Technical Field

[0001] Described herein are products in which liquid acidic reagents and, in particular, mercury - containing liquid acidic reagents are safely contained. Also described herein are diagnostic devices and methods for detecting a species indicative of a disease in a patient using the safely contained liquid acidic reagents.

Background Art

[0002] Many liquid reagents are toxic and/or corrosive and therefore need to be safely contained in order to prevent their escape. Reagents may be toxic and/or corrosive due to them containing toxic substances and/or because they are highly acidic or alkaline. One specific example of a reagent that is both toxic and highly acidic is Millon’s Reagent, which is obtained by dissolving metallic mercury in nitric acid and diluting with water. Millon’s Reagent has a number of applications, one of which is its ability to indicate the presence of phenolic compounds such as tyrosine and its metabolites in a patient’s urine, which can be indicative of the patient having a cancer such as colorectal cancer.

[0003] As Millon’s Reagent contains mercury, however, it needs to be very carefully handled. Conventionally, Millon’s Reagent is provided in a glass ampoule with a frangible portion. Immediately before use, the ampoule is broken and the urine sample is mixed with the Millon’s Reagent. However, the broken glass ampoule has sharp edges and presents a health hazard, and there is a risk that the user may be exposed to mercury (e.g. due to spillage). Furthermore, if the ampoule is not broken with a good sharp knock, it may shatter. For at least these reasons, existing tests involving Millon’s Reagent are not really suitable for non- specialised use.

[0004] The inventors recognised these limitations and invented a novel formulation for detecting a species indicative of a disease in a patient. In this formulation, an improved form of Millon’s Reagent was contained within a non-flowing carrier, such as a gel carrier or an absorbent material. As is described in international (PCT) application no. PCT/AU2016/050067, the contents of which are hereby incorporated in their entirety, upon exposure to a fluid sample from a patient (e.g. a urine sample), the non-flowing carrier quickly disintegrates, thus enabling rapid and complete mixing of the fluid sample and the mercury-containing reagent. A resultant colour change of the mixture to red is indicative of the presence of species such as tyrosine and/or its metabolites, which would prompt the patient to visit their doctor for further investigation.

[0005] Whilst the inventors’ earlier formulations are effective and have been successfully used to detect early stage cancers in many patients, the inventors have found that stability issues can occur if gel carriers are allowed to heat up too much, or if they are stored for particularly long periods of time. Whilst such storage conditions would be contrary to the formulation’s labelling, given the nature of the reagents carried in the gel carrier, the inventors recognised that providing for an improved storage of the mercury-containing reagent, as well as other toxic and/or corrosive liquid reagents, would be advantageous.

Summary of Invention

[0006] In a first aspect, the present invention provides a mercury-containing liquid acidic reagent contained by pH sensitive polymer film. The pH sensitive polymer film is configured to rapidly degrade upon contact with a fluid sample from a patient, whereupon the fluid sample and liquid acidic reagent can mix.

[0007] The inventors of the invention the subject of the present application have discovered that liquid acidic reagents (such as those containing mercury) can be contained just as safely by a pH sensitive polymer film as they can within a non-flowing carrier, such as the gel carriers and absorbent materials described in PCT/AU2016/050067, but without the risk of the liquid reagent escaping should the gel carriers be stored contrary to their indicated storage conditions. pH sensitive polymer films have also been shown to degrade when contacted with urine in a time frame that is compatible with the diagnostic applications described in PCT/AU2016/050067 and below.

[0008] As described in further detail below, physical samples produced in accordance with embodiments of the present invention have withstood temperatures of up to 40°C in stability trials, and have also worked as intended by disintegrating after 1-3 minutes of shaking after exposure to urine having pH ranging from 5.5 to 7.5.

[0009] In some embodiments, the pH sensitive polymer film may be configured to rapidly degrade when contacted with a fluid sample having a pH of above about 4.5.

[0010] In some embodiments, the the pH sensitive polymer film may be formed from one or more of the following: polyacids, polybases, natural polymers, hydrogels and multistimuli polymers. Stimuli-responsive polymer materials are responsive to multiple physical or chemical stimulus including pH, and examples of such polymers include poly(2-diisopropylaminoethyl methacrylate)-PDPA, poly(ethylene glycol)-block-poly(2-hexoxy-2-oxo-l,3,2- dioxaphospholane), Poly(N-isopropylacrylamide) (PNIPAM) and poly(lactic-co-glycolic acid) (PLGA).

[0011] In some embodiments, the pH sensitive polymer film may be formed from (or include) one or more of the following: polyvinyl derivatives such as polyvinyl acetate phthalate (PVAP), polyvinylpyrrolidone (PVP) and polyvinylpyrrolidone/vinyl acetate (PVP-VA); methacrylate copolymers such as methacrylic acid-ethyl acrylate copolymer type A (MA-EA); cellulosic polymers such as hydroxypropyl methylcellulose, methyl cellulose, hydroxy ethyl cellulose, cellulose acetate phthalates (CAP), cellulose acetate trimellitate; polyacrylic resins cellulose esters, glycans, waxes, shellacs, melamines, modified starches, resins, gums and chitosan.

[0012] In some embodiments, the pH sensitive polymer film may be configured to rapidly degrade in less than about 1 minute.

[0013] In some embodiments, the mercury-containing liquid acidic reagent may colourimetrically indicate whether the fluid sample contains a species indicative of a disease in a patient upon mixing with the fluid sample from the patient. The mercury-containing liquid acidic reagent may, for example, comprise a source of Hg ⁺ ions and a source of Hg ²⁺ ions in an acid solution. The mercury-containing liquid acidic reagent may further comprise one or more of the following: a source of Ni ²⁺ ions, a source of Cd ²⁺ ions, l-nitroso-2-naphthol, 4-aminoantipyrine, a diazonium compound and a tetrazolium salt. In some embodiments, the mercury-containing liquid acidic reagent may further comprise one or more of the following agents: dispersing agents, solubilizing agents, wetting agents and emulsifying agents.

[0014] In embodiments where the mercury-containing liquid acidic reagent colourimetrically indicates whether the fluid sample contains a species indicative of a disease upon mixing with a fluid sample from the patient, the species indicative of a disease may be an amino acid such as tyrosine (or its metabolites) and the disease a cancer (e.g. colorectal cancer). The fluid sample may be a urine sample.

[0015] In a second aspect, the present invention provides a diagnostic device for detecting a species indicative of a disease in a patient. The device comprises a cell in which a liquid acidic reagent (e.g. a mercury-containing liquid acidic reagent) is covered by a pH sensitive polymer film that is configured to rapidly degrade upon contact with a fluid sample from a patient, whereupon the fluid sample and the liquid acidic reagent can mix.

[0016] In some embodiments, the pH sensitive polymer film in the device of the second aspect may be as described herein in relation to the first aspect of the invention. [0017] In some embodiments, the liquid acidic reagent may be located at a bottom of the cell and overlayed by the pH sensitive polymer film.

[0018] In some embodiments, the cell may also include one or more glass balls or zirconia ceramic balls which, upon shaking of the device, impact on the pH sensitive polymer film. Such impacts may help to even more quickly degrade the film and enable mixing of the fluid sample and liquid acidic reagent.

[0019] In some embodiments, the liquid acidic reagent may be a mercury-containing reagent which, when mixed with the fluid sample from the patient, colourimetrically indicates whether the fluid sample contains the species indicative of a disease. In some embodiments, the mercury- containing reagent in the device of the second aspect may be as described herein in relation to the first aspect of the invention.

[0020] Similar to the first aspect, in embodiments of the second aspect where the liquid acidic reagent is a mercury-containing liquid acidic reagent, mixing of the reagent with the fluid sample from the patient colourimetrically indicates whether the sample contains tyrosine (or its metabolites). In such cases, the presence of tyrosine or its metabolites is indicative of the patient having cancer (e.g. colorectal cancer). In some embodiments, the fluid sample may be a urine sample.

[0021] In a third aspect, the present invention provides a method of detecting a species indicative of a disease in a patient. The method comprises: contacting the reagent according to the first aspect of the present invention with a fluid sample from the patient (e.g. a urine sample); causing (e.g. by shaking for about 1 minute) the fluid sample and liquid reagent to become mixed; and observing a colour of the mixture, which colour correlates to the presence of the species in the fluid sample .

[0022] In a fourth aspect, the present invention provides a method of detecting a species indicative of a disease in a patient. The method comprises: adding a fluid sample from the patient (e.g. a urine sample) to the test cell of the diagnostic device according to the second aspect of the present invention; shaking the device (e.g. for about 1 minute), whereby the fluid sample and the liquid reagent to become mixed; and observing a change in the mixture which correlates to the presence (or not) of the species in the fluid sample.

[0023] In a fifth aspect, the present invention provides a method for loading a liquid acidic reagent into a test cell of a diagnostic device for detecting a species indicative of a disease in a patient. The method comprises: adding the liquid acidic reagent to the test cell; covering the liquid acidic reagent with a substance comprising monomers capable of reacting to produce a pH sensitive polymer film; and curing the substance, whereby it hardens to form the pH sensitive polymer film covering the liquid acidic reagent.

[0024] In a sixth aspect, the present invention provides the use of the reagent according to the first aspect of the present invention for detecting a species indicative of a disease in a patient.

[0025] In a seventh aspect, the present invention provides the use of the diagnostic device according to the second aspect of the present invention for detecting a species indicative of a disease in a patient.

[0026] Other aspects, embodiments and advantages of the present invention will be described below.

Brief Description of Drawings

[0027] Embodiments of the present invention will be described in further detail below with reference to the following drawing, in which:

[0028] Figure 1 illustrates a diagnostic device for detecting a species indicative of a disease in a patient in accordance with an embodiment of the present invention.

Description of Embodiments

[0029] The overarching purpose of the present invention is to provide for the stable containment of relatively small volumes of toxic and/or corrosive liquid reagents, primarily for medical or diagnostic use, although the invention is not necessarily so-limited. The liquid reagent is a liquid acidic reagent and, in some embodiments, a mercury-containing liquid acidic reagent (e.g.

Millon’s Reagent and derivatives thereof). The present invention thus provides a diagnostic device for detecting a species indicative of a disease in a patient, the device comprising a cell in which a liquid acidic reagent is covered by a pH sensitive polymer film that is configured to rapidly degrade upon exposure to a fluid sample from a patient. Subsequent mixing of the fluid sample and liquid acidic reagent in the cell indicates the presence (or not) of the species, for example dur to the reactions and coloured precipitate formation described below.

[0030] The present invention also provides a mercury-containing liquid acidic reagent contained by a pH sensitive polymer film. The pH sensitive polymer film is configured to rapidly degrade upon contact with (e.g. immersion in) a fluid sample from a patient, whereupon the fluid sample and liquid acidic reagent can mix.

[0031] In the present invention, the reagent is contained or covered by a pH sensitive polymer film which is stable to low pH, metallic salts and other compounds in liquid acidic reagents such as Millon’s Reagent, such that it cannot leak past the barrier provided by the polymer film. As noted above, in PCT/AU2016/050067, containing the reagent either within an absorbent material or a carrier gel has been found to limit the products that can be used because of the severe reactive nature of the reagent. Although the inventors have successfully used both gels and absorbent materials as carriers for the reagent, they have also found it necessary to use storage conditions below 5 °C in order to completely ensure the stability of the gels (in particular).

Whilst such storage conditions would be indicated on the labelling, non-compliance may result in mercury-containing reagents escaping, which may be an unacceptable risk. Further, storage under the indicated conditions may be inconvenient for some users.

[0032] The reagent and diagnostic device of the present invention can be used for detecting a species indicative of a disease in a patient. Any species that can be detected (e.g. colourimetrically) upon exposure to the liquid acidic reagent (e.g. a mercury-containing reagent) may be detected in accordance with the present invention.

[0033] The invention will be described below primarily in the context of containing an improved form of Millon’s Reagent and for the detection of tyrosine (4-hydroxyphenylalanine) and/or its metabolites. The presence of tyrosine and/or its metabolites in a urine sample taken from a patient can be indicative of the patient having cancer. It is to be appreciated, however, that the invention has broader applicability, and the inventors expect that it would be suitable for use in detecting many other species indicative of disease.

[0034] The presence of elevated quantities of amino acids including tyrosine and/or its metabolites in a fluid sample obtained from a patient can be indicative of the patient having tumours, especially tumours associated with gastrointestinal cancers such as colorectal cancer. Amino acids such as tyrosine are used by cells to synthesise proteins, and elevated levels of tyrosine (and/or its metabolites, such as p-hydroxyphenylpyruvate, tyramine and L-DOPA) in the patient’s fluid sample can be indicative of their body producing a larger than usual number of cells, as is often the case with fast-growing tumours. Other tumours which might cause elevated quantities of amino acids such as tyrosine (and/or its metabolites) in a patient’s fluid sample include prostate cancer, ovarian cancer, bladder cancer, breast cancer, cervical cancer, stomach cancer, lung cancer, laryngeal cancer, oesophageal cancer, biliary cancer, hepatoma, duodenal cancer, bronchogenic cancer and bone marrow cancer.

[0035] In some embodiments, the species indicative of a disease in a patient may also include metabolites of tryptophan, such as 5 -hydroxy tryptamine (serotonin) and 5 -hydroxy indoleacetic acid (5-HIAA). Some cancer cells metabolise large amounts of tryptophan and the presence of elevated levels of tryptophan metabolites in a patient’s fluid sample may be indicative of cancer. In some embodiments, the species indicative of a disease in a patient may be melanin, the presence of which in a patient’s urine may be indicative of the patient having a melanoma.

[0036] This class of embodiments is described in further detail below, but it is important to recognise that the present invention is not limited to detecting only tyrosine or its metabolites and, in some embodiments, metabolites of tryptophan. For example, it is envisaged that embodiments of the present invention may also be used to detect raised levels of urinary nucleosides such as pseudouridine, 2-pyridone-5-carboxamide-Nl -ribofuranoside, N2, N2- dimethylguanine, 1 -methylguanosine, 2-methylguanosine and 1 -methyladenosine, the presence of which in fluid samples from patients may be used for early detection and clinical diagnosis of cancers such as colorectal, gastric, liver, lung and breast cancers.

[0037] In some embodiments, the fluid sample is a urine sample from the patient. As noted above, urine samples are relatively easy to obtain and often contain species indicative of a disease in a patient. Typically, the urine sample is obtained mid-stream (any potential contaminants will generally be present in a patient’s initial urine stream) from the patent’s first pass urination in the morning (which contains the highest concentration of markers). This class of embodiments is described in further detail below, but it is important to recognise that the present invention is not limited to detecting species in only urine. For example, it is envisaged that embodiments of the present invention may be used to detect the species in saliva, blood and, despite compliance issues surrounding its collection, faecal matter. Mercury-containing liquid acidic reagent

[0038] In some embodiments and aspects of the present invention, a mercury-containing liquid acidic reagent (also referred to below as the mercury-containing reagent) is contained/covered by the pH sensitive polymer film. When mixed with a patient’s fluid sample, the mercury- containing liquid acidic reagent can react to colourimetrically indicate whether or not the fluid sample contains a species indicative of a disease in a patient. That is, when the fluid sample is mixed with the mercury-containing reagent, a reaction that results in a colour change occurs in the event of the fluid sample containing the species. The reaction may, for example, result in the formation of a coloured precipitate. Alternatively (or in addition), the reaction may result in a colour change of the mixture.

[0039] In some embodiments, the mercury-containing reagent may comprise mercury dissolved in an acid solution. Such a reagent is known as Millon’s Reagent which, as noted above, has reliably been used to detect phenolic compounds (such as tyrosine) in fluid samples for many years. The acid solution is typically a mineral acid (preferably a strong mineral acid), such as nitric acid, sulphuric acid, or a combination thereof.

[0040] In some embodiments, the mercury-containing reagent may comprise different forms of mercury (e.g. salts having different oxidation states like Hg ⁺ and Hg ²⁺ ) or additional components that impart advantageous properties to the reagent or which make it more suitable for detecting a particular species. For example, the mercury-containing reagent may comprise a source of Hg ⁺ ions and a source of Hg ²⁺ ions (e.g. mercury(II) sulphate and mercurous nitrate), typically in an acid solution. Such a reagent is an improved form of Millon’s Reagent, and has been shown to have an even greater sensitivity to tyrosine and its metabolites. Any precipitates that form may also be more stable than would be the case for reagents containing just Hg ⁺ .

[0041] In some embodiments, the mercury containing reagent may comprise between about 10 and 90 wt% mercury, for example, between about 20 and 80 wt%, between about 30 and 70 wt%, between about 40 and 60 wt% mercury. In some embodiments, for example, the mercury containing reagent may comprise about 10 wt%, 20 wt%, 30 wt%, 40 wt%, 50 wt%, 60 wt%, 70 wt%, 80 wt% or 90 wt% mercury. In embodiments where the mercury containing reagent comprises Hg ⁺ and Hg ²⁺ , the ratio of Hg ⁺ : Hg ²⁺ may be from 0.1 to 1 : 0.1 to 1.0.

[0042] The mercury-containing reagent may also include a source of Ni ²⁺ ions, for example a nickel salt such as nickel sulphate, in order to improve the stability of the precipitate that forms in the presence of the species, and thereby improve the reliability of the test (precipitates having enhanced stability are more likely to reliably form and, once formed, last for longer, therefore making them easier to visually detect). If present, the mercury containing reagent may include nickel in an amount of between about 0.1 to 0.5 parts by weight to 1 part by weight mercury.

[0043] The mercury-containing reagent may also include a source of Cd ²⁺ ions, for example a cadmium salt such as cadmium sulphate, in order to improve the stability of the precipitate that forms in the presence of the species and thereby improve the reliability of the test. If present, the mercury containing reagent may include cadmium in an amount of between about 0.05 to 0.5 parts by weight to 1 part by weight mercury.

[0044] In embodiments where the species being detected includes tyrosine or its metabolites and, optionally, metabolites of tryptophan, the mercury-containing reagent may also include a compound such as l-nitroso-2-naphthol or 4-aminoantipyrine (or both), l-nitroso-2-naphthol and 4-aminoantipyrine can complex with 4 -hydroxylated phenolic acids such as tyrosine and the tryptophan metabolites serotonin and 5-HIAA to form a product having a red colour (the product is a different product to that formed when the species indicative of a disease reacts with the mercury-containing reagent). Elevated levels of 5-HIAA in patient’s fluid samples (especially urine samples) are well documented in gastrointestinal tumours, and the presence of l-nitroso-2- naphthol in the mercury-containing reagent may therefore help to enhance the sensitivity of the formulation to detecting species indicative of this particular disease. If present, the mercury containing reagent may include l-nitroso-2-naphthol and/or 4-aminoantipyrine in an amount of between about 0.05 to 0.5 parts by weight to 1 part by weight mercury.

[0045] The mercury-containing reagent may also include diazonium compounds, such as benzenediazonium chloride, 2,4-dichloroaniline diazonium salts, 2,6-dichlorobenzene- diazonium-tetrafluoroborate and p-nitrobenzene-diazonium-p-toulenesulfonate, which can complex with tyrosine and its metabolites, even under highly acidic conditions, in order to increase the sensitivity of the reagent.

[0046] The mercury-containing reagent may also include tetrazolium salts, such as tetrazolium diaminodiphenylamine. Such salts can form a fluorescent red dye in the presence of tyrosine and tryptophan.

[0047] The mercury-containing reagent may also include leuco chromophores, such as benzofuran derivatives, which can even further enhance a colour change.

[0048] The exact composition of the mercury-containing reagent will depend on factors such as the nature of the fluid sample and the species to be detected. It is within the ability of a person skilled in the art, using the teachings disclosed herein and in PCT/AU2016/050067, to prepare an appropriate mercury-containing reagent for use in the present invention. [0049] The amount or proportion of mercury-containing liquid acidic reagent contained in the diagnostic device or reagent will depend on factors such as the expected concentration of the species in the patient’s sample and the volume of the sample, etc. It is within the ability of a person skilled in the art to estimate the amount and proportion of mercury-containing reagent required for any given application and, if necessary, conduct routine tests to determine if the estimated proportion is appropriate. By way of example, the test cells described in further detail below contain between about 1.5 and 5 mL of the mercury-containing reagent.

[0050] The mercury-containing reagent used in the present invention may also include other components. For example, some embodiments may also include agents such as dispersing agents, solubilising agents, wetting agents or emulsifying agents to improve (or advantageously alter) properties of the pH sensitive polymer film (e.g. its disintegration properties). For example:

• dispersing agents can help to quickly disperse the mercury-containing reagent throughout the patient’s fluid sample (i.e. once the pH sensitive film has degraded), or aid to quickly disperse the degraded pH sensitive polymer film;

• solubilising agents can help to solubilise components in the reagent and patient’s fluid sample, which may enhance any reactions therebetween;

• wetting agents can help components in the reagent and patient’s fluid sample to mix, which may enhance any reactions therebetween; and

• emulsifying agents can help slightly immiscible components in the reagent and patient’s fluid sample to become more intimately mixed (and hence enhance any reactions therebetween).

[0051] The volume of the patient’s fluid sample required to detect whether the sample contains the species may vary depending on the nature of the species to be detected, its likely concentration in the sample and other factors either known to a person skilled in the art or readily ascertainable using routine experimentation. In some embodiments, the volume of the fluid sample required to detect whether the sample contains the species may vary between about 50- 150% of the volume of the reagent (e.g. the volume of the fluid sample is about 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140% or 150% of the volume of the reagent). If the volume of the fluid sample used is too low, then the amount of species present may be too low for detection and/or the pH sensitive film may not all disintegrate. If the volume of the fluid sample used is too high, then the resultant precipitate may not form or may be too dispersed for reliable detection. pH sensitive polymer film

[0052] In the present invention, the liquid acidic reagent or mercury-containing liquid acidic reagent is contained or covered by a pH sensitive polymer film. The pH sensitive polymer film is configured to rapidly degrade upon contact with (e.g. exposure to or immersion in) the patient’s fluid sample, after which the sample and reagent can mix in order to produce the effect described herein.

[0053] Any pH sensitive polymer film that will meet the functional requirements described herein may be used in the present invention. The film must be capable of containing the liquid acidic reagent for an appropriate period of time, without degrading or adversely affecting the reactions that result in the presence of the species being indicated, for example (i.e. it needs to be an acid-resistant film). The precursors to the pH sensitive polymer film (e.g. a substance or substances comprising monomers capable of reacting to produce the film) must also be stable and capable of reacting in the necessary manner in the presence of the liquid acidic reagent. Routine trial and experimentation, using the teachings contained herein, would enable a person skilled in the art to determine if a particular substance may be used to produce a pH sensitive polymer film suitable for use in the present invention.

[0054] Upon exposure to the patient’s fluid sample, the pH sensitive polymer film rapidly degrades, which enables the contained/covered liquid acidic reagent to mix, and react, with the sample. The pH sensitive polymer film may be configured using standard chemical techniques and the teachings contained herein to provide such functionality for any given liquid reagent and fluid sample.

[0055] In some embodiments, a pH of the patient’s fluid sample may cause the acid resistant film to rapidly degrade. For example, the acid resistant film may be configured to rapidly degrade when exposed to a fluid sample having a pH of above about 4.5 (e.g. above about 5.0, above about 5.5, above about 6.0, above about 6.5, above about 7.0, above about 7.5 or above about 8.0), which would encompass most bodily fluids including urine and saliva. The average pH of human urine is about 6, although can range from about 4.6 to 8.0, depending on diet. Saliva reportedly has a pH normal range of 6.2-7.6 with 6.7 being the average pH.

[0056] The inventors expect that the pH sensitive polymer film can be formed from any substance which can form (e.g. by solvent evaporation) a thin film coating over the liquid acidic regent or which can encapsulate the liquid acidic regent. For example, the inventors expect that one or more of the following: polyacids, polybases, natural polymers, hydrogels and multistimuli polymers would be suitable. [0057] As noted above, stimuli-responsive polymer materials are polymers which are responsive to multiple physical or chemical stimulus including pH, reduction, enzyme, temperature, diol moieties, reactive oxygen species, ionic strength, shear stress, and light. Examples of multistimuli polymers include poly(2-diisopropylaminoethyl methacrylate)-PDPA, poly(ethylene glycol)-block-poly(2-hexoxy-2-oxo-l,3,2-dioxaphospholane), Poly(N-isopropylacrylamide) (PNIPAM) and poly (lactic-co-gly colic acid) (PLGA).

[0058] More specifically, the pH sensitive polymer film may be formed from (or include) one or more of the following: polyvinyl derivatives such as polyvinyl acetate phthalate (PVAP), polyvinylpyrrolidone (PVP) and polyvinylpyrrolidone/vinyl acetate (PVP-VA), methacrylate copolymers such as methacrylic acid-ethyl acrylate copolymer type A (MA-EA), cellulosic polymers such as hydroxypropyl methylcellulose, methyl cellulose, hydroxy ethyl cellulose, cellulose acetate phthalates (CAP), cellulose acetate trimellitate, polyacrylic resins cellulose esters, glycans, waxes, shellacs, melamines, modified starches, resins, gums and chitosan.

[0059] In specific embodiments trialed by the inventors, polyvinyl acetate phthalate (PVAP) and methacrylic acid-ethyl acrylate (MA-EA) copolymer type A were used to produce a film that covered and was effective to contain a form of Millon’s Reagent in a test cell. In these trials, the liquid polymer was dropped onto the reagent surface and, by evaporation, formed an impervious film containing the Reagent. When a bodily fluid was added (pH greater than 5), the film disintegrated, allowing the fluid and the reagent to mix.

[0060] Other standard chemical techniques such as solvent casting may be used (as described below) to produce the pH sensitive film for use in the invention.

[0061] In some embodiments, the liquid acidic reagent contained in a cell may have a higher specific gravity than a liquid polymer precursor used to form the pH sensitive polymer film. In such embodiments, adding the liquid polymer to the surface of the reagent results in the polymer overlying the reagent as it hardens, thereby forming a completely isolating barrier over the acidic reagent.

[0062] The pH sensitive polymer film is configured to rapidly degrade upon contact with (e.g. immersion in) the patient’s fluid sample in order for mixing to occur and the resultant effect achieved. In some embodiments, the acid resistant film may be configured to rapidly degrade in less than about 5, 4, 3, 2 or 1 minutes.

[0063] The pH sensitive polymer film may have any suitable thickness, provided that it achieves the functionality described herein - i.e. thin enough that it can quickly degrade upon exposure to the patient’s fluid sample, but thick enough that it safely and reliably contains the liquid acidic reagent, even should accidental (minor) shaking or other disturbance occur. An appropriate thickness will depend on factors such as whether the film needs to completely encase the reagent or simply cover the reagent at the bottom of a test cell, the nature of the reagent and fluid sample and the rapidity with which the film would ideally disintegrate. It is within the ability of a person skilled in the art, based on the teachings contained herein and no more than routine trial and experimentation, to determine an appropriate film thickness for a given application.

[0064] In some embodiments of the diagnostic device, the liquid acidic reagent is located at a bottom of the cell and overlayed by the pH sensitive polymer film, as described in further detail below. Such a configuration is likely to ensure a more rapid and through mixing.

[0065] In some embodiments of the diagnostic device, the test cell also includes one or more glass balls or zirconia ceramic balls which, upon shaking of the device, impact on the pH sensitive polymer film and help to break it into smaller fragments for even more rapid degrading.

[0066] Any suitable device or cell may be used in the invention, such as those described in further detail below or in the applicant’s other patent specifications including

PCT/AU2016/050067 and Australian innovation patent no. 2020100739, for example. The device needs to be able to stably contain the mercury-containing reagent (in these cases) for its shelf-life (e.g. the material from which the device/test cell is formed itself also needs to be stable with highly acidic substances).

[0067] The diagnostic device (particularly the test cell) may be adapted to receive the fluid sample from the patient (e.g. by a patient either directly or indirectly urinating onto the container). In some embodiments, for example, the device may be adapted to receive a predetermined volume of the patient’s fluid sample. As would be appreciated, this would help to improve the accuracy and consistency of test results.

[0068] As described herein, it would be advantageous to provide a reliable test kit that can be used by the patient themselves, preferably in the comfort of their own home, for example. As such, embodiments of the device of the present invention may be provided in a form that is simple enough for patients to use at home (e.g. with freshly collected a urine sample), with instructions provided to emphasise the importance of the patient seeking specialised medical advice in the event of a positive result.

[0069] In some embodiments, the diagnostic device may include two (or more) cells, with at least one of the cells containing the liquid acidic (e.g. mercury-containing) reagent; and another cell containing a control sample that does not contain the reagent. The cells are configured to allow a visual comparison of the colour in each of the cells. In embodiments such as that described below, the device may be configured to provide a positive result in one of the cells, for direct comparison with the test cell that contains the reagent and patient’s fluid sample.

[0070] In such embodiments, a direct visual comparison can be made between at least two of the cells of the device in order to provide an even more reliable result because factors such as the colour of a patient’s urine (for example) or potential colour blindness of the patient (or person assessing the results) are accounted for.

[0071] In some embodiments, different amounts of liquid acidic (e.g. mercury-containing) reagent may be contained in each of the cells. After addition of the patient’s fluid sample and shaking, the gradation in colour across these cells may provide results indicative of a quantity of the species (e.g. tyrosine) in the sample, which may be correlatable with a severity of the disease.

[0072] A specific embodiment of a diagnostic device in accordance with an embodiment of the present invention will now be described with reference to Figure 1. Shown in Figure l is a test device 10, which has three cells 12, 14, 16. Cells 12 and 14 are for receiving a patient’s urine sample, and cell 16 is an overflow chamber which, as will be described below, ensures that the correct volume of urine remains in cells 12 and 14.

[0073] At the bottom of each of cells 12 and 14 is a mercury-containing liquid acidic reagent, depicted in the Figure as reagents 18 and 22 respectively. Reagents 18 and 22 are overlaid by a thin film of a pH sensitive material, depicted in the drawings as films 20 and 24 respectively. Films 20 and 24 prevent reagents 18 and 22 from escaping the device 10, even if it were to be shaken.

[0074] Notches 26 and 26 may be provided in the protrusion which separates cells 12, 14 and 16. Excess urine poured into cells 12 and 14 thus overflows, through a respective notch 26, into cell 16. This overflow ensures that cells 12 and 14 contain the same volume of urine. The device 10 also includes a lid 28, which can be folded over in order to seal the device and its cells 12, 14 and 16. Initially, the lid 28 will ensure that the cells 12, 14 and 16 cannot be contaminated with extraneous matter and, subsequently, so that urine (for example) cannot escape the device when it is shaken to produce the test result.

[0075] Cell 14 also includes a quick-dissolving tablet 30. Tablet 30 sits in the cell 14 on the opposite side of the film 24 from reagent 22, where it will be immersed in urine during use of the device 10, as described below.

[0076] In use, a patient’s urine sample (not shown) is poured into the cells 12 and 14 until they are full, with any excess urine overflowing through notches 26, 26 and into cell 16. The lid 28 is then put on the device 10, whereupon the cells are isolated from one another, and the device shaken. Immediately upon the urine making contact with the films 20 and 24, the films start to degrade because of their pH sensitivity. Tablet 30 also quickly dissolves. In some embodiments (not shown), cells 12 and 14 may include glass balls, or the like, which play no chemical role but which physically help to break the films 20 and 24 upon shaking, hence causing the films to even more rapidly degrade.

[0077] Once films 20 and 24 have disintegrated, the urine is able to mx with the reagents 18 and 22, whereupon a colorimetric indication occurs if the patient’s urine sample includes species indicative of a disease for the reasons described above and in PCT/AU2016/050067. Cell 12 contains just the patent’s urine sample and the reagent 18, whilst cell 14 contains the patent’s urine sample, the reagent 22 and the recently dissolved tablet 30. Tablet 30 contains species that will ensure that a colorimetric reaction occurs in cell 14 that is indicative of a positive result, in order to provide a clear comparison with the colorimetric result (if any) that occurs in cell 12. For example, tablet 30 may include tyrosine, or its metabolites which, as described above, will react with the inventors’ improved Millon’s Reagent and result in the formation of a clearly visible red precipitate in cell 14. Should the patient’s urine sample also contain tyrosine or its metabolites (which is indicative of the patient having a disease), then cell 12 will turn a similar colour to cell 14. A positive result in this test would prompt the patient to attend their physician to undergo further testing.

[0078] Notwithstanding the benefits described in the preceding paragraph regarding a positive result being shown, in alternative embodiments (not shown), cell 14 may be empty. The colour comparison between cells 12 and 14 is therefore between the colour of the patient’s urine (i.e. in cell 14) with the colour in cell 12 post-reaction between the urine and the reagent 18. This may, particularly for striking colour changes, be sufficient.

[0079] In another aspect, the invention provides a method of detecting a species indicative of a disease in a patient. The method comprises: contacting the reagent of the invention, as described above, with a fluid sample from the patient (e.g. a urine sample); causing (e.g. by shaking for about 1 minute) the fluid sample and liquid reagent to become mixed; and observing a colour of the mixture, which colour correlates to the presence of the species in the fluid sample. [0080] In another aspect, the invention provides a method of detecting a species indicative of a disease in a patient. The method comprises: adding a fluid sample (e.g. a urine sample) from the patient to the test cell of the diagnostic device of the invention, as described above; shaking the device (e.g. for about 1 minute), whereby the fluid sample and liquid reagent become mixed; and observing a change in the mixture which correlates to the presence of the species in the fluid sample.

[0081] The patient’s urine sample may obtained mid-stream from the patient’s first pass urination, for the reasons described above.

[0082] In a further aspect, the invention provides a method for loading a liquid acidic reagent into a test cell of a diagnostic device for detecting a species indicative of a disease in a patient. The method comprises: adding the liquid acidic reagent to the test cell; covering the liquid acidic reagent with a substance comprising monomers capable of reacting to produce a pH sensitive polymer film; and curing the substance, whereby it hardens to form the pH sensitive polymer film covering the liquid acidic reagent.

Examples

[0083] Proof of concept experiments have been carried out by the inventors, some of which are described below. The inventors have produced physical samples and carried out stability trials up to 35°C with no significant stability issues being observed. The inventors have also carried out trials with urine ranging from pH 5.5 to 7.5 (pH 5.5, 6.0, 6.5, 7.0 and 7.5), which resulted in the polymer films disintegrating after 1-3 minutes shaking.

[0084] The trialled polymers were polyvinyl acetate phthalate (PVAP) and methacrylic acid- ethyl acrylate (MA-EA) copolymer type A, which are polymers conventionally used in the manufacture of enteric coatings in the pharmaceutical industry. The polymers were dissolved in methanol and different concentrations of the polymers in various volume ratios were tested. The polymer solutions were applied on top of a sample of the inventors’ mercury-containing reagent (PCT/AU2016/050067), located at the bottom of a test cell (e.g. as shown in Figure 1). The samples were then allowed to dry at least overnight to allow the organic liquid to evaporate, leaving a solid film of polymer on top of the reagent. This film holds the reagent in place and prevents spillage in the event that the test kit is tipped over. The experimental conditions were varied (not all results are described here) in order to determine the optimal polymer- solvent mix and volume that forms a solid film that entirely covers the reagent, but which still disintegrates upon mixing with urine in an acceptable time period.

[0085] In a first series of trials, PVAP in amounts ranging from 1.5-3.5 g was dissolved in methanol and the resulting mixture had a weight of solute in g and volume of solvent in mL that totalled to 10 (e.g. 1.5 g PVAP in 8.5 mL methanol). Various polymer- solvent mixes successfully formed a film on top of the reagent, with 0.75 mL of a 2.5 g PVAP + 7.5 mL methanol mixture being found to produce the thinnest film without holes. The dissolution of this film was tested with urine pH = 5.41. Two glass balls (5 mm diameter) were included in each cell to assist film breakage. On this occasion, the film did not dissolve/break after 3 minutes of shaking. The experiment was therefore repeated using a urine sample having a pH of 7.0, with the film completely disintegrated after 3 minutes of shaking.

[0086] In a second series of trials, MA-EA in amounts ranging from 1.0-2.0 g was dissolved in methanol and the resulting mixture had a weight of solute in g and volume of solvent in mL that totalled to 10 (e.g. 1.0 g MA-EA in 9.0 mL methanol). 0.75 mL of a 1.0 g MA-EA + 9.0 methanol mixture produced the thinnest film without holes. The dissolution of this film was tested with urine with a pH range of 5.38-6.68. The film broke at pH > 6.38 without the use of an aid. For pH < 6.38, two glass balls were included in each well and all the tests were successful. Upon the addition of urine, the film’s surface appeared to begin disintegration. The film broke at around 2 minutes of shaking, allowing the reagent to mix with the urine.

[0087] The dried film formed using 0.75 mL of the MA-EA polymer solution was quite thin and the inventors appreciate that reproducing in large quantities might also be difficult and that the film may be too delicate for some handling conditions. Therefore, 1.0, 1.25, 1.5, and 1.75 mL of the 1.0 g MA-EA + 9.0 methanol mixture was also tested using the urine samples. 1.0 mL of the 1.0 g MA-EA + 9.0 methanol mixture was also tested using the urine samples in the pH range 5.38-6.68. The film broke after around 2 minutes of vigorous shaking with the aid of two glass balls (5 mm diameter) in each cell. However, 1.0 mL polymer solution was still too thin as samples taken from the stability trials (stored at 4 and 25°C) leaked between the interface of the container and the film after being manually inspected.

[0088] The amount of MA-EA solution was therefore increased to 1.25 mL and 1.5 mL, was tested for ease of breakage with urine and subjected to stability tests. The glass balls used to aide in film breakage were replaced by a higher density yttria stabilised zirconium (Zr) balls (5mm diameter). Two Zr balls were placed in each cell of the samples and tested using urine with pH ranging pH 5.51-6.46. The film of both cells containing either 1.25 mL or 1.5 mL MA-EA broke within 2 minutes of vigorous shaking at all pH levels in the range. Additionally, the integrity of the MA-EA polymer film remained intact for both volumes used after stability trials over prolonged periods.

[0089] 8.0 and 10.0 mm diameter Zr balls were also tested with samples prepared using 1.25, 1.5 and 1.75 of the MA-EA-methanol mixture. One ball was placed per cell and urine of pH 5.76 was used. The films broke under 1 minute of vigorous shaking when using the 8.0 mm ball and broke under 30 seconds using the 10.0 mm ball.

[0090] In conclusion, both MA-EA and PVAP produce films that can contain the mercury- containing reagent in the test kit. MA-EA produced the thinnest films that were effective to contain the reagent and disintegrate in the presence of urine. Films with volume of 0.75 mL will break when exposed to urine alone at pH > 6.38. However, at higher volumes and lower pH, using an aide (such as the glass balls) to break the film may be beneficial, in order to enhance the disintegrating effect of the urine. Finally, 0.75 mL of the MA-EA-methanol mixture is sufficient to form a film but may not withstand manual handling and transportation conditions. Using 1.25 mL or more of the wet mixture will provide a more stable and durable film to contain the liquid reagent.

[0091] Another series of trials were conducted to determine the minimum volume of methanol in the polymer- solvent mix that forms a solid film. 3 mL methanol was able to dissolve 1 g of MA- EA completely while allowing the mixture to be thin enough to work with. Any less methanol would be too viscous and difficult to apply a film over the reagent. 0.85 g of this mixture was found to be the minimum amount needed to form a solid film.

[0092] Further experiments were conducted to establish the stability of exemplary polymeric films in the device shown in Figure 1 over extended periods of time when stored at 25 °C (for about 14 months) and 35°C (for about 12 months). In these experiments (results not shown), a number of devices were prepared containing a sample of the inventors’ mercury -containing reagent (PCT/AU2016/050067) overlaid with a polymer film. Every month, one device was randomly selected from the ones stored at 25 °C and 35 °C and urine was added as described above. A freshly prepared device was used as a control sample and similarly tested with the same urine. Regardless of whether the devices were tested using urine from a healthy patient, or urine from a person with a known condition (i.e. gives a positive result), the same result was observed for the three devices (i.e. those stored at 25°C and 35°C and the freshly prepared device).

[0093] It will be appreciated that the present invention provides a number of new and useful results and advantages over existing diagnostic tests. For example, specific embodiments of the present invention may provide one or more of the following advantages:

• mercury-containing reagents are safely contained by the polymer film, which is less susceptible to stability issues;

• reliable results can be obtained due to the rapid mixing of the mercury-containing reagent and fluid sample (no second-guessing);

• testing can be performed by people with relatively little (or no) medical training - e.g. the patient themselves.

[0094] It will be understood to persons skilled in the art of the invention that many modifications may be made without departing from the spirit and scope of the invention.

[0095] In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word “comprise” or variations such as “comprises” or “comprising” is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.