum, Bacillus subtilis, or a combination thereof. In an example, the bilirubin oxidase is from Myrothecium verrucaria. In another example, the bilirubin oxidase is from Magnaporthe oryzae. In another example, the bilirubin oxidase is from Pleurotus ostreatus. In another example, the bilirubin oxidase is from Trachyderma tsunodae. In another example, the bilirubin oxidase is from Penicillium janthinellum. In another example, the bilirubin oxidase is from Bacillus subtilis. In another example, the bilirubin oxidase is from Bacillus licheniformis . In another example, the bilirubin oxidase is from Bacillus pumilus. In another example, the bilirubin oxidase is from Myrothecium verrucaria and Magnaporthe oryzae.

In another example, the bilirubin oxidase is selected from the group consisting of Myr. verrucaria MT1, Myr. verrucaria 3.2190, Myr. verrucaria B03, Myr. verrucaria IMER1, Myr. verrucaria FERM-P 5918, Myr. verrucaria IFO 6113, Myr. verrucaria IFO 6133, Myr. verrucaria IFO 6351 and Myr. verrucaria IFO 9056, Myr. cinctum IFO 9950, and Myr. roridum IFO 9531, or a combination thereof. In another example, the bilirubin oxidase is Myr. verrucaria MT1. In another example, the bilirubin oxidase is Myr. verrucaria 3.2190. In another example, the bilirubin oxidase is Myr. verrucaria B03. In another example, the bilirubin oxidase is Myr. verrucaria IMER1. In another example, the bilirubin oxidase is Myr. verrucaria FERM-P 5918. In another example, the bilirubin oxidase is Myr. verrucaria IFO 6113. In another example, the bilirubin oxidase is Myr. verrucaria IFO 6133. In another example, the bilirubin oxidase is Myr. verrucaria IFO 6351 and Myr. verrucaria IFO 9056. In another example, the bilirubin oxidase is Myr. cinctum IFO 9950. In another example, the bilirubin oxidase is Myr. roridum IFO 9531. In another example, the bilirubin oxidase is Magnaporthe oryzae PI 31.

Amino acid sequences of the above referenced enzymes and nucleic acids sequences encoding the same can be obtained from publically available databases as required. For example, one of skill in the art may search UniProt (http://www.uniprot.org/; www dot uni prot dot org), National Centre for Biotechnology Information (NCBI) - Protein (https://www.ncbi.nlm.nih.gov/protein/; www dot ncbi dot nlm dot nih dot gov/protein/) or NCBI - gene (https://www.ncbi.nlm.nih.gov/gene; www dot ncbi dot nlm dot nih dot gov/gene). In an example, the bilirubin oxidase comprises the amino acid sequence set forth in SEQ ID NO: 1. In another example, the bilirubin oxidase comprises the amino acid sequence set forth in SEQ ID NO: 2. Bilirubin Oxidase

One of skill in the art will appreciate that the various absorbent articles discussed herein such as nappies, diapers and training pants may need to be stored for a number of months before use. Accordingly, in an example, the present disclosure encompasses a bilirubin oxidase that is stable at room temperature (i.e. about 20 - 25 °C) for an extended period of time. The term "stable" is used in this context to describe a bilirubin oxidase that is capable of catalysing a colorimetric reaction in the presence of bilirubin (i.e. catalyse conversion of bilirubin to biliverdin). In an example, the colorimetric reaction catalysed by a stable bilirubin oxidase defined herein can be observed by the naked eye. In an example, the bilirubin oxidase is stable at room temperature for at least three months. In another example, the bilirubin oxidase is stable at room temperature for at least six months. In another example, the bilirubin oxidase is stable at room temperature for at least 12 months. In another example, the bilirubin oxidase is stable at room temperature for at least 18 months. In another example, the bilirubin oxidase is stable at room temperature for at least 24 months. In another example, the bilirubin oxidase is stable at room temperature for at least three to 24 months. In another example, the bilirubin oxidase is stable at room temperature for at least six to 12 months.

In another example, the bilirubin oxidase can detect bilirubin in urine at 8 mg/dL. The term "detect" is used in this context to refer to the capacity of a bilirubin oxidase disclosed herein to catalyse a colorimetric reaction in the presence of the referenced level of bilirubin (mg/dL). In an example, the colorimetric reaction can be observed by the naked eye. In another example, the bilirubin oxidase can detect bilirubin in urine at 6 mg/dL. In another example, the bilirubin oxidase can detect bilirubin in urine at 5 mg/dL. In another example, the bilirubin oxidase can detect bilirubin in urine between 8 and 5 mg/dL. In another example, the bilirubin oxidase can detect bilirubin in urine between 7 and 5 mg/dL. In an example, the bilirubin oxidase is stable for an above referenced period and can detect an above referenced level of bilirubin in urine. In an example, the bilirubin oxidase is stable for at least 12 months at room temperature and can detect bilirubin in urine at 5 mg/dL.

Bilirubin oxidase may be provided in various formulations to achieve the above referenced properties such as stability and/or capacity to detect bilirubin in urine. For example, bilirubin oxidase may be provided as a protein - polymer conjugate such that bilirubin oxidase is covalently bound to one or more polymers.

Any suitable polymer or combination thereof can be used. In some embodiments, the polymer is water soluble. In an example, the polymer comprises acid groups. Examples of acid groups include, but are not limited to, carboxylic acid groups (i.e.,— COOH) and sulfonic acid groups (i.e. — SCbH). In another example, the polymer is a polyacid, such as a polycarboxylic acid or a polysulfonic acid. For example, the polymer can be a polycarboxylic acid. Non-limiting examples of polycarboxylic acids include, polyacrylic acid, poly(acrylic acid-co-maleic acid), poly(methyl vinyl ether-alt-maleic acid), poly(aciylamide-co-acrylic acid), poly(lactic acid), poly(glycolic acid) and salts thereof. Suitable exemplary salts include alkali and alkaline earth metal salts such as, for example sodium salts.

In some embodiments, the polymer contains amine groups, for example a primary amine. In some embodiments, the polymer is a polyamine or a salt thereof. Non-limiting examples of polyamines include polyvinyl amine, polyallyl amine, polylysine, or combinations thereof. In some embodiments, the polyamine is polyethyleneimine.

The present inventors have identified that the molecular weight of the polymer may be important. Without wishing to be bound by any particular theory, it is thought that the polymer wraps around the bilirubin oxidase and restricts the number of conformational states the protein can achieve. In an example, the polymer has a molecular weight between about 500 and about 100,000. In another example, the polymer has a molecular weight between about 500 and about 50,000. In another example, the polymer has a molecular weight between about 500 and about 20,000. In another example, the molecular weight is between about 1,000 and about 20,000. In another example, the molecular weight is between about 1,000 and about 10,000. In another example, the molecular weight is between about 1,000 and about 8500. In another example, the molecular weight is between about 5,000 and about 10,000. In another example, the molecular weight is between about 5000 and about 8500. In another example, the molecular weight is about 1200. In another example, the molecular weight is about 5100. In another example, the molecular weight is about 8000.

In another example, the polymer is polyacrylic acid or a salt thereof. In an example, the polyacrylic acid has a molecular weight between about 500 and about 100,000. In another example, the polyacrylic acid has a molecular weight between about 500 and about 50,000. In another example, the polyacrylic acid has a molecular weight between about 500 and about 20,000. In another example, the polyacrylic acid has a molecular weight between about 1,000 and about 20,000. In another example, the polyacrylic acid has a molecular weight between about 1,000 and about 10,000. In another example, the polyacrylic acid has a molecular weight between about 1,000 and about 8500. In another example, the polyacrylic acid has a molecular weight between about 5,000 and about 10,000. In another example, the polyacrylic acid has a molecular weight between about 5000 and about 8500. In another example, the polyacrylic acid has a molecular weight of about 1200. In another example, the polyacrylic acid has a molecular weight of about 5100. In another example, the polyacrylic acid has a molecular weight of about 8000.

One of skill in the art can form the protein-polymer conjugate disclosed herein using methods known in the art. In some embodiments, the protein - polymer conjugate is formed by crosslinking. Crosslinking may occur on a specific location within the protein and/or polymer, or across the entire protein and /or polymer chain. For example, the polymer may be attached at a specific amino acid residue on bilirubin oxidase (such as a lysine, cysteine, aspartic acid or glutamic acid residue) or at a random site (i.e. anywhere in the protein). In an example, the attachment of bilirubin oxidase to the polymer is through a lysine side chain of bilirubin oxidase. In another example, the attachment of bilirubin oxidase to the polymer is through a glutamic acid or aspartic acid side chain. In another example, the attachment of bilirubin oxidase to the polymer is through a cysteine side chain. In another example, the attachment of bilirubin oxidase to the polymer is at one or more sites that are randomly located on bilirubin oxidase. In another example, bilirubin oxidase is covalently attached to a polymer at one or more sites on the protein and at one or more sites on the polymer.

Examples of crosslinking reactions suitable for providing protein - polymer conjugates disclosed herein include, but are not limited to, esterification, amidation, addition, or condensation reactions. Crosslinking can be induced by light, temperature, crosslinking reagents, and/or a catalyst. Alternatively, crosslinking can be spontaneous (i.e. where the crosslinking reaction is not induced by light or by temperature, or where the crosslinking reaction is in the absence of catalyst, external crosslinker, protein, or enzyme). In some embodiments, cross-linking is induced by the addition of a crosslinking reagent (also referred to as a crosslinker). The crosslinking reagent may form part of the final crosslink or can cause crosslinking without forming part of the final crosslink. Any suitable crosslinking reagent can be used and the crosslinking reagent is selected based on the desired attachment point in the protein and/or polymer. Suitable exemplary crosslinking reagents include, but are not limited to, carbodiimide compounds, such as, Ν,Ν'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, and l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (otherwise referred to as N-(3- dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride or "EDC"). Carbodiimides are often referred to as zero-length crosslinkers; as they cause direct conjugation of carboxylates (-COOH) to primary amines (- H2) without forming part of the final crosslink (amide bond) between target molecules. Other examples of crosslinking reagents that can be used to cross-link polyacrylic acid and other polymers disclosed herein, include, but are not limited to, difunctional or polyfunctional alcohol (e.g. ethylene glycol, ethylenedioxy-bis(ethylamine), glycerol, polyethylene glycol), difunctional or polyfunctional amine (e.g, ethylene diamine, JEFF AMINE®), polyetheramines (Huntsman), poly(ethyleneimine)).

In an example, the crosslinker is EDC. Crosslinking using EDC can be separated into two steps. In the first step, the polymer is activated with EDC. This prevents the formation of any protein-protein crosslinking. In the second step, the EDC activated polymer is combined with the protein (and optionally BSA) resulting in the formation of amide bonds between the polymer and bilirubin oxidase and the formulation is applied to the absorbent article. In an example, BSA is added to help stabilise the bilirubin oxidase and the resulting bilirubin oxidase-polymer conjugate. In another example, the polymer comprises carboxyl groups and the attachment of bilirubin oxidase to the polymer is through lysine side chains of bilirubin oxidase. In an example, the polymer is polyacrylic acid and the attachment of bilirubin oxidase to the polymer is through lysine side chains of bilirubin oxidase. In another example, the polymer comprises amine groups and the attachment of bilirubin oxidase to the polymer is through a glutamic acid or aspartic acid side chains of bilirubin oxidase.

The degree of crosslinking between the protein and polymer can be modified by the person skilled in the art. For example, the amount of crosslinking reagent can be varied, the reaction time can be varied and/or the reaction temperature can be varied.

One of skill in the art can assess stability of bilirubin oxidase disclosed herein alone or when incorporated into absorbent articles of the present disclosure using various methods. One example is provided below in Example 2. Briefly, an absorbent article incorporating bilirubin oxidase can be subjected to elevated temperatures (such as 40 °C and 45 °C) for a period of time and the ability of the bilirubin oxidase incorporated in the absorbent article to catalyse the oxidation of bilirubin to biliverdin is assessed. Other methods for testing enzyme stability, include, but are not limited to treating the absorbent article incorporating bilirubin oxidase with a denaturant (for example, heat, extremes of pH, or chemical denaturants such as urea, guanidine HC1, SDS) and assessing the ability of the treated bilirubin oxidase to catalyse the oxidation of bilirubin to biliverdin.

One of skill in the art can also assess capacity of a bilirubin oxidase disclosed herein to detect bilirubin in urine using various methods. One example is provided below in Example 3. For example, colorimetric assay can be used to determine whether a bilirubin oxidase disclosed herein can convert an amount of bilirubin (e.g. 5 mg/dL) to a visually detectable level of biliverdin.

In the above exemplary methods stability and/or capacity to detect bilirubin in urine can be compared with an unconjugated or native bilirubin oxidase in side by side experiments. Results can be compared to determine whether stability and/or capacity to detect bilirubin in urine is improved in conjugated bilirubin oxidase relative to unconjugated or native bilirubin oxidase. In an example, capacity of conjugated bilirubin oxidase to detect bilirubin is improved relative to unconjugated or native bilirubin oxidase when the conjugated bilirubin oxidase can detect lower levels of bilirubin in urine. In this example, the conjugated bilirubin oxidase may produce more biliverdin than unconjugated or native bilirubin oxidase when the enzymes are contacted with equal levels of bilirubin.

Formulations

Bilirubin oxidase disclosed herein may be formulated as a composition. In an example, the composition is suitable for application to an absorbent article disclosed herein. Exemplary compositions may provide bilirubin oxidase alone or in combination with an acceptable carrier, diluent or excipient. In these compositions bilirubin oxidase is provided in an amount sufficient to detect bilirubin in urine from a subject.

Exemplary compositions may also comprise sterile aqueous or nonaqueous solutions, dispersions, suspensions, or emulsions. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, and injectable organic esters such as ethyl oleate. Such compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents or antibacterial and antifungal agents. Absorbent Article

The present disclosure relates to absorbent articles that absorb and contain body exudates such as urine. The term "exudates" is used to refer to any material separated, eliminated or discharged from the body. In an example, the exudate is urine.

Examples of absorbent articles encompassed by the present disclosure include nappies or diapers, training pants, absorbent inserts, nappy or diaper liners, incontinence briefs, incontinence undergarments and wipes. In an example, the absorbent article is a nappy or diaper. The terms "nappy" and "diaper" are used interchangeably in the context of the present disclosure to refer to an absorbent article worn by a subject in the place of underwear. These articles are generally worn about the lower torso and secured at the waist of the subject. In another example, the absorbent article is a training pant. In an example, the absorbent article is disposable. The term "disposable" is used in the context of the present disclosure to refer to absorbent articles that generally are not intended to be laundered or otherwise restored or reused as absorbent articles (e.g. they are intended to be discarded after single use).

In an example, the absorbent article is an absorbent insert. The term "absorbent insert" is used in the context of the present disclosure to refer to a modular unit that is adapted to be installable and removable in an external outer cover. In an example of the present disclosure, the absorbent insert may be a nappy or diaper liner. For example, the nappy or diaper liner may be inserted into a cloth nappy or diaper to be worn by the subject.

In an example, an absorbent article of the present disclosure comprises attachments and/or fasteners to fasten around the waist and legs of a subject. For example, the absorbent article can comprise hook and loop fasteners. In another example, the absorbent article comprises adhesive tabs.

In an example, an absorbent article of the present disclosure comprises a liquid permeable bodyside liner, an outer cover, and an absorbent body positioned between the bodyside liner and the outer cover. Various examples of absorbent articles having such features are known in the art. Suitable examples include, but are not limited to, those disclosed in US Patent No. 6,120,485, US Patent No. 5,545,158, US Patent No. 4,705,513, US Patent No. 3,315,676, and US Patent No. 4,753,646.

In an example, the liquid permeable bodyside liner may be compliant, soft to feel, and non-irritating, and may be employed to assist in isolating exudates (e.g. urine) from the wearer's skin. The term "liquid permeable" is used in the context of the present disclosure to describe the body side liners ability to permit the passage of liquids, usually by pores or openings. In an example, the liquid permeable bodyside liner is selectively permeable, in that it may permit the passage of some liquids and restrict the passage of other liquids. In another example, the liquid permeable bodyside liner is permeable to urine.

In another example, the liquid permeable bodyside liner is liquid permeable on one side (i.e. face) and liquid impermeable on the opposing side (i.e. opposing face). In this example, the liquid permeable bodyside liner permits the passage of liquids in one direction, and restricts the passage of liquids in the opposite direction. One of skill in the art will appreciate that in these examples, such a bodyside liner will be positioned relative to the subject to facilitate passage of liquid away from the subject and restrict passage of liquid towards the subj ect.

In an example, the liquid permeable bodyside liner comprises a material less hydrophilic than the absorbent body to present a relatively dry surface to the subject. In another example, the bodyside liner is sufficiently porous to be liquid permeable, permitting liquid exudates (e.g. urine).

Liquid permeable bodyside liners may be manufactured from a wide selection of exemplary materials, such as porous foams, reticulated foams, aperture plastic films, natural fibres (for example, wood or cotton fibres), synthetic fibres (for example, polyester or polypropylene fibres) and combinations thereof.

In other examples, the liquid permeable bodyside liner can comprise various woven and nonwoven materials such as a meltblown or spunbound web (e.g. of polyolefin fibres), a bond-carded web (e.g. of natural and/or synthetic fibres), a substantially hydrophobic material (e.g. treated with a surfactant or otherwise processed to impart a desired level of wetablity and hydrophilicity) as well as combinations comprising at least one of these materials. In an example of the present disclosure, the liquid permeable bodyside liner comprises polypropylene.

In another example, the liquid permeable bodyside liner may be partially or substantially transparent so that the absorbent body can be assessed through the bodyside liner. In an example of the present disclosure, the liquid permeable bodyside liner is partially transparent. In another example, the liquid permeable bodyside liner is substantially transparent. In an example, the liquid permeable bodyside liner is transparent.

In an example, the outer cover is substantially impermeable to liquid to substantially reduce or entirely prevent exudates (e.g. urine) from leaking from the absorbent article. In an example, the outer cover comprises a nonwoven fibrous web constructed to provide the required level of liquid impermeability. In an example, such a nonwoven fibrous web comprises spunbound and/or meltblown polymer fibres selectively treated with a water repellent coating. In another example the outer cover is laminated with a liquid impermeable, vapour permeable polymer film. In another example, the outer cover may include a vapour permeable nonwoven layer that has been partially coated or otherwise configured to be substantially liquid impermeable.

The absorbent article of the present disclosure may additionally comprise any number of materials and/or layers of materials between the liquid permeable bodyside liner and the outer cover. Exemplary additional materials and/or layers of materials include further absorbent bodies, liquid permeable layers, and liquid impermeable layers. In an example of the present disclosure, the absorbent article comprises additional materials and/or layers of materials between the liquid permeable bodyside liner and the outer cover.

In an example, the absorbent article comprises a liquid permeable bodyside liner, outer cover, and absorbent body as sheets of material.

In an example, absorbent articles of the present disclosure comprise an absorbent body capable of collecting and retaining exudates (e.g. urine). In an example, the absorbent body can comprise any material which is generally compressible, conformable, non-irritating to the wearer's skin, and capable of collecting and retaining exudates (e.g. urine). In an example, the material is a matrix of hydrophilic fibres. For example, the absorbent body may comprise a fibrous web of cellulosic fibres (e.g. wood pulp) mixed with particles of a high absorbency material (such as the material commonly known as super-absorbent material). In this example, the wood pulp may be exchanged with synthetic, polymeric and meltblown fibres, as well as combinations thereof. In an example, the super-absorbent particles can be substantially homogeneously mixed with the hydrophilic fibres. In another example, the super-absorbent particles can be non-uniformly mixed. Various examples of super- absorbent material are known in the art. Suitable examples include, but are not limited to, those disclosed in US Patent No. 7,179,851 and US Patent No. 6,720,073.

In another example, the absorbent body includes a laminate of fibrous webs or another suitable matrix for maintaining a super-absorbent material in a specified area of the absorbent article.

Absorbent Article Including Bilirubin Oxidase

In an example, bilirubin oxidase disclosed herein is incorporated into absorbent articles of the present disclosure. The positioning of bilirubin oxidase in absorbent articles according to the present disclosure is not particularly limited so long as the bilirubin oxidase is able to detect the presence of bilirubin in bodily fluid absorbed by the article. For example, bilirubin oxidase may be positioned in a liquid permeable bodyside liner, an outer cover, an absorbent body, or a combination thereof. In an example, bilirubin oxidase is positioned in a liquid permeable bodyside liner. In another example, bilirubin oxidase is positioned in an outer cover. In another example, bilirubin oxidase is positioned in an absorbent body.

In an example, an absorbent article is impregnated with bilirubin oxidase. For example, bilirubin oxidase can be impregnated into the fibres of an absorbent article. The term "impregnated" is used in the context of the present disclosure to refer to the soaking or saturation (partial or complete) of a substrate with a substance comprising bilirubin oxidase. In another example, bilirubin oxidase is embedded in an absorbent article. For example, bilirubin oxidase can be embedded into the fibres of an absorbent article. The term "embedded" is used in the context of the present disclosure to refer to a substance comprising bilirubin oxidase being fixed firmly and deeply in an absorbent body.

In an example, bilirubin oxidase can be incorporated into the absorbent article by covalent bonding. In this example, a covalent bond is formed between one or more fibres in the absorbent article and bilirubin oxidase. In another example, bilirubin oxidase can be incorporated into the absorbent article by non-covalent bonding. Suitable exemplary non-covalent bonds include hydrophobic bonds, hydrogen bonds, ionic bonds and/or van der Waals bonds being formed between one or more fibres in the absorbent article and bilirubin oxidase.

In an example, lyophilised bilirubin oxidase is incorporated into the absorbent article. Lyophilising bilirubin oxidase prior to its incorporation into the absorbent article may improve the stability of the bilirubin oxidase.

In another example, bilirubin oxidase is encapsulated in a shell via microencapsulation prior to its incorporation into the absorbent article. In this example, the bilirubin oxidase is provided in a microcapsule. Microencapsulation is a process in which tiny particles or droplets of an active agent are surrounded by a coating to give small capsules that will dissolve, disintegrate, rupture or otherwise break down upon contact with the urine, to allow for the release of the active agent. Microencapsulation is commonly employed as a method of increasing stability and/or decreasing degradation of the active agent. In an example, bilirubin oxidase is encapsulated prior to being impregnated or embedded in the absorbent article.

The bilirubin oxidase may be microencapsulated in any material so long as upon contact with urine, the bilirubin oxidase is exposed so as to allow a colorimetric reaction to be catalysed in the presence of bilirubin. In an example, the microencapsulation material is soluble in urine. Suitable microencapsulation materials are known in the art and include cellulose-based polymeric materials (e.g. ethyl cellulose), lactic acid-based aliphatic polyesters, carbohydrate-based materials (e.g. cationic starches and sugars), and materials derived therefrom (e.g. dextrins and cyclodextrins), as well as other materials compatible with human tissues. In an example of the present disclosure, the microencapsulation material comprises a polysaccharide. In another example, the polysaccharide is a starch- or cellulose-based material.

The thickness of the microencapsulation shell may vary depending upon the material used and the formulation required, and is generally manufactured to allow the encapsulated active agent to be covered by a thin shell, which may be a monolayer or thicker laminate layer, or may be a composite layer. The encapsulation shell may also made of multiple layers, and such layers may be of the same or of differing materials. The microencapsulation shell should be thick enough to resist cracking or breaking of the shell during handling or shipping of the product. The microencapsulation shell should be constructed such that humidity from atmospheric conditions during storage, shipment, or wear, will not cause a breakdown of the microencapsulation shell and result in a release of the active agent.

In an example, microcapsules containing the bilirubin oxidase is applied directly to the absorbent article (e.g. by impregnating the absorbent body or embedding in the absorbent body) are of a size such that the user cannot feel the encapsulated shell on the skin during use. In this example, there no "gritty" or "scratchy" feeling if the microcapsules come into contact with the skin.

In an example, the microcapsules are about 1, 2, 5, 10, 15, 20, or 25 μιη in diameter. In another example, the microcapsules are less than about 50, 30, 25, 20, 15, 10, 5, or 2 μιη in diameter.

In an example, contact of urine with an absorbent article comprising bilirubin oxidase according to the present disclosure produces a coloured product that is visible following visual assessment of the absorbent article. In an example, the coloured product is yellow. In another example, the coloured product is green. In another example, the coloured product is purple. In another example, the coloured product is green.

In an example, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100% of the total surface area of the absorbent article comprises bilirubin oxidase. Put another way, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100% of the total surface area of the absorbent article can detect bilirubin in urine. In other examples, about 5 to 95%, 10 to 90%), 30 to 80%), or 40 to 70% of the total surface area of the absorbent article comprises bilirubin oxidase.

In an example, the absorbent article comprises bilirubin at about 0.001,

0.0015, 0.002, 0.0025, 0.003, 0.0035, 0.004, 0.0045, 0.005, 0.01, 0.1, 0.2, or 0.4 units/mg protein. In another example, the absorbent article comprises bilirubin oxidase less than about 0.4, 0.2, 0.1, 0.01, 0.005, 0.0045, 0.004, 0.0035, 0.003, 0.0025, 0.002, 0.0015, or 0.001 units/mg protein. In another example, the absorbent article comprises bilirubin oxidase ranging from about 0.001 to 0.4, 0.0015 to 0.1, 0.002 to 0.01, or 0.0025 to 0.005 units/mg protein. In an example, the absorbent article comprises bilirubin oxidase at about 0.0025 units/mg protein. In another example, the absorbent article comprises bilirubin oxidase at about 0.005 units/mg protein. In another example, the absorbent article comprises bilirubin oxidase ranging from about 0.0025 to 0.005 units/mg protein. "Units/mg of protein" is used in the context of the present disclosure to refer to the amount of the enzyme that produces a certain amount of enzymatic activity, that is, the amount of enzyme that catalyses the conversion of 1 micromole of substrate per minute. In an example, the outer cover, the absorbent body, the bodyside liner or a combination thereof comprises bilirubin oxidase. In another example, the absorbent body comprises bilirubin oxidase.

In another example, the absorbent article comprises about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.75, 1.0 μg of bilirubin oxidase. In another example, the absorbent article comprises less than about 1.0, 0.75, 0.5, 0.45, 0.4, 0.35, 0.3, 0.25, 0.2, 0.15, or 0.1 μg of bilirubin oxidase. In another example, the absorbent article comprises about 0.1 to 1.0 μg, 0.15 to 0.75 μg, 0.2 to 0.5 μg, 0.2 to 0.45 μg, or 0.2 to 0.4 μg bilirubin oxidase. In another example, the absorbent article comprises about 0.2 to 0.5 μg bilirubin oxidase.

In an example, the absorbent article of the present disclosure may be divided into one or more compartment zones. "Compartment zones" are designed to contain bilirubin oxidase and urine. The absorbent article may comprise one or more compartment zones. For example, the absorbent article may comprise 2, 3, 4, 5, 10, 25, 50, 100, 1000 or more compartment zones. In an example, the compartment zones are present in the absorbent body. In other examples, compartment zones are present in the outer cover and/or bodyside liner of the absorbent article.

The amount of bilirubin oxidase in a compartment zone is sufficient for the catalysis of the colorimetric reaction visible when contacted with urine containing bilimbin. The relevant amount can be easily calculated by one of skill in the art. An exemplary method is described in Example 1 below. Example levels of bilirubin oxidase for a compartment zone are also discussed above.

In an example of the present disclosure, each compartment zone comprises bilirubin oxidase at about 0.0025 units/mg protein. In another example, each compartment zone comprises bilirubin oxidase at about 0.005 units/mg protein. In another example, each compartment zone comprises bilirubin oxidase at about 0.001 units/mg protein. In another example, each compartment zone comprises bilirubin oxidase at about 0.4 units/mg protein. In another example, each compartment zone comprises bilirubin oxidase at about 0.0025 to 0.005 units/mg protein. However, in another example, compartment zones may comprise varying levels of bilirubin oxidase.

In an example of the present disclosure, the colorimetric reaction catalysed by bilirubin oxidase is confined to a compartment zone comprising bilirubin oxidase. In another example, the colorimetric reaction catalysed by bilirubin oxidase is confined to each compartment zone that comprises bilirubin oxidase.

In another example, the absorbent article comprises one or more control zones. For example, the absorbent article may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 25, 50, 100, 1000 or more control zones. In an example, the control zones are present in the absorbent body. In other examples, control zones are present in the outer cover and/or bodyside liner of the absorbent article. In one example, the control zones comprise bilirubin oxidase but are not accessible to urine. In this example, the amount of bilirubin oxidase in the control zone is not considered to count towards the total bilirubin oxidase in the absorbent article. In another example, the control zones do not comprise bilirubin oxidase. After an absorbent article according to the present disclosure has been used, control zone(s) may be compared to a compartment zone(s) comprising bilirubin oxidase to provide a comparative visual colour signal. For example, the coloured product produced by the colorimetric reaction in a compartment zone comprising bilirubin oxidase may be compared to the colour of a control zone that does not comprise bilirubin oxidase. Such a control zone may be particularly useful when the colour change is subtle (e.g. clear to yellow).

The compartment zones and control zones may be of any suitable size, shape and configuration sufficient to receive urine. The location of the compartment zones and control zones is not particularly limited. However, one of skill in the art would appreciate that they are generally positioned to receive urine when the absorbent article is in use. Further, the compartment zones and control zones are usually located in an area visible to the user so that a simple, accurate, and rapid visual detection may be made. The visibility of such zones(s) may be accomplished in a variety of ways. For example, the absorbent article may include a transparent or translucent portion (e.g. window, film, etc.) that allows the compartment zone and/or control zone to be readily viewed without the removal of the absorbent article from the subject and/or without disassembly of the absorbent article. In another example, the compartment zone and/or control zone may extend through a hole or aperture in the absorbent article for visual detection. In another example, the compartment zone and/or control zone may be located on a surface of the absorbent article for visual detection. For example, the compartment zone and/or control zone may be located on a surface of an absorbent body.

In another example, the absorbent article comprises one or more channels. The channels may be of any suitable size, shape and configuration. The location of the channels is not particularly limited, though again, one of skill in the art would appreciate that they are generally positioned to receive urine when the absorbent article is in use. For example, the absorbent article can include a single channel or a series of channels. In an example, the compartment zones and/or control zones are separated by a series of channels. The channels may serve to separate the compartment zones and/or control zones. A channel may serve one or both of two functions; (1) to direct urine to the relevant compartment zone and/or control zone, and (2) to confine the colorimetric reaction to a compartment zone (i.e. separate to a control zone). This means that the channels may serve to confine the bilirubin oxidase to a particular compartment zone. A channel may be made of any material suitable to perform such functions. For example, a channel may be made of any of the materials described herein from which the outer cover, absorbent body or liquid impermeable bodyside liner may be made. The channel may be made of a substantially hydrophobic material, and may be treated with a surfactant or other material to impart a desired level of wettability and hydrophilicity/hydrophobicity. For example, a channel may be made of a material that is comparatively more hydrophobic than the compartment zone and/or control zone so as to direct urine through the channels to the zone(s). Such a hydrophobic channel may also be used to confine the colorimetric reaction to one or more compartment zones. In an example, compartment zones and or/control zones are separated by a series of channels that are made of a material that is comparatively more hydrophobic than the material of the compartment zones and/or control zones.

In an example, the absorbent article of the present disclosure also comprises a colourant to enhance the colour change or to provide a more significant visual change in colour in the presence of bilirubin. The colourant may be any colourant suitable to enhance the colour change and or provide a more significant visual signal. In an example, the colourant may correspond to a change in colour to a different hue of the same colour. For example, the colour change could be a progression from light to dark wherein the hue of the colour indicates the presence of bilirubin. The colourant may comprise any suitable material such that the visual signal of the colorimetric reaction is enhanced. For example, the colourant may be a dye.

In an example, the colorimetric reaction of the present disclosure is optimised through control of the pH of the reaction. In various examples, a colorimetric reaction according to the present disclosure is catalysed at a pH of about 7, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, or 9. In an example, the colorimetric reaction is catalysed at a pH of about 8.4. The pH of the reaction may be adjusted through the use of a buffer. In an example, the absorbent article comprises a buffer. In another example, compartment zones comprise a buffer. In these examples, the buffer also comprises bilirubin oxidase. The term "buffer" is used in the context of the present disclosure to refer to a solution which resists change in pH when acid or alkali substances are added to it.

In an example, the present disclosure encompasses an absorbent article disclosed herein comprising a bilirubin oxidase disclosed herein, wherein the bilirubin oxidase is stable at room temperature (i.e. about 20 - 25 °C) for an extended period of time. In an example, the absorbent article comprises a bilirubin oxidase that is stable at room temperature for at least three months. In another example, the absorbent article comprises a bilirubin oxidase that is stable at room temperature for at least six months. In another example, the absorbent article comprises a bilirubin oxidase that is stable at room temperature for at least 12 months. In another example, the absorbent article comprises a bilirubin oxidase that is stable at room temperature for at least 18 months. In another example, the absorbent article comprises a bilirubin oxidase that is stable at room temperature for at least 24 months. In another example, the absorbent article comprises a bilirubin oxidase that is stable at room temperature for at least three to 24 months. In another example, the absorbent article comprises a bilirubin oxidase that is stable at room temperature for at least six to 12 months.

In another example, the absorbent article comprises a bilirubin oxidase disclosed herein having improved capacity to detect bilirubin in urine. For example, the absorbent article can comprise a bilirubin oxidase that can detect bilirubin in urine at 8 mg/dL. In another example, the absorbent article can comprise a bilirubin oxidase that can detect bilirubin in urine at 6 mg/dL. In another example, the absorbent article can comprise a bilirubin oxidase that can detect bilirubin in urine at 5 mg/dL. In another example, the absorbent article can comprise a bilirubin oxidase that can detect bilimbin in urine between 8 and 5 mg/dL. In another example, the absorbent article can comprise a bilirubin oxidase that can detect bilirubin in urine between 7 and 5 mg/dL. In another example, the absorbent article can comprise a bilirubin oxidase that is stable for an above referenced period and can detect an above referenced level of bilirubin in urine. In an example, the absorbent article can comprise a bilirubin oxidase that is stable for at least 12 months at room temperature and can detect bilirubin in urine at 5 mg/dL.

Method of Detecting the Presence of Bilirubin

In an example, an absorbent article of the present disclosure is used for a method of detecting the presence of bilirubin in a subject's urine. In another example, an absorbent article of the present disclosure is used to identify jaundice in a subject. For example, an absorbent article of the present disclosure can be used to identify jaundice in an infant. In another example, an absorbent article of the present disclosure is used to identify jaundice in a 0 - 3 week old infant. In another example, an absorbent article of the present disclosure is used to identify jaundice in a 0 - 2 week old infant. In another example, an absorbent article of the present disclosure is used to identify jaundice in a 0 - 1 week old infant. In another example, an absorbent article of the present disclosure is used to identify jaundice in a person classified as a geriatric.

In performing the above exemplified methods, the absorbent article of the present disclosure may be fitted to the subject by any conventional means. For example, the absorbent article can be fastened about the waist of a subject. In another example, the absorbent article is placed in a nappy or diaper to be worn by the subject.

In an example, the absorbent article or part thereof becomes green in the presence of bilirubin or urine containing bilirubin. In another example, the absorbent article or part thereof becomes yellow in the presence of bilirubin or urine containing bilirubin. In another example, the absorbent article or part thereof becomes purple in the presence of bilirubin or urine containing bilirubin.

In an example, detection of bilirubin in urine from the subject of the absorbent article is indicative of jaundice. Accordingly, in an example, an absorbent article of the present disclosure is used for diagnosing jaundice.

In an example, the subject is referred to a clinician if a colorimetric reaction is observed in an absorbent article according to the present disclosure. In this example, the clinician may confirm a diagnosis of jaundice via blood test or other suitable means. If a positive diagnosis of jaundice is found, the subject may receive the appropriate medical intervention. For example, the subject may receive light therapy or

intravenous immunoglobulin (IVIg).

Compositions/Kits

In one example, the present disclosure relates to a kit for detecting bilirubin in urine of a subject, the kit comprising a bilirubin oxidase and an absorbent article. In this example, the bilirubin oxidase can be added to the absorbent article before being fitted to a subject. In an example, the kit is used for diagnosing jaundice in a subject.

In an example, the bilirubin oxidase is packaged in or with a suitable solvent or in lyophilised form. In another example, the bilirubin oxidase is packaged with a preservative or other suitable stabilising agent. Examples of suitable preservatives or stabilising agents include, but are not limited to, chelating agents, protease inhibitors, buffering agents, and salts.

In an example, the kit components are packaged in a suitable container with written instructions for performing the methods of the present disclosure.

EXAMPLES

EXAMPLE 1 - Detection of Bilirubin

Bilirubin oxidase was purchased from Sigma-Aldrich as a dry powder (bilirubin oxidase from Myrothecium verrucaria, lyophilised powder, 15-65 units/mg protein).

Bilirubin (5.0 μΕ), adjusted to pH 8.4 using Tris-hydrochloride (193 mM) and diluted four times with water, was added to a 3.0 ml reaction vessel (See Figure 1A).

Bilirubin oxidase (0.0025 units/mg protein) was added and a colour change of the solution from yellow to green was observed (See Figure IB).

No colour change was observed when 5.0 μΐ_^ of water was added to a 3.0 ml reaction vessel comprising bilirubin oxidase (0.0025 units/mg protein).

The ability of BOx to detect bilirubin in a dry fabric strip was then assessed via colorimetric assay. Briefly, a dry fabric strip or nappy liner was cut into 0.5 cm by 1.5 cm pieces. 100 μΐ of sensor mixture (BOx and BOx/PAA) was applied to a piece of fabric strip and allowed to completely dry. Once the fabric strip was dry, 100 μΐ of bilirubin (in varying amounts) was added to the strip and the strip incubated at 37°C for

3 h. After incubation, reacted and unreacted bilirubin was extracted from the fabric strip by shaking the strip with 500 μΐ RO water and 20 μΐ sodium hydroxide. The absorbance of the extract sample was measured at 450 nm. EXAMPLE 2 - Enzyme Stability

Polyacrylic acid (PAA) of varying molecular weight (1,200, 5,100 Da and 8,000 Da) in PBS buffer was activated by the addition of l-Ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDC) followed by mixing for 15 minutes. Bilirubin oxidase was then conjugated to activated PAA in the presence of Bovine Serum Albumin (BSA) before being added to supporting material strips (Example strip composition analysis shown in Table 1). The ability of BOx/PAA to detect bilirubin in a dry fabric strip was assessed via colorimetric assay as described above.

Accelerated aging studies were conducted at room temperature (23 °C), 40 °C and 45 °C to assess stability of material strips comprising bilirubin oxidase crosslinked with activated PAA (BOx/PAA) (Table 2). Currently available data support enzyme stability over at least 12 months. The stability data also suggest that PAA of varying chain length can be used to stabilise bilirubin oxidase.

Table 2.

EXAMPLE 3 - Enzyme Activity

The ability of BOx and BOx/PAA to detect bilirubin in a dry fabric strip was assessed via colorimetric assay as described above. Interestingly, improved bilirubin detection was observed using BOx/PAA. BOx was able to detect bilirubin at 10 mg/dL while BOx/PAA was able to detect bilirubin as low as 5 mg/dL (Figure 2).

BOx and BOx/PAA activity was then assessed in solution over a 21 day time course. BOx/PAA achieved greater activity compared to BOx after two weeks in solution (Figure 3). Activity of BOx/PAA was 40% greater at day 14 (p-value < 0.0001) and 50% greater at day 21 (p-value < 0.0001).

Finally, activity of BOx and BOx/PAA was assessed in clinical urine samples spiked with bilirubin. Both BOx and BOx/PAA were able to detect bilirubin in these urine samples (Figure 4).

These data suggest that EDC mediated cross linking of bilirubin oxidase can provide an enzyme complex capable of detecting bilirubin in urine with a shelf life that is suitable for use in consumer goods.

EXAMPLE 4 - Direct Continuous Spectrophotometric Analysis

In some examples, direct continuous spectrophotometric analysis was performed using a flat 96 microwell plate. All reagents were pre-warmed at 37°C. A microplate spectrophotomer (Elx808, BioTek in Vermont, United States of America) was used to measure the absorbance at 450 nm of the solution in each microwell. The plate reader was configured to take readings once per minute for the duration of the assay (usually 45 minutes), and to briefly agitate the plate to ensure the wells were thoroughly mixed before the first read. 450nm was chosen as the best available wavelength near the maximum spectral absorption of bilirubin (~440nm) (Lee, K.S. and L.M. Gartner, Spectrophotometric characteristics of bilirubin. Pediatr Res, 1976. 10(9): p. 782-8). Measurements were recorded as "absorbance". Once the assay was complete, an image was taken of the assay plate.

EXAMPLE 5 - Image Analysis Technique

MATLAB (version R2016a, Mathworks in Massachusetts, United States of America) was used to analyse the differences in colour as a result of the colorimetric reaction and quantify these differences. Briefly, the user selects regions of interest, capturing each well or area on the fabric. Masks are created to isolate these regions and the image is converted to CIELab colour space (CIE/ISO, 1976 Lab Colour Space. 1976. The Euclidean distance between two colour points is calculated as a numerical measure of the difference in colour in CIELab colour space (Schuessler, Z., Defining Delta E, in Learn, Z. Schuessler, Editor. 2016: GitHub). In some example, an additional step of the alternation on saturation in the Hue Saturation Value colour space was required to remove non-coloured regions.

EXAMPLE 6 - Detection of Jaundice

A nappy comprising bilirubin oxidase is fitted to an infant. Urine from the infant passes through the liquid permeable bodyside liner to the absorbent body. If bilirubin is present in the infants urine, bilirubin oxidase catalyses a colorimetric reaction (e.g. oxidation of bilirubin present in the urine to biliverdin). The resulting development of colour serves as a visual signal that bilirubin is present in the infants urine. The infant can then be referred to a clinician for further diagnosis and confirmation of jaundice at an early stage.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the disclosure as shown in the specific embodiments without departing from the spirit or scope of the disclosure as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

The present application claims priority from AU 2017900289 filed 1 February 2017, the disclosure of which is incorporated herein by reference.

All publications discussed above are incorporated herein in their entirety. Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present disclosure. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed before the priority date of each claim of this application.

Claims

1. An absorbent article, comprising:

a liquid permeable bodyside liner;

- an outer cover; and

an absorbent body positioned between the bodyside liner and the outer cover;

2. The absorbent article according to claim 1, wherein the bilirubin oxidase is from Myrothecium sp., Magnaporthe sp., Pleurotus sp., Trachyderma sp., Penicillium sp., Bacillus sp., or a combination thereof.

3. The absorbent article according to claim 2, wherein the bilirubin oxidase is from

Myrothecium verrucaria, Magnaporthe oryzae, Pleurotus ostreatus, Trachyderma tsunodae, Penicillium janthinellum, Bacillus subtilis, or a combination thereof.

4. The absorbent article according to claim 3, wherein the bilirubin oxidase is selected from the group consisting of Myr. verrucaria MTl, Myr. verrucaria 3.2190, Myr. verrucaria B03, Myr. verrucaria IMERl, Myr. verrucaria FERM-P 5918, Myr. verrucaria IFO 6113, Myr. verrucaria IFO 6133, Myr. verrucaria IFO 6351 and Myr. verrucaria IFO, Magnaporthe oryzae P131 or a combination thereof.

5. The absorbent article according to claim 1, wherein the bilirubin oxidase comprises the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2.

6. The absorbent article according to any one of claims 1 to 5, wherein the bilirubin oxidase is encapsulated in a microcapsule.

7. The absorbent article according to claim 6, wherein the microcapsule is a starch- based material, a cellulose-based material, or a combination thereof.

8. The absorbent article according to any one of claims 1 to 5, wherein the bilirubin oxidase is covalently bound to one or more polymers.

9. The absorbent article according to claim 8, wherein the polymer is a polyacid.

10. The absorbent article according to claim 9, wherein the polyacid is polyacryclic acid.

11. The absorbent article according to any one of claims 8 to 10, wherein the polymer has a molecular weight between about 500 and about 100,000.

12. The absorbent article according to any one of claims 1 to 11, wherein about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%), about 90%, about 100% of the total surface area of the absorbent article comprises bilirubin oxidase.

13. The absorbent article according to any one of claims 1 to 12, wherein the absorbent article comprises a total amount of bilirubin oxidase less than about 0.4 units/mg of protein.

14. The absorbent article according to any one of claims 1 to 12, wherein the absorbent article comprises a total amount of bilirubin oxidase between about 0.001 units/mg of protein and about 0.4 units/mg of protein.

15. The absorbent article according to any one of claims 1 to 12, wherein the absorbent article comprises a total amount of bilirubin oxidase between about 0.025 units/mg of protein and about 0.05 units/mg of protein.

16. The absorbent article according to any one of claims 1 to 15, wherein the absorbent body comprises the bilirubin oxidase.

17. The absorbent article according to any one of claims 1 to 16, wherein the absorbent body comprises a urine permeable compartment zone, and wherein the compartment zone comprises the bilirubin oxidase.

18. The absorbent article according to claim 17, wherein the absorbent body comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 25, at least 50, at least 100, at least 1000 urine permeable compartment zones.

19. The absorbent article according to any one of claims 1 to 18, wherein the colorimetric reaction is confined to a compartment zone comprising the bilirubin oxidase.

20. The absorbent article according to any one of claims 1 to 19, wherein the absorbent body further comprises a control zone.

21. The absorbent article according to any one of claims 17 to 20, wherein the amount of bilirubin oxidase in a compartment zone is between about 0.001 units/mg of protein and about 0.4 units/mg of protein.

22. The absorbent article according to any one of claims 17 to 20, wherein the amount of bilirubin oxidase in a compartment zone is between about 0.025 units/mg of protein and about 0.05 units/mg of protein.

23. The absorbent article according to any one of claims 1 to 22, wherein the absorbent article comprises a colourant to enhance the colorimetric reaction visible when contacted with urine containing bilirubin.

24. The absorbent article according to any one of claims 1 to 23, wherein the absorbent article is a nappy or diaper or training pant.

25. The absorbent article according to any one of claims 1 to 23, wherein the absorbent article is an absorbent liner.

26. The absorbent article according to claim 25, wherein the absorbent liner is a nappy or diaper liner.

27. The absorbent article according to any one of claims 1 to 26, wherein the bilirubin oxidase remains stable for at least 3 months at room temperature.

28. The absorbent article according to any one of claims 1 to 26, wherein the bilirubin oxidase remains stable for at least 6 months at room temperature.

29. The absorbent article according to any one of claims 1 to 26, wherein the bilirubin oxidase remains stable for at least 12 months at room temperature.

30. A bilirubin oxidase, conjugated to a polyacid, wherein the bilirubin oxidase has improved capacity to detect bilirubin in urine compared to an unconjugated bilirubin oxidase.

31. The bilirubin oxidase of claim 30, wherein the polyacid has a molecular weight between about 1,000 and 100,000.

32. The bilirubin oxidase of claim 30, wherein the polyacid has a molecular weight between about 1,000 and 10,000.

33. The bilirubin oxidase according to any one of claims 30 - 32, wherein the polyacid is polyacrylic acid.

34. The bilirubin oxidase according to any one of claims 30 - 33, wherein the bilirubin oxidase is stable for 6 months at room temperature.

35. The bilirubin oxidase according to any one of claims 30 - 33, wherein the bilirubin oxidase is stable for 12 months at room temperature.

36. The bilirubin oxidase according to any one of claims 30 - 35, wherein the bilirubin oxidase can detect bilirubin in urine at 8 mg/dL.

37. The bilirubin oxidase according to any one of claims 30 - 35, wherein the bilirubin oxidase can detect bilirubin in urine at 5 mg/dL.

38. A bilirubin oxidase, conjugated to a polyacid, wherein the polyacid has a molecular weight between about 1,000 and 10,000.

39. The bilirubin oxidase of claim 38, wherein the polyacid is polyacrylic acid.

40. Method of detecting presence of bilirubin in urine from a subject, the method comprising: fitting a subject with the absorbent article according to any one of claims 1 to 29, visually assessing the article after the subject has urinated therein for a colorimetric change, wherein a colorimetric change indicates the presence of bilirubin in the subject's urine.

41. Method of diagnosing jaundice in a subject, the method comprising: fitting a subject with the absorbent article according to any one of claims 1 to 29, visually assessing the article after the subject has urinated therein for a colorimetric change, wherein a colorimetric change indicates that the subject has jaundice.

42. The method according to claim 38 or 39, wherein the subject is an infant.