The present invention relates to an indicator, a method for applying an indicator to a printing substrate, and products derived therefrom. In particular, the present invention relates to an oxygen and/or water sensitive ink, and to a method for applying the ink to a blank for a package, or to a printing substrate for forming a blank for a package. The products derived therefrom include, for example: a printing substrate, a blank, and a package to which the ink has been applied. Further products may include, for example, a time-temperature history indicator. The indicator composition can be used to detect oxidising agents, oxygen, water, reducing agents, temperature and the passage of time. Perishable goods, and in particular drinks and foodstuffs, are often provided in some form of air-tight packaging. This packing can be applied in a modified atmosphere (known as modified atmosphere packaging (MAP)), which limits the presence of oxygen. Alternatively, perishable goods may be packaged in normal atmospheric conditions.

Items that are packaged in air-tight packaging include: food, beverages, works of art, pharmaceuticals, medical diagnostic kits and sterilised packages. As mentioned, it is particularly desirable in the food industry to package goods such that their exposure to oxygen after packaging is minimised. This can be used to effectively extend the shelf life of many perishable items.

Background Art

It is useful to be able to determine the length of time for which a package containing perishable goods has been opened. To this end, many different sensors for detecting oxidising agents, and in particular for detecting oxygen, have been produced. Several of these sensors have been adapted for attachment to packages containing perishable goods. For example, GB 2419868, FR 2836677, WO 2006/077413, GB2344101 and US 2006/01 10835 disclose the use of oxygen sensitive dyes that are covered by a seal, the seal being broken by the opening of a package, and the dye changing colour over a set period of time to indicate the duration for which the package has been opened. WO 03/021252 (incorporated herein by reference) discloses a sensor for oxidising agents which is activated using UV light. This patent application discusses the use of a particular chemical composition which can be in the form of an ink, and which may be printed onto a variety of supports.

However, the indicator disclosed is necessarily sensitive to oxygen. It is not apparent from reading the application how such an indicator may be effectively integrated with, or printed onto, packaging in the reduced form. In addition, it is unclear as to how such an indicator may be applied in the reduced form in atmospheric conditions. The composition described in WO 03/021252 comprises at least one redox-sensitive material, at least one semiconductor material and at least one electron donor. The intimate contact of the various components of the indicator allows the redox sensitive material to undergo a redox reaction wherein there is a transfer of electrons from the photogenerated reduced form of the semiconductor material to the redox sensitive material.

The redox-sensitive material can be a dye such as a thiazine dyestuff, an oxazine dyestuff, an azine dyestuff, a triphenylmethane dyestuff, an indophenol dyestuff, an indigo dyestuff, viologen and/or mixtures thereof. The electron donor has the ability to donate electrons, preferably irreversibly. The electron donor may, for example, be an amine (e. g. NaEDTA and TEOA), reducing saccharide (such as glucose and fructose), readily oxidisable polymer (such as polyvinyl alcohol), a polyol (such as glycol) and other general antioxidants or an easily oxidizable material (such as glycerin) and/or mixtures thereof. The electron donor must not have sufficient strength (or reduction potential) to reduce the redox active material.

The electron donor is a sacrificial electron donor (SED). The SED is selected on the basis that, at the levels employed in the oxygen indicator, it: (a) does not reduce the redox-sensitive dye at a significant rate under either aerobic or anaerobic conditions; and (b) does not reductively quench the electronically excited state of the redox-sensitive dye under either aerobic or anaerobic conditions. These conditions being satisfied, the combination of redox-sensitive dye and mild reducing agent is stable and long-lived under ambient atmospheric and typical room-light conditions. Note that no strong alkaline material is present in the oxygen indicator since the latter turns agents such as some sugars, TEOA and

NaEDTA into strong reducing agents, and conditions (a) and (b) would no longer be satisfied.

A near UV-absorbing semiconductor photoactive material (SC) is also present in the oxygen indicator. The semiconductor material has the ability to form an excited electronic state that is sufficiently oxidising to oxidize the sacrificial electron donor and has a reduced form that is able to reduce the redox sensitive material. The semiconductor material may be an oxide of titanium (such as titanium (IV) oxide; Ti0 ₂ , and strontium titanate; SrTiOa), tin (such as tin (IV) oxide; SnC> ₂ ), tungsten (such as tungsten (VI) oxide; WO ₃ ) and zinc (such as zinc (II) oxide; ZnO) and mixtures thereof.

The role of the semiconductor is to initiate the process of indicator activation by absorbing some of the burst of near UV light that the indicator is exposed to. Absorption of a photon of near UV light by the semiconductor material in particle, film (micro or nanocrystalline) or single crystal form leads to the creation of a photogenerated electron-hole pair.

The semiconductor material is selected so that the photogenerated electron is sufficiently reducing in power that it can reduce the redox- sensitive dye present and the hole is sufficiently oxidising that it can oxidise the mild reducing agent present. The net effect upon UV activation of the combination of semiconductor material/redox-sensitive

dye/sacrificial electron donor that goes to make the oxygen indicator is that the dye is converted to its differently coloured, or fluorescent, reduced, oxygen-sensitive, form. For example, methylene blue (which is blue) is reduced to leuco-methylene blue (which is colourless) and the sacrificial electron donor, or mild reducing agent, is oxidised, i.e. SED to SEDox; both latter species are usually colourless.

The indicator may further comprise a binder which binds all the

components together. The binder may be a polymeric material such as gelatin, hydroxyethyl cellulose (HEC), polyvinyl alcohol (PVA), ethyl cellulose (EC), cellulose acetate (CEA), polypyrolidone (PVP),

polyethylene oxide, and polymethylmethacrylate (PMMA). Many modern day printing processes, and many types of modern packaging, require the use of organic solvent based inks. Whilst it is stated in WO 03/021252 that the indicator compositions can be combined with an organic solvent to provide an ink or printable solution, the inventors have found that the redox active species tends to leach out of such solutions. For example, the polymers described as binders are generally water soluble (such as polyvinyl alcohol), as are the redox active species (such as methylene blue). When these compositions are added to organic solvents the dye (redox active species) and the polymer do not dissolve well. Furthermore, on exposure of the indicator compositions described in WO 03/021252 to water or moisture (as is normally present to at least some degree in organic solvents and in the atmosphere), the dye in particular tends to leach out of solution. As the dye leaches out of solution the ink degrades and becomes much less effective (less coloured) and more difficult to bleach. Furthermore, the lifetime of the ink becomes more difficult to predict, limiting the use of the ink in, for example, time temperature indicators. Therefore, the indicator compositions described in WO 03/02 252 tend to break down on addition to an organic solvent, and on contact with water. Such compositions are therefore inherently unstable.

Generally a composition comprising the indicator compositions of WO 03/021252 will be unstable upon addition to, for example, food packaging. Typically, such a composition will break down upon addition to food packaging as the dye contained therein, upon contact with water contained in the food packaging or the atmosphere, will tend to leach out of the composition. Furthermore, the redox active species can leach out of solution following storage under ambient conditions, meaning that a solution of the composition is not suitable for use in printing. The inks and indicator compositions described in WO 03/021252 are therefore not suitable for use in many modern day printing processes, which require the use of organic solvent based inks, or in the application to many types of modern packaging, which require that the compositions are stable (i.e., resistant to leaching) on exposure to water. Moreover, it is not apparent how a charged, water soluble, redox active species (such as methylene blue) may be incorporated with a high degree of permanence into an organic solvent soluble indicator composition suitable for use in many modern day printing processes.

Furthermore, it can be impractical and expensive to activate indicator compositions (incorporated into inks) using UV light, particularly when using existing printing machinery and set-ups. For example, it can be difficult to expose the printed indicator composition to the correct amount of UV light in a controlled fashion, particularly as most printing processes do not have the time window to allow such exposure. Also, exposing the indicator composition to UV light after printing and sealing requires additional specialised equipment to be used in the printing process, and makes the process more complicated and time-consuming. Known UV activatable inks are unsuitable for activation before printing as they will change colour (i.e., oxidise) before or during the printing process. In addition, inks that are not activated using UV light are also unsuitable for activation before printing as they also change colour before or during the printing process. Furthermore, such inks are not normally soluble in organic solvents or mixtures thereof due to the ionic nature of the materials involved.

Therefore, it is an object of the present invention to obviate or mitigate at least some of the disadvantages of the prior art. A further object of the invention is to provide a method for applying an oxygen sensitive ink to a printing substrate.

A still further object of the invention is to provide an indicator ink that can be used in modern day printing processes.

Disclosure of Invention

According to a first aspect of the invention there is provided an indicator for detecting at least one of an oxidising agent and water, the indicator comprising an organic solvent soluble polymer and a redox sensitive material which displays different visible properties in the oxidised and reduced forms, wherein the redox sensitive material in the oxidised form is bound to the organic solvent soluble polymer by ionic bonding, and wherein the indicator further comprises a reducing agent and/or the oxidation product therefrom.

The indicator is in the form of a composition. The oxidising agent may be oxygen. In the context of the invention, a "reducing agent" is a material that has sufficient strength (or reduction potential) to directly reduce the redox sensitive material without the aid of further compounds, such as semiconductor materials, or external stimuli, such as UV light. The oxidation product or products of a reducing agent are the compounds produced when a reducing agent acts as such, providing electrons to another species. For example, when the reducing agent is dithionite, the oxidation products therefrom may include thiosulfate and bisulfite.

The indicator of the present invention contains an ion-paired dye (the oxidised, positively charged dye can ion pair with the negatively charged polymer). Surprisingly, the inventors have found that this ion-paired dye bleaches (or reduces) more completely and more quickly than the same dye which is not ion paired. This allows the indicator composition of the present invention to be activated more quickly and more completely than known indicators incorporating redox dyes.

The inventors have surprisingly found that the redox active material in the indicator of the present invention can be reduced directly using a reducing agent. Ordinarily, this would not be possible as the combination of materials would generally be incompatible insofar as they exhibit such different polarities that they would not all dissolve in an organic, aqueous, or mixed solvent system. Thus, previously it has been impossible to produce an indicator (and a related ink) of this type, which show sufficient longevity when reduced and stability to be usable in a printing process.

The indicator of the present invention also allows a large amount of dye to be incorporated, yet shows significant resistance to leaching, even in the presence of water. Furthermore, the indicator is soluble in, and compatible with, organic solvents such as ethanol, acetone and ethyl acetate. Surprisingly, the ink is also soluble in, and compatible with, organic/aqueous solvent mixes such as mixtures comprising ethanol and water. Therefore the ink is more stable when stored for relatively long periods of time, and more suitable for use with modern day printing processes.

Furthermore, once reduced, the indicator composition of the present invention takes typically hours or days (depending on the ambient conditions, especially temperature) to oxidise back to its original colour. Furthermore, the rate of oxidation of the composition of the present invention is highly predictable and controllable. These aspects mean that the indicator composition is particularly useful in indicating the freshness of foodstuffs and the like.

Also, the inventors have found that when the ink of the present invention is applied to packaging as an indicator composition, the composition shows very low levels of migration through the substrate to which it is applied. This is advantageous for the packaging industry, and particularly the food packaging industry, as the composition has less tendency to migrate through packaging, and therefore is less likely to contaminate the food or drink contained therein.

In addition, the inventors have found that the ink of the present invention is sufficiently stable and long lasting in the reduced form, that it can be reduced before being used in the printing process, obviating the need to integrate a reduction step into the printing process or to use UV light.

Water may be in the form of moisture in the atmosphere, typically having a Relative Humidity > 10 % at 20 °C. According to one aspect of the invention, the indicator consists essentially of the organic solvent soluble polymer, the redox sensitive material, and the reducing agent and/or the oxidation product therefrom.

According to another aspect of the invention, the indicator consists of the organic solvent soluble polymer, the redox sensitive material, and the reducing agent and/or the oxidation product therefrom.

The reducing agent may have sufficient strength to reduce the redox sensitive material. In particular, the reducing agent may have sufficient strength to reduce the redox sensitive material in the absence of a 01728

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semiconductor material. More specifically, the reducing agent may have sufficient strength to reduce the redox sensitive material in the absence of a semiconductor material and UV light. The strength of the reducing agent may be expressed in terms of its reduction potential. Thus the reducing agent may have sufficient reduction potential to reduce the redox sensitive material. Typically, the reducing agent has a reduction potential of at least 0.1 1 V at pH 7 vs NHE (normal hydrogen electrode).

The reducing agent may be selected from one or more of the group consisting of: dithionites, sulphites, ascorbic acid, an ascorbic acid

hydrochloric acid mixture; or acceptable salts thereof; and/or the oxidation products therefrom.

Typically the reducing agent is a dithionite; or an acceptable salt thereof; and/or the oxidation products therefrom.

Preferably the organic solvent soluble polymer comprises a hydrophobic backbone and a plurality of electronically charged sidechains.

The organic solvent soluble polymer is at least partially-sulfonated

polystyrene. Typically the at least partially-sulfonated polystyrene is from about 10% to about 30% sulfonated. The at least partially-sulfonated polystyrene may have the formula ([sulfonated polystyrene unit]o.i to o.3x[polystyrene unit]i _-x ) _n .

The redox sensitive material may be selected from one or more of the group consisting of: a thiazine dyestuff, an oxazine dyestuff, an azine dyestuff, a triphenylmethane dyestuff, an indophenol dyestuff, an indigo dyestuff and viologen. Typically the redox sensitive material is methylene blue.

The indicator in both the activated and non-activated forms may be insensitive to UV light, typically having a wavelength of about 200-400 nm.

In one embodiment the indicator does not include an electron donor, said electron donor having insufficient strength to reduce the redox sensitive material. Typically the electron donor has insufficient reduction potential to reduce the redox sensitive material. Most typically the electron donor has a reduction potential less than 0. 1 V vs NHE (normal hydrogen electrode).

Typically the indicator does not include an amine, a reducing saccharide, a readily oxidisable polymer, a polyol, glycerol, trihydroxyhexane or a general anti-oxidant.

In one embodiment the indicator does not include a semiconductor material. Typically the semiconductor material is specifically sensitive to light having a wavelength of about 200-400 nm.

Typically the indicator does not include an oxide of: titanium, tin, tungsten, zirconium, zinc or mixtures thereof. The indicator may comprise about 79.1 % by weight organic solvent soluble polymer, about 6.3 % by weight redox sensitive material, and about 14.6 % by weight reducing agent. The % by weight figures are in the absence of solvent. The indicator may be supported on an inert material such as glass, paper, fabric, plastic, ceramic or metal.

Optionally the inert material comprises a coating configured to seal at least a portion of the inert material having the indicator applied thereto.

The seal may have a very low oxidising agent permeability thereby preventing oxidation of the indicator. Alternatively, the coating may provide a semi-permeable seal configured to allow controlled flow of oxidising agent to the indicator.

The seal may be selected from one or more of the group consisting of: PET, EVOH, PVDC, PVA and regenerated cellulose. The seal may be configured to be selectively removable.

In one embodiment, the indicator is dissolved in a solvent to form an ink.

The solvent may comprise an organic solvent. The organic solvent may be selected from one or more of the group consisting of: alcohols, including methanol and ethanol; acetone and ethyl acetate.

Optionally the solvent further comprises an aqueous solvent. The organic solvent may be ethanol and the aqueous solvent may be water. The solvent may comprise around 80 % by weight ethanol.

The solvent may comprise around 20 % by weight water.

According to a fourth aspect of the invention there is provided an ink comprising the indicator as described herein and a solvent. According to a further aspect of the invention there is provided an ink consisting essentially of the indicator as described herein and a solvent. According to a still further aspect of the invention there is provided an ink consisting of the indicator as described herein and a solvent.

Typically the solvent comprises an organic solvent. The organic solvent may be selected from one or more of the group consisting of: alcohols, including methanol and ethanol; acetone and ethyl acetate.

Optionally, the solvent further comprises an aqueous solvent.

The organic solvent may be ethanol and the aqueous solvent may be water.

The solvent may comprise around 80 % by weight ethanol. The solvent may comprise around 20 % by weight water.

The ink may comprise from about 5.6 % to about 6.6 % by weight organic solvent soluble polymer, from about 0.45 % to about 0.5 % by weight redox sensitive material, from about 1.05% to about 1.2 % by weight reducing agent, and from about 91.7 % to about 92.9 % by weight solvent.

The ink may comprise from about 5.6 % to about 6.6 % by weight organic solvent soluble polymer, from about 0.45 % to about 0.5 % by weight redox sensitive material, from about 1.05 % to about 1.2 % by weight reducing agent, from about 56.3 % to about 65.5 % by weight organic solvent and from about 26.2 % to about 36.6 % by weight aqueous solvent.

Generally the indicator is > 17 weight percent (wt %) soluble in an organic solvent such as acetone, ethanol or ethyl acetate. Typically the ink of the present invention is stable upon storage. Generally the ink is stable upon storage for at least six months under dark, but otherwise ambient, conditions. Suitably at least 99% of the redox sensitive material remains in solution following such storage, more suitably substantially all of the redox sensitive material remains in solution.

According to a seventh aspect of the invention there is provided a printing substrate for use in the manufacture of a blank for a package or a package, wherein the printing substrate comprises the indicator as described herein or the ink as described herein.

According to a further aspect of the invention there is provided a blank prepared from a printing substrate as described herein. According to a still further aspect of the invention there is provided a package prepared from a printing substrate or a blank as described herein.

The indicator and/or the ink described herein can be applied to printing substrates (such as paper and card) used in the manufacture of blanks and packages.

According to a further aspect of the invention there is provided a method of detecting exposure to at least one of an oxidising agent and water, comprising the steps of: a) providing an indicator as described herein; and

b) subsequently detecting a visible change in the indicator

whereby exposure to an oxidising agent or water is revealed. The method may comprise the further step of removing a seal from the indicator to expose the indicator to at least one of an oxidising agent and water.

The indicator may be exposed to both an oxidising agent and water.

Water can be in the form of moisture, water vapour, or relative humidity, the relative humidity typically being≥ 10 %.

According to a further aspect of the invention there is provided an indicator for detecting when an object has been subject to an increase in

temperature for a set period of time, comprising the indicator as described herein.

According to a still further aspect of the invention there is provided a method of detecting when an object has been subject to an increase in temperature for a set period of time comprising the steps of:

a) providing to the object the indicator as described herein; and b) subsequently detecting a visible change in the indicator, whereby exposure to an increase in temperature is revealed.

When the indicator is frozen (i.e., subject to temperature < -20 °C), it loses its ability to respond to oxidising agents, but the sensitivity to oxidising agents is restored on exposing the indicator to warmer temperatures. This enables the indicator to be used in time-temperature indicators. Time-temperature indicators operate by showing (once activated) no optical effect over a desired period at, or below, a desired temperature (say 4 °C for an indicator for a refrigerated item, and -20 °C for an indicator for a frozen item). However, time-temperature indicators do show an optical effect over the same desired period if the temperature of the indicator (and the item to which it is applied) exceeds a predetermined minimum temperature. Thus, time-temperature indicators can be used to show an item has been exposed to a temperature above the

recommended minimum temperature for storage. For example, a tirne- temperature indicator can show that an item has been defrosted and re- frozen.

According to a further aspect of the invention there is provided a method of applying an oxidising agent and/or water indicator to a printing substrate, the method comprising the steps of:

a) providing a printing substrate to a printing assembly;

b) applying to the printing substrate the indicator as described herein; and

c) providing a coating configured to seal at least a portion of the printing substrate having the indicator applied thereto.

Under very dry conditions (typically < 10 % relative humidity at 20 °C), the indicator ceases to respond to oxidising agents, but the sensitivity to oxidising agents is restored on exposure to air with a relative humidity≥ 10 %. The indicator can be reduced, or activated/bleached, at very low humidity (< 10 % relative humidity). However, under these low humidity conditions (such as found in dry foods, or in vacuum packed foods) the indicator will not effect a visible change in the presence of an oxidising agent, such as oxygen. Thus water, usually as a vapour, can be used to trigger the indicator. For example, a colourless (i.e., activated) film in air, at a relative humidity < 10 % at 20 °C will change colour (i.e., oxidise) on exposure to ambient air with a relative humidity≥ 10 %.

This method enables the stable compositions of the present invention to be activated before printing. Due to their stable nature, the compositions of the present invention can be used in the reduced state, and in a normal atmosphere printing process, without appreciably oxidising. Therefore, there is no requirement to apply UV light to printed packages, as the ink contained therein is already activated. This removes the need for additional specialist equipment during printing, and obviates the problem of fitting an activation step into the printing process.

The printing methods may be carried out in the presence of oxygen, and in particular may be carried out in atmospheric conditions. This is in contrast to known methods of applying indicators for detecting oxidising agents. Such known methods must generally be carried out in an atmosphere having a reduced oxygen content, or require activation using UV light. This increases the cost and inconvenience of such known methods.

Furthermore, if the amount of oxygen present in the atmosphere is not sufficiently low, the reliability of the indicator may be adversely affected. Therefore, the method disclosed herein is more cost effective than known methods. Also, the printing methods enable activation of the indicator at any time as chosen by the manufacturer. For example, the activation may take place sometime after the printing process, and printed packages can be stored and transported in normal atmospheric conditions. Alternatively, using the first printing method, the indicator can be activated before printing commences.

The method may comprise the further step of activating the redox sensitive material by combining: i) a first composition comprising the organic solvent soluble polymer and the redox sensitive material; and ii) a second composition comprising the reducing agent. Optionally the indicator is applied to the printing substrate in the form of a LOGO or text.

The printing substrate may be configured to prepare a blank for use in the manufacture of a package.

The method may comprise the further step of arranging the blank to form a package.

According to another aspect of the invention there is provided a printing substrate prepared by the method as described herein.

According to a further aspect of the invention there is provided a blank prepared from a printing substrate as described herein. According to a still further aspect of the invention there is provided a package prepared from a printing substrate or a blank as described herein.

A two part composition for detecting at least one of an oxidising agent and water, said composition comprising the indicator as described herein, and wherein said composition comprises:

i) a first composition comprising the organic solvent soluble polymer and the redox sensitive material; and ii) a second composition comprising the reducing agent. The indicator and ink as described herein can be stored as two separate compositions which are combined just before use. For example, a first composition can include a redox active material and an organic solvent soluble polymer, and a second composition can include a reducing agent. When the two compositions are combined, the redox material becomes reduced. The so-formed indicator or ink will stay reduced for sufficient time to allow printing to take place. In this way, the indicator of the present invention can be stored indefinitely. In one embodiment, a printing substrate is prepared wherein a portion of the seal covering the indicator is detachable from the printing substrate by a user. For example, when the printing substrate is configured as a fruit drink package, the portion of the seal is attached to the lid of the package, and is automatically removed from the package when the package is opened by a user. Therefore, the seal is configured to be selectively removable.

The method of the present invention enables application of an oxygen sensitive indicator in the oxidised form, and potentially in the presence of oxygen. Once applied to a printing substrate, the indicator is sealed using a material that inhibits oxygen, or oxidising agents, from reacting with the indicator. After sealing, the indicator is exposed to UV light which converts the indicator to the reduced form. The indicator remains sealed from the atmosphere until such time that the package is opened, at which point the seal is broken or removed thus exposing the indicator to the atmosphere. Once exposed to the atmosphere, the indicator will change colour at a particular rate dependent on the particular composition of the indicator, and optionally the temperature. This same method can be used to apply a water sensitive indicator, a time temperature indicator or a security indicator.

Alternatively, the seal may be semi-permeable, and may be configured to allow ingress of an oxidising agent at a predetermined rate, without the requirement of removing or breaking the seal.

In one embodiment, the present invention enables the application of an oxygen sensitive ink to a printing substrate using commonplace printing techniques. There is no requirement for the atmosphere to be modified, as is the case with modified atmosphere packaging. However, it will be appreciated that the technique could also be used in a modified atmosphere packaging process. The printing methods described herein are used in the manufacture of printing substrates (such as paper and card), blanks and packages.

Optionally the printing substrate is configured to prepare a blank for use in the manufacture of a package.

According to a further aspect of the present invention there is provided a printing substrate obtainable according to the printing methods as described herein. The present invention will now be described by way of example only. Modes for Carrying out the Invention

Most colourimetric oxygen indicators rely on a reaction between a redox- sensitive indicator dye, such as methylene blue, and a strong reducing agent, such as glucose in a strongly alkaline (pH > 12) and aqueous environment. This reaction leads to the reduction of the redox-sensitive dye and concomitant colour change. The redox dye is readily reoxidised back to its original colour upon exposure to oxygen. Such inks are unsuitable for most printing processes as they are water based. Known indictors comprising a redox-sensitive dye, a sacrificial electron donor and a semiconductor photoactive material have been described for the detection of oxygen in WO 03/021252. However, such dyes are not usable in most organic solvents, as the redox-sensitive dye used tends to leach out of solution. Also, such dyes are designed to be activated using UV light.

Although the following discussion is limited to that of oxygen it should be realised that the discussion is applicable to any other type of oxidising agents.

The novel oxygen indicator described herein utilises at least one redox- sensitive dye (eg. a thiazine dyestuff, oxazine dyestuff, azine dyestuff, triphenylmethane dyestuff, indophenol dyestuff, indigo dyestuff, viologen or a mixture thereof). The redox-sensitive dyestuff is also chosen so that its reduced form has a different colour, and/or fluorescence, to its oxidised form and is oxidised to the latter by oxygen.

The indicator of the present invention also comprises an organic solvent soluble polymer which can bind the redox-sensitive material by ionic bonding, at least when the redox-sensitive material is in the oxidised form. The polymer can have a hydrophobic backbone and electronically charged sidechains. For example, the polymer can be partially-sulfonated polystyrene or another suitable polymer that has at least some

electronically charged, or sufficiently polar, sidegroups that have sufficient affinity for the redox dye to mitigate leaching of the dye under storage or on exposure to organic solvents or water, to produce an ink that is useable in the printing process.

The oxygen indicator can be re-used simply by reactivating with a suitable reducing agent. Note that the oxygen indicator is not selective towards oxygen, but will also respond to most strong oxidising agents if they are present, such as chlorine, nitrogen dioxide and ozone.

This lack of selectivity towards oxygen is also a feature of almost all other oxygen indicators. Fortunately, in most packages (especially food) and the ambient environment, there are no, or very little, oxidising agents other than oxygen. The sensitivity of the oxygen indicator towards oxidising gases other than oxygen may be exploited to create indicators for these other gases.

The oxygen sensing action of the oxygen indicator is irreversible in that it only works after it has been activated by exposure to a reducing agent of sufficient strength. Once oxidised, it cannot be reactivated unless it is deliberately exposed again to a reducing agent.

The dyestuff brings about the colour change exhibited. The dyestuffs that can be used include: thiazine dyestuffs (such as: methylene blue, thionin and toluidine blue), oxazine dyestuffs (such as: resazurin, safranine O, and celestine blue), azine dyestuffs, (such as: and cresyl violet acetate and azure A), indophenol dyestuffs (such as dichloroindophenol), indigo dyestuffs (such as; indigo and indigo carmine), viologens (such as heptyl and benzyl viologen) and mixtures thereof.

The organic solvent soluble polymer binds with the dyestuff to ensure that the dyestuff remains soluble in organic solvent. Organic solvent soluble polymers that can be used include partially-sulfonated polystyrene or another suitable hydrophobic, anionic, organic solvent soluble polymer.

When combined with a suitable solvent, the initial form of the oxygen indicator is as an ink or castable solution that can be printed or cast on a wide variety of supports. Examples of typical solvents that can be used include: ketones (such as acetone), alkylhalides (such as chloromethane), esters (such as ethyl acetate), alcohols (such as methanol and ethanol) and aromatics (such as toluene). Mixtures of organic solvents and aqueous solvents can also be used, such as ethanol and water.

General Example

A mixed solvent (organic and aqueous) based ink, according to one embodiment of the present invention, and comprising sulfonated- polystyrene (a polymer binder), sodium dithionite (a reducing agent) and methylene blue (a dye) was prepared as described below.

Preparation of sulfonated polystyrene

The sulfonated polystyrene (SPS) used was prepared in-house by the direct sulfonation of polystyrene as described in previous literature (C. R. Martins et al, Journal of the Brazilian Chemical Society, 14 (2003) No. 5; Makowski et al, US Patent 3870841 (1975); R. A. Weiss et al, Journal of Polymer Science: Polymer Chemistry Edition, 23 (1985) pp 525-533). Initially, 52 g of polystyrene (average molecular weight 250,000, supplied by Acros Organics) was placed in a 3-necked, round-bottomed flask and to it was added 245 mL of dichloromethane (DCM). The solution was stirred vigorously on a magnetic stirrer to promote dissolution of the polymer, typically requiring 1 to 2 hours for complete dissolution. Whilst the polystyrene was dissolving, acetyl sulphate (which acts as the sulfonating agent) was prepared. 49 mL of DCM was placed in a conical flask sealed with a rubber septum stopper, and to it was carefully added 9.5 mL of acetic anhydride via a syringe. The resulting solution was immediately placed under an argon atmosphere and cooled using an ice bath. Once sufficiently cool, and once the polystyrene had dissolved in the DCM, 3.5 mL of 95 % sulphuric acid was added dropwise to the DCM/acetic anhydride mixture, thus converting the acetic anhydride to acetyl sulphate. Once all of the sulphuric acid had been added, the acetyl sulphate was removed from the argon atmosphere. 35 mL of acetyl sulphate was then extracted and subsequently added to the stirred polystyrene solution.

After addition of the acetyl sulphate, two of the three necks of the round- bottomed flask were sealed with rubber septum stoppers, and the flask was transferred to an oil bath on a hotplate stirrer. A reflux condenser was attached to the free neck of the round-bottomed flask, and the hotplate was set to a constant 40 °C using a fuzzy logic temperature control attachment. Once at 40 °C, the stirring solution was left under reflux for 4 hours. During this time the solution typically changes colour from colourless to a pale blue. Also note that, whilst not strictly necessary, the degree of sulfonation of the polystyrene tends to increase if the refluxing solution is bubbled gently with argon over the 4 hour period. By increasing the degree of sulfonation, the SPS is found to be more soluble in both acetone and ethyl acetate.

After 4 hours, the solution was removed from reflux (and from the argon atmosphere if applicable) and to it was added 50 mL of ethanol. Upon addition, a white precipitate was visible. The solution was gently swirled to aid the distribution of the ethanol throughout the round-bottomed flask before the mixture was poured slowly and carefully into 1.75 L of boiling water. As it was added, the sulfonated polystyrene precipitated rapidly, hence it was necessary to stir the boiling water, preferably through the use of both a magnetic stirrer and a glass/plastic rod operated manually. It was also necessary to add the sulfonated polystyrene solution in stages, removing the precipitate at regular intervals to increase yield. Once complete, the precipitate was washed with water several times before being transferred to a vacuum desiccator for drying. For best results, the precipitate was left overnight to dry. Typically the synthesis, as outlined above, yields ca. 50 g of SPS.

A batch of enhanced polarity sulfonated polystyrene was prepared for use in making indicator compositions that could be used to prepare ethanol based inks. The method used was identical to that described above, except the amount of acetyl sulphonate used in the sulfonation step was increased to 105 mL (i.e., 3 times as much acetyl sulfonate was used). The resulting enhanced polarity sulfonated polystyrene was soluble in ethanol, but insoluble in both ethyl acetate and water. The degree of sulphonation for the SPS and enhanced polarity SPS is between 10% and 30%.

Preparation of solvent-based ink

A solvent based oxygen sensitive ink, in accordance with one embodiment of the present invention, was prepared as follows.

200 mg of SPS was weighed into a small sample vial and to it was added 2 g of ethanol, 800 mg of water and 16 mg of methylene blue. The solution was stirred and nitrogen purged until all the polymer had dissolved to give a deep blue solution. To the solution was then added 0.5 ml of 0.43 M sodium dithionite solution, and the solution immediately changed colour, turning transparent light yellow. The solution was allowed to stir overnight, and remained a transparent light yellow colour. Further ethanol based inks were prepared as follows. 40 mg of methylene blue was added to a sample bottle, to which was added 5 g of ethanol and 2 g of deionised water. The bottle was then closed and sonicated for 10 minutes before the solution was left to gently stir until all the methylene blue had mixed well. After all the methylene blue had dissolved, 500 mg of the enhanced polarity SPS was then added to the solution. The solution formed a gel like substance within the sample bottle as soon as the enhanced polarity SPS was added. The sample bottle was sonicated for approximately 40 minutes, then left to stir gently to facilitate the gel reforming into a solution. A five-fold excess (based on the amount of methylene blue) of sodium dithionite was then added to the solution, effecting a colour change from blue to colourless/light yellow.

A further ethanol based ink was prepared as follows. 250 mg of enhanced polarity SPS was weighed into a small sample vial and to it was added 2 g of ethanol. The solution was gently stirred until all the polymer had dissolved. A five-fold excess (based on the amount of methylene blue) of sodium dithionite was then added to the solution, effecting a colour change from blue to colourless/light yellow. A further ethanol based ink was prepared using the techniques described above. The amounts of materials used in the ink were: 3 g of ethanol; 1 g of water; 500 mg of enhanced polarity SPS; 30 mg of methylene blue; and a five-fold excess (based on the amount of methylene blue) of sodium dithionite. Note that the amount of methylene blue used depends on the desired intensity of "blue" colour. For example, from around 2.5 mg to 40 mg of methylene blue can be used for the solvent quantities discussed above. Alternative solvents that can be used in place of ethanol are acetone and/or ethyl acetate.

Activation of the inks

The inks were activated before they were prepared as films on substrates, and before any experimental studies were carried out. The ink is sufficiently stable that it can be prepared substantially as described above to provide an activated (i.e., reduced) ink which can be stored in inert conditions without becoming oxidised. Alternatively, the ink may be provided in two separate compositions, one comprising the SPS and the methylene blue (and optionally a solvent) and another comprising a sodium dithionite (and optionally a solvent). The two parts of the two part composition are combined prior to use to provide an activated ink suitable for printing. Film preparation

Several experiments were carried out in order to test the redox properties of the ink under different conditions.

In order to apply inks to polypropylene, a K-Bar hand application coater, for example, as supplied by RK Print Coat Instruments Ltd, Litlington,

Royston, Herts, SG8 0QZ, United Kingdom, is used as follows. A plastic sheet is clamped tightly to a flat surface and a line of ink is deposited at the head of the sheet. The ink is then drawn down the sheet using the desired gauge of K-Bar, before being left to dry. The ink thickness can be varied from 6 pm to 00 μιη as necessary using K-Bar 1 to K-Bar 8. Typically, K-Bar 8 is used producing an ink with a thickness of 100 μητι when wet. In flexography printing, typically K-Bar 1 or K-Bar 2 is used to coat the ink, which produces a wet layer thickness of 6 μιτι or 12 pm respectively.

The ink can was also cast on to coverslips, for example on to 24 mm glass coverslips, using an Electronic Micro Systems Model 4000-1 spin coater. The coverslip was set spinning at 3500 rpm for 30 seconds and 5 drops of the ink were applied, resulting in a blue film. The film can then be used directly without any further treatment (owing to the high spin speed, the film is dry after spin-coating, thus negating the need for a drying step). In the ethyl acetate based examples, the film thickness of the ink is approximately 25 pm, unless otherwise stated. Barrier layers can be applied on top of an ink film substantially as described above. For example, an ink coated coverslip is spun at a rate of between 500 and 3500 rpm for 30 seconds after the application to the film surface of 1 to 5 drops of the desired barrier layer in liquid form or as a solution.

Monitoring of the film compositions

The bleaching (reduction) of the film compositions and their subsequent recovery (oxidation) was recorded photographically. In addition, or alternatively, the recovery of the film compositions was monitored by diffuse reflectance spectroscopy using a hand-held Konica-Minolta CM- 2500d Spectrophotometer.

The reflectance measurements taken were reported using the following readings and nomenclature, and using an instrument that emits D65 light (i.e., light that is related to daylight with the same amount of UV light as in a winter day):

L ^* , which is a measure of perceived lightness on a scale of 0 to 100;

a ^* , which is a measure of the hue on the red/green axis, where a positive value means red, and a negative value means green; and b*, which is a measure of the hue on the yellow/blue axis, where a positive value means yellow, and a negative value means blue. Example 1

An ethanol based ink was prepared using the technique described in the general example. A film of the ink was then drawn down onto a polypropylene polymer film using a K-Bar to give a colourless coated polymer film. Over time, the coated polymer film changed colour, turning blue. The so formed film composition was monitored using the diffuse electron spectroscopy technique as described above. The so formed film composition was monitored using the diffuse electron spectroscopy technique as described above. Example 2

An ethanol and water based ink was prepared using the technique described in the general example. A film of the ink was then drawn down onto a polypropylene polymer film using a K-Bar to give a colourless coated polymer film. The ink composition comprised 2 g ethanol, 800 mg water, 200 mg sulfonated polystyrene and 6 mg methylene blue. A fivefold excess of sodium dithionite was added (relative to the methylene blue concentration) to achieve activation/reduction of the ink.

The recovery of the activated/reduced drawn-down ink containing sodium dithionate was then tested in various conditions. Samples of the ink were drawn down onto a polypropylene polymer film and the recovery was monitored at room temperature (approximately 20 °C) and in a dry refrigerator (approximately 5 °C). The latter environment provided far lower humidity and was expected to slow down the recovery of the ink, particularly in combination with the lower temperature. In addition, parallel studies were carried out wherein the drawn-down films were rinsed quickly in water after the ink had dried.

The recoveries of these films were monitored at regular intervals and were charted both numerically and pictorially at regular intervals by diffuse reflectance spectroscopy using a hand-held Konica-Minolta CM-2500d Spectrophotometer. The results obtained from the above studies, and using the ink form Example 2, are illustrated in Tables 1 to 4 below.

Table 1 - Recovery at room temperature; film not washed

Time (hours) L*(D65) a*(D65) b ^* (D65)

0 89.03 3.29 -14.22

1 79.93 -15.60 -25.50

2 79.09 -16.81 -26.33

3 78.21 -18.48 -27.48 4 77.83 -19.13 -28.06

6 76.13 -19.46 -26.53

Table 2 - Recovery at room temperature; washed film

Table 3 - Recovery in refrigerator; film not washed

Table 4 - Recovery in refrigerator; washed film Humidity and temperature were noted to have a large influence on recovery. For example, the complete recovery time for a bleached film of ink containing sodium dithionite went from six hours at room temperature to over a week when placed in a dry refrigerator. Other reducing agents, such as ascorbic acid, were studied and were noted to provide a film that would reduce and recover. The recovery was, however, noted to be extremely slow. Furthermore, the ink was unstable when washed with water, and was noted to solubilise.

In contrast, films made from inks containing sodium dithionite can be washed with water to provide a faster recovery with no impact on the quality of the draw-down. Irrespective of humidity such films give a clear, gradual and consistent recovery.

In one embodiment the indicator is incorporated into an oxygen indicator for informing a user when a food stuff or the like is no longer fresh, or no longer considered to be fit for consumption. The indicator may be displayed as a dot inside a coloured ring. When the dot changes colour to the extent that it is darker than the ring, the product is no longer fit for consumption.

The indicator can be printed on to the reverse side of an oxygen barrier film. Another film, which is oxygen permeable can be applied such that the ink is sandwiched between the two films. The oxygen permeable film delays the ingress of oxygen to the indicator, and thus increases the amount of time taken for the indicator to change colour. Various reference colours can be placed next to the indicator such that the freshness of the item to which the indicator is applied can be determined. Depending on whether the indicator is placed inside or outside the packaging, this can be used to determine if a package has been inadvertently opened, to determine how long a package has been opened for, or to establish for how long a package has been stored. The indicator of the present invention can be used as a "best before" type indicator as follows. The indicator is applied to the outside of food or drink packaging by those packaging food (for example, in an in-store bakery or butchers in a supermarket). The ink is activated at the same time as, or just after, application to the packaging. A seal or barrier can be used to delay the colour change of the ink, although this is not always necessary. For example, a suitable varnish or oxygen barrier material can be combined with the ink to delay the colour change. The ink can therefore be used as a visual indicator to indicate to staff working in a supermarket that goods are nearing, or beyond, the best before date. This will allow staff to quickly identify such goods, thus allowing them to mark them for reduction, or to otherwise dispose of them. In a further embodiment, barcodes can be printed onto a material onto which has already been applied the indicator ink as described herein. For example, the indicator ink can be applied to the background area of a package onto which a barcode will be printed. The ink is activated so that it is colourless. Over time, the ink oxidises and becomes coloured. As the ink becomes coloured, it obscures the barcode, thus preventing the barcode from being read by barcode scanners. Therefore, in this embodiment, "out of date" goods are highlighted at the point of sale, which enables the prevention of sale of such goods. In further embodiment, the indicator is used to indicate when a "consume within a certain time from opening" period has passed. Such periods vary from a few days to several months depending on the nature of the perishable item. The ink can be, for example, applied to the inside of vacuum or controlled atmosphere packaging (thus keeping the ink free from exposure to oxygen). When the packaging is opened, the indicator will be exposed to oxygen and will change colour over a set period of time.

Alternatively, the consume within indicator can be applied to the outside of packaging along with a seal or barrier (if, for example, the oxygen content inside the packaging is too high for the indicator to remain clear). When the packaging is opened, the seal or barrier is also broken allowing the indicator to be exposed to the atmosphere. The indicator will then change colour over a set period of time.

In one embodiment the indicator is incorporated into a water indicator for informing a user when an item has been exposed to moisture. The ink can be applied to the inside of moisture sensitive goods in substantially moisture-free packaging such as dried foods, electrical equipment and pharmaceuticals. If the packaging is compromised, moisture will enter the package and the indicator will change colour thus indicating to the end consumer that the goods that they have received have not been in the required atmospheric conditions and therefore may be spoiled or damaged. In this embodiment, the indicator can also be used to warn manufacturers or distributors that their goods are being exposed to moisture, and that they may need re-packaged, or that they may not be fit for sale.

In one embodiment the indicator ink is incorporated into a time- temperature indicator. Oxidation (but seemingly not reduction) of the indicator composition is temperature sensitive. Once activated, the reduced composition will remain clear if kept at typical domestic freezer temperatures (less than -20 °C). Therefore, the indicator can be used to demonstrate when frozen items have been subject to an increase in temperature. For example, the activated indicator composition can be applied to a frozen foodstuff. If the item defrosts, the indicator will change colour, thereby illustrating that the item has been at too high a

temperature. If the item is re-frozen, the indicator remains coloured. This can be useful for the consumer, the manufacturer, the distributor and the seller. In particular, the time temperature indicator can be useful for frozen seafood, frozen dairy products (such as ice cream), and frozen poultry, all of which are associated with exponential bacterial growth when exposed to high temperatures (particularly when subsequently re-frozen and defrosted).

In a further embodiment, the indicator ink is incorporated into an anti- counterfeiting device.

The indicator can be applied to the inside of substantially air-free and/or substantially oxygen-free packaging for high value, or often counterfeited goods. When the packaging is opened, a logo or message appears indicating that the goods are genuine. This could, of course, also be applied to currency and/or documentation. Examples of ink in use

The inks as described above are suitable for use in the printing methods of the present invention, which can be carried out using conventional printing apparatus, for example as used in Gravure printing, flexography printing or screen printing.

In one embodiment, the activated indicator is provided to a Gravure printer in the form of a printable ink. The Gravure printer is equipped with laminated paper and the printing process is commenced. The printable indicator is applied to the laminated paper in the form of a LOGO or text in the conventional manner using an etched printing cylinder and an impression cylinder as is common in the Gravure process. Once printed, the area of the laminated paper printed with the indicator is sealed by application of polyethylene terephthalate (PET). In a further embodiment, 2 kg of ink in a glass flask is purged with nitrogen for 15 minutes before activation with a five-fold excess of sodium dithionate (relative to the amount of methylene blue), whilst stirring. The ink is then provided to a flexographic printer in the form of a printable ink. The flexographic printer is equipped with paper and the printing process is commenced. The printable indicator is applied to the paper in the form of a LOGO or text in the conventional manner as is common in the flexographic process. Once printed, the paper is rolled up on itself, thus preventing the ink from exposure to the atmosphere. The roll of printed paper is then unwound and laminated using conventional laminating techniques. The laminated paper is then used in the manufacture of packing for, for example, foodstuffs.

It will be appreciated that the method can be applied using different types of printing assembly and alternative printing regimes such as web offset printing. Whilst paper and laminated paper are used in the examples given, other suitable printing substrates known in the art can be used. Also, it will be understood that the printable indicator may be applied to the whole, or only a section of, the printing substrate, and that the seal may be applied to the whole, or only a section of, the printing substrate and/or section of the printing substrate to which the indicator is applied. In this embodiment, the printing substrate contains blanks which can be folded to manufacture packages.

The method described can be used to manufacture a blank for a package. In addition, the method described can be used to manufacture a package by folding or arranging a printing substrate or a blank produced using the method.

In the example given, the indicator is sealed using PET. However, it will be appreciated that any suitable seal having a very low oxidising agent permeability (thereby preventing oxidation of the indicator) can be used. Alternatively, the seal may be chosen to provide a semi-permeable seal configured to allow controlled flow of oxidising agent to the indicator.

Examples of suitable materials for providing a seal are polyethylene terephthalate (PET), ethylene-vinyl alcohol copolymer (EVOH),

polyvinylidene chloride (PVDC), polyvinyl alcohol (PVA) and regenerated cellulose.

The rate of reaction between the reduced form of the redox dye, Red, and oxygen may be rendered mass-transfer dependent, by making the rate of diffusion of oxygen from the ambient air through the film the rate determining step.

The latter can be achieved by making the diffusion process very slow through the use of polymers with low oxygen permeabilities such as polyethylene terephthalate, either as the polymer encapsulating medium or as a film covering the film indicator. Through the use of such a diffusion barrier it is possible to create a type of indicator film that exhibits a recovery time (when exposed to air) that depends upon the thickness of the diffusion barrier film; the thicker the oxygen barrier, the slower the film recovery. When used as an oxygen indicator, in an oxygen-free package, the colour recovery times of this type of indicator can be made sufficiently long (i.e. hours and/or days) that it can provide an indication of how long a modified atmosphere package has been opened, after it is opened and air allowed in. Various modifications and variations to the described embodiments of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes of carrying out the invention which are obvious to those skilled in the art are intended to be covered by the present invention.