Colorimetric sensor for oxygen and use thereof

Abstract

NOT VISIBLE DUE TO STATUS OF PATENT

Description

[0001] COLORIMETRIC SENSOR FOR OXYGEN AND USE THEREOF

[0002] Technical Field

[0003] The present invention relates to a non-toxic, luminescence-based colorimetric sensor and to methods of using the sensor in food packaging and other applications. The sensor can be used to assess oxygen concentration, in particular in controlled oxygen atmospheres such as food packaging.

[0004] Background

[0005] In recent years, the use of protective atmosphere packaging has increased substantially due to a number of factors, including stringent food safety requirements, increased demand for fresh produce, the importation of non-native and non-seasonal foodstuffs, and the drive towards reducing food waste and associated CO2 and CCh-equivalent emissions.

[0006] Protective atmosphere packaging generally includes gas-free (i.e. vacuum packaging) and modified atmosphere packaging (MAP). A gas-free environment is made by expelling all of the air, and therefore the oxygen, from the packaging under vacuum. Gas-free environments are favoured for the packaging of products that are susceptible to oxidative degradation, which include electronic components, pharmaceuticals, and foodstuffs. MAP is created by altering the normal gas composition of air (78% nitrogen, 21% oxygen, 1% trace gases) within the packaging. These processes provide atmospheres that inhibit the primary food spoilage processes, thereby extending shelf-life of the products and enhancing food quality and safety.

[0007] Oxygen (O2) and carbon dioxide (CO2) are the most commonly used gases for MAP. Generally, the modified atmosphere is tailored to the product to be packaged, with fruit and vegetables as well as cooked meats typically being stored under low oxygen atmospheres (0-20%) and raw red meat and poultry products typically being stored under higher oxygen atmospheres (20-80%). The partial pressures of these gases (pCCh, pCh) within the packaging headspace can fluctuate over time and are influenced by factors such as the product type, microbial activity, storage conditions, and the packaging material and integrity. Since any leaks or damage to the packaging will induce loss of the modified atmosphere and a return to ambient air conditions, and improper gas flushing can lead to an incorrect atmosphere being created within the packaging, the composition of the gas in the packaging headspace, and in particular the O2 concentration, can provide a useful indication of the packaging integrity and reliability of the gas flush, as well as of the freshness and safety of the packaged foodstuff.

[0008] Thin film oxygen sensors for the assessment of oxygen in food packaging are known. In these known sensors, the sensor response is typically based on a change in an optical property, such as absorbance or luminescence, as a function of oxygen concentration. Luminescence-based sensors can be particularly advantageous, as they provide a non-destructive means of quantitative detection. Incorporating these sensors into supply chains can allow individual damaged or spoiled goods to be removed from the supply chain, without necessitating disposal of the entire batch, thereby retaining any remaining fresh and safe to eat produce. On a commercial scale, this can result in increased efficiencies and lead to significant reductions in food waste, and food waste packaging.

[0009] However, in order for sensors to monitor real time conditions within a closed environment, such as modified atmosphere packaging, the sensor has to come into direct contact with the atmosphere, i.e., it needs to be positioned within the packaging headspace, and in direct contact with the foodstuffs or other ingestible products the MAP is designed to preserve. This is problematic, as known lumophores tend to exhibit various toxicities, including organ toxicity, cytotoxicity, and / or genotoxicity, and are therefore inherently unsuitable for applications where they come into direct contact with foodstuffs or other ingestible products. Rigorous testing of proposed food contact materials is also required before legislative approval to exploit these materials is received, which can be burdensome from a time and cost perspective. This is particularly the case if multi-component systems are being developed, with comprehensive testing of each individual component required before market approval is granted. These, among other constraints, have to-date limited the wide-spread adoption of luminescence-based sensors into modified atmosphere packaging for foodstuffs or other ingestible products such as pharmaceuticals and veterinary medicines.

[0010] It is an aim of the invention to obviate or mitigate one or more of the disadvantages associated with the prior art. Ideally, it would be advantageous to provide a non-toxic colorimetric oxygen sensor which, in use, can be positioned in direct contact with foodstuffs and / or other ingestible products, for instance in the headspace of modified atmosphere packaging. A tuneable colorimetric sensor which can deliver a colour change over a relevant oxygen concentration range, for instance the oxygen levels required for preservation of specific food items such as meat or vegetables in modified atmosphere packaging, would be particularly advantageous.

[0011] Summary of the Invention

[0012] According to the present invention there is provided a colorimetric oxygen sensor comprising a first lumophore and a second lumophore, each lumophore being dispersed in a polymer matrix, wherein the first lumophore is selected from ruthenium (II) 4,7-diphenyl-l,10'- phenanthroline [Ru(dpp)s]2+; platinum octaethylporphyrin (PtOEP); platinum 5,10,15,20- tetrakis(2,3,4,5,6-pentafluorophenyl)porphyrin (PtTFPP) and platinum meso- tetraphenylporphyrin (PtTPP); and the second lumophore is a naturally-occurring compound or a synthetic analogue thereof selected from a diarylheptanoid, flavonol, carotenoid, coenzyme, minor flavonoid, or which is paeonol or ellagic acid.

[0013] The inventors have advantageously discovered that certain naturally-occurring compounds have surprising utility in luminescence-based oxygen sensors. While some naturally-occurring compounds are known to exhibit fluorescence, these generally demonstrate low colour intensity and fastness and poor emission brightness. Their inherent fluorescence characteristics are therefore generally considered disadvantageous, and tend to make detection and analysis more, rather than less, difficult. This is due to a number of factors, including the low levels of fluorescence, which make independent exploitation of the fluorescence characteristics unfeasible; the oftentimes presence of numerous competing analytes in intact foodstuffs in which they can occur; and the interference of the food endogenous autofluorescence with external detection systems. Furthermore, these naturally- occurring compounds typically exhibit poor photostability and / or poor chemical stability. These characteristics mean that naturally-occurring products, such as the diarylheptanoids, flavonols, carotenoids, co-enzymes, minor flavonoids, and other compounds described herein, have not previously been incorporated in luminescence-based sensors, and would generally be considered wholly unsuitable for exploitation in commercial sensor applications.

[0014] In another aspect of the present invention there is provided a method of determining the oxygen content of an atmosphere, the method comprising exposing the colorimetric sensor described above to the atmosphere; applying a source of UV or UV-visible excitation to the sensor, and observing the colour of the sensor.

[0015] The atmosphere may be a food packaging atmosphere. Advantageously, the sensor of the invention is suitable for direct contact with foodstuffs and other ingestible products, such as within the headspace in MAP, due to its natural and non-toxic nature. Alternatively, the atmosphere may be any environment in which a vacuum or controlled oxygen concentration is preferred; such as in the packaging of pharmaceutical or veterinary products, disposable medical products or electronic components; or in packaging or storage in the areas of conservation / preservation (e.g. artworks or antiquities etc.).

[0016] Also described herein are laminate films and labels comprising a printed colorimetric oxygen sensor, and the use of such laminate films and labels in packaging applications.

[0017] Various further features and aspects of the invention are defined in the claims.

[0018] Brief Description of the Drawings

[0019] Embodiments of the present invention will now be described by way of example only with reference to the accompanying drawings where like parts are provided with corresponding reference numerals and in which:

[0020] Figure 1 shows colour response of oxygen sensors (a) PtOEP: Curcumin (1:3.0) (b) [Ru(dpp)3]2+:Curcumin (1:2.8) and (c) [Ru(dpp)3]2+:Curcumin (1:7.5).

[0021] Detailed Description

[0022] The present invention relates to a colorimetric oxygen sensor comprising a first lumophore and a second lumophore, each lumophore being dispersed in a polymer matrix, wherein the first lumophore is selected from ruthenium (II) 4,7-diphenyl-l,10'-phenanthroline [Ru(dpp)s]2+; platinum octaethylporphyrin (PtOEP); platinum 5,10,15,20-tetrakis(2,3,4,5,6- pentafluorophenyl)porphyrin (PtTFPP) and platinum meso-tetraphenylporphyrin (PtTPP); and the second lumophore is a naturally-occurring compound selected from a diarylheptanoid, a flavonol, a carotenoid selected from bixin, capsanthin and capsorubin; a co-enzyme, a minor flavonoid, or which is paeonol or ellagic acid.

[0023] Throughout this specification, the term "lumophore" is used synonymously with "luminophore" to mean a chemical species which spontaneously emits light upon radiative relaxation (luminescence) from a higher energy electronic excited state (unstable) to its lowest energy ground state (stable). The higher energy electronic state is formed when the lumophore absorbs radiation, which is usually, but not always, at an energy greater than that of the subsequently emitted light. The colorimetric sensor is oxygen-sensitive, i.e. it exhibits an observable colour change in response to a change in the oxygen concentration of an atmosphere or local environment to which it is exposed.

[0024] The colorimetric sensor is luminescence-based. The colour change arises due to preferential quenching of the light emitted by at least one of the lumophores in the sensor. The colour change is visible to the naked eye only when the sensor is exposed to UV or UV-visible light. This can have advantages in terms of monitoring consumer products, as the sensors can be concealed from general view, if preferred.

[0025] The colorimetric sensor is an oxygen sensor. Throughout this specification, "oxygen sensor" is intended to mean a single parameter sensor which is sensitive to oxygen as an analyte.

[0026] In the colorimetric sensor of the invention, each of the at least two lumophores is dispersed in a polymer matrix. The at least two lumophores may be dispersed in the same polymer. In this embodiment, the sensor comprises a single lumophore layer. Alternatively, the at least two lumophores may be dispersed in different polymers. In this embodiment, the sensor comprises two lumophore layers.

[0027] Throughout this specification, the term "naturally-occurring" is used to mean a compound that can be found in nature or can be isolated from nature or a synthetic analogue thereof. This term encompasses compounds that are derived from nature, as well as those same compounds when prepared synthetically. As would be evident to one skilled in the art, any synthetic analogue of a naturally-occurring product would be required to be of food-grade purity.

[0028] In some embodiments, the naturally-occurring compound is naturally-derived, i.e. has been extracted or isolated from nature. Examples of naturally-derived compounds include, for instance, curcumin, capsorubin, capsanthin, bixin and safflower extract. As a skilled person will appreciate, when the naturally-occurring compound is a naturally-derived extract, such as safflower extract, the extract may contain minor components in the extract alongside the major component (e.g. for safflower, it is Hydroxysafflor Yellow A / (6E)-2,5-dihydroxy-6-[(E)-l- hydroxy-3-(4-hydroxyphenyl)prop-2-enylidene]-2,4-bis[(2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6- (hydroxymethyl)oxan-2-yl]cyclohex-4-ene-l, 3-dione) which functions as the lumophore in the sensor. For natural curcumin extracts, demethoxycurcumin (13-20%) and bisdemethoxycurcumin (1-4%) may be present in the extract alongside curcumin which functions as the second lumophore. When the extract is a carotenoid extract from chili pepper, capsanthin and capsorubin may both be present and may both function as the second lumophore.

[0029] Alternatively, the second lumophore may be a synthetic analogue of a naturally-occurring compound, such as synthetic curcumin (typically 98+% pure).

[0030] The first lumophore is an oxygen-sensitive lumophore, i.e. its emission is quenched in the presence of oxygen. The emission of the first lumophore is typically insensitive to pH and temperature.

[0031] According to the invention, the first lumophore is ruthenium (II) 4,7-diphenyl-l,10'- phenanthroline [Ru(dpp)s]2+(also known as ruthenium tris (II) 4,7-diphenyl-l,10'- phenanthroline [Ru(dpp)s]2+); platinum octaethylporphyrin (PtOEP); platinum 5,10,15,20- tetrakis(2,3,4,5,6-pentafluorophenyl)porphyrin (PtTFPP); or platinum meso- tetraphenylporphyrin (PtTPP). These lumophores are non-toxic, and ruthenium (II) 4,7- diphenyl-l,10'-phenanthroline [Ru(dpp)s]2+and platinum 5,10,15,20-tetrakis(2,3,4,5,6- pentafluorophenyl)porphyrin (PtTFPP) are known to meet the regulations for use in food packaging applications in the EU. The second lumophore is an oxygen-insensitive lumophore, i.e. its emission is not quenched significantly in the presence of oxygen. The second lumophore typically absorbs UV and / or UV-visible light substantially in the same spectral region as the first lumophore. This is advantageous as it allows a single excitation source to be used. However, if the first and second lumophores do not absorb in substantially the same spectral region, multiple excitation sources can be used. The second lumophore emits substantially in a different spectral region to the first lumophore. By 'substantially' it is meant that the maximum emission of each lumophore should differ by at least ~ 20 nm in order to allow a colour change to be observed. The emission of the second lumophore in the sensor is also typically insensitive to pH and temperature.

[0032] According to the invention, the second lumophore is a naturally-occurring compound selected from a diarylheptanoid, flavonol, carotenoid, co-enzyme, or minor flavonoid, or the second lumophore is paeonol or ellagic acid.

[0033] To date, naturally-occurring compounds have typically not been widely exploited for use in commercial luminescence-based sensors due to a combination of factors including low levels of fluorescence, poor photostability, and poor formulation characteristics. The inventors have, however, demonstrated that some naturally-occurring lumophores are surprisingly effective in colorimetric oxygen sensors, allowing them to be used in a host of oxygen sensor applications where non-toxicity is critical. Advantageously, as these naturally-occurring compounds are in most cases approved for use in other food-safe applications, such as for example, as food colourings (e.g. curcumin, Bixin and FMN), the lumophores can be exploited commercially for food-related applications, without undergoing significant toxicity testing. This paves the way towards widespread adoption of the colorimetric sensors of the invention on a commercial scale.

[0034] In the colorimetric sensor of the invention, each lumophore is dispersed directly in a polymer matrix and the lumophores are not encapsulated or otherwise embedded (e.g. in a gel) before dispersion in the polymer matrix. The lumophores are not incorporated on the surface of microbeads. The lumophores are not incorporated within mesoporous structures. The lumophores are not incorporated into microspheres at any point in time during the formation of the polymer matrix. When incorporated into a sensor label or laminate film, for instance, the lumophores can be dispersed in a single lumophore-polymer layer, or can be dispersed individually in separate contiguous polymer layers. The sensor can therefore be a single layer or a multi-layer structure, depending on the dispersion of the lumophores. When the sensor comprises multiple layers, the layers preferably have the same thickness.

[0035] The sensor changes colour in response to a change in the oxygen concentration in an atmosphere or local environment to which it is exposed due to a preferential and quantitative reduction (quenching) in the luminescence of the oxygen-sensitive lumophore. Advantageously, the colour change is reversible, enabling colour changes to proceed in the forward and reverse directions of the response range in line with an increase or decrease in the local oxygen concentration. This affords the oxygen sensor broad applicability for commercial applications.

[0036] The emission of each of the lumophores is typically insensitive to pH and temperature, allowing the sensor to be applied directly in a wide variety of environments and ensuring that the colorimetric change is directly attributable to the change in oxygen concentration.

[0037] The second lumophore in the sensor is a naturally-occurring compound selected from a diarylheptanoid, a carotenoid, a co-enzyme, a flavonol, a minor flavonoid, or the second lumophore is paeonol or ellagic acid.

[0038] The term "diarylheptanoid" refers to a polyphenol consisting of two aromatic rings joined by a C7 chain, and which can have various substituents. Diarylheptanoids can be linear (curcuminoids) or cyclic. Suitable diarylheptanoids for use in the invention include curcumin.

[0039] The term "carotenoid" is a tetraterpenoid formally derived from the acyclic parent, carotene I by hydrogenation, dehydrogenation, cyclisation, oxidation, or combination of these processes. The term "carotenoid" includes carotenes, xanthophylls and certain compounds arising from the rearrangement of the skeleton of I or by loss of part of this structure. Suitable examples of carotenoids for use in the invention include bixin, capsanthin and capsorubin. Capsanthin and capsorubin may both occur in the same extract and may be used in combination as the second lumophore. Alternatively, they can be used independently, i.e. where capsanthin only or where capsorubin only is used as the second lumophore.

[0040] The term "co-enzyme" refers to an organic, non-protein compound which binds with an enzyme to catalyse a reaction, and in some embodiments, may be a vitamin. Suitable coenzymes according to the invention include riboflavin, nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN). Riboflavin, NADH, FAD and FMN can be used in their acid form or their salt (sodium or disodium) forms.

[0041] The term "flavonoid" refers to natural products derived from 2-phenylchromen-4-one (flavone). According to the invention, the second lumophore may be a flavonol or a minor flavonoid. Flavonols are a class of flavonoids comprising a 3-hydroxyflavone backbone. Suitable examples of flavonols include quercetin, fisetin, kaempferol, myricetin, 3- hydroxyflavone and morin. The term "minor flavonoids" refers to five minor classes of flavonoids, namely chaicones, aurones, dihydrochalcones, flavanones and dihydroflavonols. Suitable examples of minor flavonoids for use in the invention include safflower extract.

[0042] Further compounds for use in the invention include the phenol, paeonol, and the polyphenol ellagic acid.

[0043] According to an embodiment of the invention, the second lumophore is a naturally-occurring compound, which is selected from: a diarylheptanoid selected from curcumin; a carotenoid selected from bixin, capsanthin and capsorubin; a co-enzyme selected from riboflavin, NADH, FAD and FMN; a flavonol or minor flavonoid selected from quercetin, fisetin, kaempferol, myricetin, 3- hydroxyflavone, morin and safflower extract; paeonol; and ellagic acid.

[0044] The use of these naturally-occurring compounds in luminescence-based oxygen sensors is novel, and provides significant advantages over the prior art.

[0045] In an embodiment, the naturally-occurring compound is a diarylheptanoid, or a flavonol. In an embodiment, the naturally-occurring compound is curcumin, quercetin, fisetin, kaempferol, myricetin, 3-hydroxyflavone or morin. These compounds in particular have been demonstrated to have surprising utility in the luminescence-based oxygen sensors of the invention, with those sensors exhibiting good emission and brightness characteristics and visible colour changes in the oxygen concentration ranges of interest.

[0046] According to an embodiment of the invention there is provided a colorimetric oxygen sensor as previously described, wherein the sensor further comprises an antioxidant.

[0047] Advantageously, the addition of an antioxidant to the sensor of the invention has been demonstrated to improve the photostability of the sensor. In an embodiment, the antioxidant is a carotenoid, a phenolic compound, or a vitamin.

[0048] In some examples, the second lumophore itself may be an antioxidant compound, which can confer photostability on the sensor. For instance, bixin, capsanthin and capsorubin are nonlimiting examples of the second lumophore which may be considered as antioxidant compounds in their own right. In this embodiment, there may be no need for an additional antioxidant; however, an antioxidant compound can still be added to the sensor to further improve photostability. In this case, the second lumophore and the antioxidant compound may be the same, or may be different compounds. In an embodiment, the second lumophore and the antioxidant are different compounds. As a non-limiting example, fisetin can be used as the second lumophore and bixin as the antioxidant.

[0049] The antioxidant can be added into the polymer alongside the lumophore or lumophores.

[0050] When an antioxidant is added to a multi-layer sensor, the antioxidant may be added into the polymer comprising the second lumophore.

[0051] In an embodiment, the molar ratio of the second lumophore to the antioxidant compound is from 1:1 to 1:10, preferably from 1:1 to 1:5. In an embodiment, the molar ratio of the second lumophore to the antioxidant compound is 1:2.

[0052] In an embodiment, the antioxidant may be selected from vitamins (Vitamin A, C or E), carotenoids (Lutein, beta-carotene, capsanthin, capsorubin, bixin, norbixin), flavonoids (quercetin, fisetin, morin, catechin, epicatechin, luteolin, baicalein) and / or phenolic acids (caffeic acid, vanillin, ferulic acid).

[0053] In an embodiment, the antioxidant is bixin, quercetin or vitamin E (DL-a-tocopherol).

[0054] In an embodiment, the antioxidant is DL-a-tocopherol.

[0055] In an embodiment of the invention, the second lumophore may be complexed with aluminium, boron or zinc. The inventors have advantageously discovered that when the second lumophore is complexed with Al(lll), B(lll), or Zn(ll), the photostability of the sensor formulation is surprisingly improved, compared with the use of the second lumophore in its uncoordinated state. Even more surprisingly, complexation has been shown to lead to sensor formulations with increased emission and enhanced sensitivity to oxygen.

[0056] In an embodiment, quercetin, curcumin, fisetin, kaempferol, morin, safflower extract, ellagic acid, paeonol, riboflavin, FMN or FAD can be complexed with Al(lll), B(lll) or Zn(ll).

[0057] In particular, quercetin, curcumin, fisetin, kaempferol, morin, safflower extract, ellagic acid and paeonol show appreciable advantages when complexed with Al(lll), B(lll) or Zn(ll).

[0058] In an embodiment, the second lumophore is curcumin, which is complexed with Al(lll).

[0059] According to the present invention, each lumophore in the colorimetric oxygen sensor is dispersed in a polymer matrix.

[0060] In an embodiment, the polymer is selected from polystyrenes, polyvinyls, polyamides, polyurethanes, acrylates, shellac, rosin, rosin esters, silicones, celluloses, and cellulosederivatives; and mixtures thereof.

[0061] The polymer or polymers may be, for example, polystyrene, a silicone, or a cellulose-based polymer or polymers. Suitable silicones include polydimethylsiloxane (PDMS). Suitable cellulose-based polymers include but are not limited to ethyl cellulose (EC), cellulose acetate (CA), cellulose acetate propionate (CAP), cellulose acetate butyrate (CAB), nitrocellulose (NC), hydroxypropyl cellulose (HPC), hydroxypropyl methyl cellulose (HPMC) or carboxymethyl cellulose (CMC).

[0062] In embodiments, the first and second lumophores are dispersed in the same polymer, e.g. in a single polymer layer. In other embodiments, the first and second lumophores are dispersed in different polymers, e.g. in multiple contiguous polymer layers.

[0063] In an embodiment, the polymer or polymers is one or more of polystyrene, PDMS, EC, CAP or CAB, HPMC and HPC.

[0064] In embodiments of the invention, the first lumophore is a red-emissive lumophore and the second lumophore is a blue-emissive, green-emissive or orange-emissive lumophore. However, coordination of the second lumophore with Al(lll), B(lll) or Zn(ll) can lead to shifts in both absorption and emission, as would be understood by one skilled in the art.

[0065] In an embodiment, the second lumophore is curcumin or fisetin.

[0066] In an embodiment, the second lumophore is curcumin or fisetin which has been complexed, preferably with Aluminium.

[0067] Curcumin and fisetin are particularly useful for the sensors of the invention due to factors such as their ready availability, relatively low cost, and good photophysical properties.

[0068] In an embodiment, the first lumophore is ruthenium (II) 4,7-diphenyl-l,10'-phenanthroline [Ru(dpp)s]2+or platinum octaethylporphyrin (PtOEP).

[0069] In an embodiment, the first lumophore is ruthenium (II) 4,7-diphenyl-l,10'-phenanthroline [Ru(dpp)s]2+or platinum octaethylporphyrin (PtOEP) and the second lumophore is curcumin or fisetin. The curcumin or fisetin may be complexed. In an embodiment, the curcumin or fisetin is complexed with Al(lll). Complexation with Al(lll) can lead to enhanced emission, enhanced colour response and stronger absorption. Advantageously, this means that lower quantities of the second lumophore are needed to achieve similar brightness output to their uncomplexed counterparts.

[0070] In an embodiment, the first lumophore is ruthenium (II) 4,7-diphenyl-l,10'-phenanthroline [Ru(dpp)s]2+and the second lumophore is curcumin.

[0071] In an embodiment, the first lumophore is ruthenium (II) 4,7-diphenyl-l,10'-phenanthroline [Ru(dpp)s]2+and the second lumophore is fisetin.

[0072] In an embodiment, the molar ratio of lumophore 1: lumophore 2 is from 1:1.9 to 1:10.

[0073] In an embodiment, the sensor comprises [Ru(dpp)s]2+, curcumin, and DL-a-tocopherol. Optionally, the curcumin may be complexed, preferably with Al(lll). In this embodiment, the molar ratios of the components may be L1:L2:AO 1:2.8:5.6. The polymer(s) may be EC, CAB, CAP, or polystyrene. Preferably, the polymer is EC. In this embodiment, the sensor is preferably formed of a single layer comprising [Ru(dpp)s]2+, curcumin (optionally complexed with Al(lll)), and DL-a-tocopherol; dispersed in EC.

[0074] In an alternative embodiment, the sensor comprises [Ru(dpp)s]2+, fisetin, and DL-a- tocopherol. Optionally, the fisetin may be complexed, preferably with Al(lll). In this embodiment, the molar ratios of the components may be L1:L2:AO 1:3:6. The polymer(s) may be EC, CAP, CAB or polystyrene. Preferably, the polymer is EC. In this embodiment, the sensor is preferably formed of a single layer comprising [Ru(dpp)s]2+, fisetin (optionally complexed with Al(lll), and DL-a-tocopherol; dispersed in EC.

[0075] The colorimetric sensor comprises a first and second lumophore, each being dispersed in a polymer matrix. The polymer matrix or matrices comprising the lumophore(s) are formulated to be printable on a substrate. In an embodiment, the colorimetric oxygen sensor comprises a first lumophore and a second lumophore, each lumophore being dispersed in a polymer matrix, wherein the polymer matrix is printed on a substrate, or, where different polymers are used, the polymer matrices are printed contiguously on a substrate so that the lumophore- containing polymer layers are in direct contact. The substrate may be a polymeric substrate, such as a PET, PE, or PP substrate.

[0076] The colorimetric sensor may be in the form of an ink or inks, and specifically a printable ink. In embodiments, the sensor is made up of a single ink formulation, in which the lumophores are dispersed in a polymer, optionally along with printing additives. As would be clearly understood, however, where the lumophores are in different polymer matrices, the sensor may comprise two ink formulations, that are printed sequentially to form contiguous layers, i.e. in which the two layers are in direct contact. Either or both inks may comprise printing additives.

[0077] Throughout this specification, the term "ink" means a fluid or viscous substance that can be printed using a suitable printing technique. Suitable printing techniques include, for instance, inkjet printing such as piezo-based inkjet or thermal inkjet, flexographic printing, gravure printing, offset lithography, screen-printing and letterpress. In an embodiment, the printing is performed by inkjet printing, flexographic printing, or gravure printing.

[0078] The ink may consist of the first lumophore and / or second lumophore dispersed in a polymer matrix, optionally along with any solvents used in the preparation process. Alternatively, the ink may comprise further lumophores and / or additional components. Thus, the polymer acts as a binder and any solvents act as vehicles for the lumophores to facilitate printing.

[0079] The ink formulation(s) may additionally comprise one or more printing additives. Such additives include, for instance, rheology modifiers, dispersants, weting agents, plasticisers etc., as would be well known to a person skilled in the art.

[0080] The rheological properties of the ink may be controlled to ensure compatibility with the printing process used, i.e. to enable high-throughput of printing and to minimise ink bleed and, in the case of inkjet printing, to minimise nozzle blockages. For flexographic and gravure printing the viscosity of the ink is typically controlled within a range of from 20-70 mPa-s, whereas for inkjet printing the viscosity of the ink is typically controlled within a range of from 2-10 mPa-s. Further properties of the ink formulation can be controlled in the later case in order to obtain formulations with ideal drop formation, continuous jeting and to prevent nozzle clogging and "skinning" effect, which lead to poor quality printing and / or jetting interruption. The rheological properties of the ink are important for achieving good quality print using flexographic or gravure printing. Furthermore, a correct selection of printing plates design, anilox roll, materials (flexographic) and / or cylinder cells geometry and depth (gravure) ensures optimal transfer of the ink to the substrate. Finally, independent of the printing technique, the surface tension of the ink should be lower than the surface energy of the substrate to allow good weting. These properties could be readily optimised by one skilled in the art.

[0081] The invention also relates to a method of determining the oxygen content of an atmosphere. This method comprises the steps of exposing the colorimetric sensor described above to the atmosphere; applying a source of UV or UV-visible excitation to the sensor, and observing the colour of the sensor. The step of exposing the colorimetric sensor to the atmosphere may include positioning the sensor within modified atmosphere packaging. Advantageously, due to its non-toxic nature, embodiments of the invention include positioning the sensor where it can come into direct contact with food or other ingestible products, such as, for instance, on the internal surface of food packaging.

[0082] As would be generally understood by a skilled person, the UV region is from 190 nm to 400 nm and the visible region is from 400 nm to 700 nm.

[0083] In an embodiment, the excitation wavelength is from 340 nm to 465 nm.

[0084] The step of observing the colour of the sensor can be performed via the naked eye, via the use of spectrometry, or via the use of a digital imaging apparatus / machine vision system.

[0085] Advantageously, the colour change is visible to the naked eye only upon the application of a UV / UV-visible light source to the sensor. This means that the sensor can be concealed within food packaging, for example, which can be beneficial when supplying consumer products. The non-toxic nature of the sensor makes it particularly suitable for such applications, as the sensor can be concealed within the headspace of modified atmosphere packaging.

[0086] As another advantage, the colour change takes place very rapidly, and typically within milliseconds of exposure to the MAP. This allows the sensor to be used for real-time monitoring.

[0087] The colour of the sensor when exposed to the atmosphere can be compared with a reference colour to determine the oxygen content. For instance, the colour of the sensor can be visibly observed by the naked eye and compared with a reference colour, a colour chart, or a colour indicator for the specific sensor being used, which can give a rapid, semi-quantitative result about the oxygen concentration in the atmosphere. Alternatively, it can be observed using a camera and compared with a calibration curve for a fully quantitative result. In embodiments, therefore, the method further comprises comparing the colour of the sensor with a reference colour to make an assessment of the oxygen content. Advantageously, the sensor can be tailored to the specific atmosphere being monitored, with both [Ru(dpp)s]2+: curcumin-AI(lll) and [Ru(dpp)s]2+: fisetin being suitable for high oxygen concentration atmospheres (e.g., those typically used to package raw red meat and poultry).

[0088] Alternatively, fully quantitative analysis can be achieved using fluorescence spectroscopy.

[0089] The invention also relates to automated methods of assessing the oxygen concentration of an atmosphere using the sensor described above. For example, in an embodiment of the invention, a camera can be used to obtain a digital image of the colour signature of the sensor. The digital image can then be processed, and the colour converted into a numerical value, which can be automatically correlated with a pre-measured calibration curve, to yield an oxygen content value. The software can be designed to locate the sensor (for example in the packaging of a product or in the headspace of food product packaging), and to identify and eliminate optical variations (e.g. reflections, scattering etc.) in the image before the image is processed against the empirical calibration file for the specific sensor combination and attributed an oxygen percentage. This approach can enable real-time, non-destructive, high throughput and remote analysis of multiple samples, for instance each packaged product on a manufacturing line. The oxygen percentage determined following this approach can then be used to eliminate packages which fall outside the prescribed specifications. For instance, the manufacturing / packaging line can incorporate a linked rejection mechanism which can remove such eliminated packages from the line, thereby preventing these products from moving down the supply chain. This has significant advantages in terms of food-waste and food-waste packaging reduction, as it can isolate individual damaged or spoiled food products and remove them from the supply chain, without removing the unspoiled or undamaged remainder of the batch.

[0090] Advantageously, the sensor of the present invention allows a colour change in many cases to be observed visually, i.e. the colour change is visible to the naked eye once exposed to UV / UV- visible light. This can negate the need for expensive external detection equipment (such as a spectrometer) and also allows the detection to be performed by a lay person (i.e. no specific technical skills required). A further advantage is that the detection can be performed using standard vision systems. In many cases, these are already present in production lines, for instance for use in label inspection, damage inspection, product colour etc., thereby avoiding the need in these cases to install expensive detection systems. Comparison of the colour with a database of calibration curves or colour charts, specific to the lumophore pair (or lumophore trio etc.) being used, allows a quantitative determination to be made. The use of a camera and comparison of the colour change with a calibration curve provides a sensitive methodology which can allow even small changes in the oxygen concentration to be determined. This may be particularly beneficial for the monitoring of oxygen concentration in modified atmosphere packaging, where, as an example, the difference between 70% and 80% oxygen may be critical to determining if a packaged food item is in-specification or out- of specification when it comes to its headspace gas mixture.

[0091] However, the colour change can be monitored via spectrometry, if required, and a full quantitative analysis performed. Embodiments of the invention also relate to electronic means of detecting the colour change, i.e. via the use of software, which may be coded into an "app", and which can detect the colour of the atmosphere and compare it with a preprogrammed database, to yield a result on the freshness or safety of the packaged item or the packaging integrity. Such a result can be binary (i.e. fresh = yes / no; safe = yes / no) or it can be a quantitative value, indicating the degree of freshness of the packaged product (for example to indicate to a seller to "maintain stock as normal", "reduce the product price", "discard product", etc.).

[0092] In an embodiment, the atmosphere is a food packaging atmosphere.

[0093] As indicated above, the sensor of the invention is particularly suitable for use in the context of food packaging atmospheres, and is ideally suited for the packaging of foodstuffs and other ingestible products due to its non-toxic nature. However, the invention is not specifically limited thereto, and it may find applicability in other areas in which oxygen concentrations are monitored, such as in pharmaceutical storage, packaging of disposable sterile medical products, conservation of artworks and cultural works or packaging of electronic components. For all of these applications an oxygen-free or controlled oxygen environment may be favoured (i.e. 0% O2). In addition, the luminescent-based colorimetric sensors of the invention could find application in the monitoring of gas supply products, such as in pre-mixed gas cylinders, where controlled oxygen concentrations (~0-100%) are required.

[0094] As noted above, the colorimetric sensor can be tuned to the application of interest, that is, by controlling the lumophore selection and their molar ratio, a colorimetric response can be obtained within the O2 concentration ranges of interest. In an embodiment, the colorimetric sensor is configured to provide a colour change across O2 concentration ranges of from 0-20% (applicable to fruit, vegetables, and oxygen-sensitive foodstuffs such as cooked meats, nuts, fish, and cheese) or from 20-80% (applicable to meat products and other foodstuffs requiring high O2 MAP). Other defined concentrations ranges can be tailored based on the application of interest.

[0095] Also envisaged herein is a method of preparing a laminate film or label comprising a colorimetric oxygen sensor, the method comprising printing the sensor onto a substrate film. The sensor may be printed in the form of an ink, in a single layer, or inks, in two or more contiguous layers. The substrate film may be a polymer substrate film. The lamination may be performed by thermal means, by the use of an adhesive, or by a combination thereof.

[0096] The substrate film may itself be a laminate film and may comprise a plurality of layers or may be a mono-layer film which is printed with the sensor ink or inks to form a sensor-printed laminate or mono-layer film. For instance, the printed polymer substrate film may be laminated with a second polymer substrate film such that the ink layer or layers are entrapped between the polymer substrate films. However, it is generally envisaged that the sensor will not be entrapped between polymer substrate films, but will be exposed to, and directly in the headspace of MAP, such as food packaging. As an example, the sensor may be printed (in a single ink layer, or multiple ink layers) directly onto the inner surface of the food packaging itself. For instance, the sensor may be printed as single multiple-lumophore containing layer, or multiple contiguous single lumophore layers, onto the internal surface of a conventional food packaging polymer film, such that at least one layer of the sensor is in direct contact with the atmosphere. Preferably, the layer comprising the first lumophore is in direct contact with the atmosphere.

[0097] The polymer substrate may be subjected to surface treatment prior to printing in order to improve the wettability of the sensor ink or inks on the film. Such surface treatment steps include, for example, corona discharge, flame activation, low-pressure oxygen plasma, laser beam, focused ion beam or electron beam processing, as well as chemical treatment via the application of an organic or inorganic coatings.

[0098] The integration of the sensor ink formulation(s) into packaging material is a key component of the invention and allows the sensor to be provided within the packaging for in-situ detection and / or monitoring of oxygen concentration within the controlled or modified atmosphere. Due to its non-toxic nature, the sensor formulation or formulations can be printed within the modified atmosphere packaging even when it brings the sensor into direct contact with foodstuffs and other ingestible products. Accordingly, the polymer substrate should be compatible with conventional / commercially-available laminate polymer films used to seal the trays of MAP foodstuffs, or with the plastic film coatings typically applied to vacuum-packed food trays or to packaging of electronic / medical products.

[0099] The polymer substrate film may be a layerof a laminate film. For instance, a polymer substrate film with a high O2 transmission rate can be printed with the sensor ink formulation(s), with the printed polymer substrate film subsequently being adhered to a lidding film, which is preferably a gas impermeable film such as PA or PET, so that the sensor ink(s) are entrapped between the polymer substrate film and the lidding film.

[0100] Alternatively, the sensor ink formulation or ink formulations can be reverse-printed onto a polymer film substrate that has a low or very low O2 transmission rate before lamination to a high O2 transmission substrate like PE or PP. Suitable polymer film substrates having low, or very low O2 transmission rates include polyamide (PA), which includes the likes of nylon, cast nylon etc., polyvinylidene chloride (PVDC), polyester (PET), and oriented polyester (OPET). A low or very low O2 transmission layer can retain the modified atmosphere within the packaging. In this embodiment, the oxygen sensor is in direct contact with the product the MAP is designed to protect, e.g., food or other ingestible products, which is facilitated by its non-toxic nature. Advantageously, this direct contact between the atmosphere and the sensor leads to a robust sensor response.

[0101] Suitable commercial lidding films with which these techniques can be used include, for instance, polyamide (PA) / polyethylene (PE) films, polyester (PET) / polyethylene (PE) films, polyester (PET) / ethylene vinyl alcohol(EVOH) / polyethylene (PE) films, polypropylene (PP) / ethylene vinyl alcohol(EVOH) / polyethylene (PE) films and polypropylene (PP) / polyamide(PA) / polyethylene (PE) films. In an embodiment, the film is a PP or PE film.

[0102] Advantageously, the sensor is not visible to the naked eye when printed onto laminate films or labels. This means that integrated or embedded sensors, used for the purposes of monitoring the integrity of packaging on a manufacturing line, for example, are not visible to consumers. This is particularly applicable to sensors embedded in food packaging, for instance, which are positioned within the headspace of MAP for foodstuffs, due to the nontoxic nature of the formulation(s). This allows for non-destructive, real-time monitoring of the freshness of the foodstuff within the packaging.

[0103] Also envisaged is a system for monitoring the oxygen content in modified atmosphere packaging. The system may comprise a laminate packaging film or label comprising a colorimetric oxygen sensor as described in detail above, a UV or UV-visible light source, and an image reader or vision system. The invention will now be described by way of reference to the following examples, which are intended to be illustrative only.

[0104] Throughout the examples, where ethyl cellulose is used, it is EC standard 4 (i.e. a 5% solution in toluene:ethanol 80:20 (v / v) which has a viscosity of 4 cP). Where curcumin is used, it is a natural extract from Curcuma longa L ((turmeric rhizomes). This natural extract comprises over 70% curcumin, alongside demethoxycurcumin (13-20%) and bisdemethoxycurcumin (1- 4%).

[0105] Examples:

[0106] 1.1 Ruthenium (II) 4,7-diphenyl-l,10'-phenanthroline and (lE,6E)-l,7-bis(4-hydroxy-3- methoxyphenyl)hepta-l,6-diene-3, 5-dione

[0107] [Ru(dpp)3]2+:Curcumin (1:7.5 molar ratio) in ethyl cellulose (EC)

[0108] 28.8 g of dry ethyl cellulose polymer (4 cP) was added to a 69:31 (v / v) mixture of ethanokethyl acetate (140.9 ml ethanol; 63.9 ml ethyl acetate) and stirred overnight to ensure full dissolution of the polymer. 146.6 mg (1.088xl0-4mol) of ruthenium (II) 4,7-diphenyl-l,10'- phenanthroline ([Ru(dpp)s]2+, Lumophore 1) and 300.5 mg (8.158xl0-4mol) of (1E,6E)-1,7- bis(4-hydroxy-3-methoxyphenyl)hepta-l,6-diene-3, 5-dione (Curcumin, Lumophore 2), was then added sequentially to the solution in powder form and the solution stirred until complete homogenisation was achieved and no sedimentation of the lumophores was observed.

[0109] In order to assess the sensor performance, the formulation was then printed onto a self- adhesive PE substrate (EZ-Pierce™) using a 20 pm spiral coating bar from Elcometer™ . The coated substrate was then dried for 2hrs at 40 °C in a thermostated oven, before being cut into 1 cm x 1 cm squares and stored in the dark at room temperature until needed. The colour change response of the printed sensor formulation was measured as outlined in Example 2 below.

[0110] 1.2 Ruthenium (II) 4,7-diphenyl-l,10'-phenanthroline and (lE,6E)-l,7-bis(4-hydroxy-3- methoxyphenyl)hepta-l,6-diene-3, 5-dione

[0111] [Ru(dpp)3]2+:Curcumin (1:2.8 molar ratio) in ethyl cellulose (EC)-based ink

[0112] 720 g of dry ethyl cellulose polymer (4cP) was added to a 69:31 (v / v) mixture of ethanokn- propyl acetate (3.18 L ethanol; 1.44 L n-propyl acetate) and stirred mechanically to ensure full dissolution of the polymer. Tributyl 2-acetyloxypropane-l,2,3-tricarboxylate (ATBC, 25.0 g) and polyethylene wax, 40% dispersion in isopropanol (50.0 g), were then added to the polymer solution as printing additives. 3.66 g (2.720xl0-3mol) of [Ru(dpp)s]2+was dissolved in a 69:31 (v / v) mixture of ethanokethyl acetate (345 ml ethanol; 155 ml ethyl acetate) to prepare a first lumophore solution, while separately 2.85 g ( 7.615xl0-3mol) of Curcumin was dissolved in a 69:31 (v / v) mixture of ethanokethyl acetate (345 ml ethanol; 155 ml ethyl acetate) to prepare a second lumophore solution. Once fully dissolved, the two lumophore solutions were added to the polymer solution prepared above.

[0113] After ensuring complete homogenisation of the formulation, a pattern consisting of 1 cm x 1 cm squares was printed onto the inside of a multilayer LINTOP PE HB film from Klbckner Pentaplast using a rotogravure printing press. The printed film passed through a drying tunnel set at 75 °C) before being rewound at the end of the line. The 1,000m long reels were protected from light using a protective foil and stored at room temperature. The printed film was mounted onto a reel-to-reel feeder and hundreds of meters of printed film were tested using a prototype vision system. After completion of the testing, the printed film was used fortrials of MAP food products. The colour change response ofthe printed sensor formulation was measured as outlined in Example 2 below.

[0114] 1.3 Ruthenium (II) 4,7-diphenyl-l,10'-phenanthroline and 2-(3,4-dihydroxyphenyl)-3,7- dihydroxychromen-4-one

[0115] [Ru(dpp)3]2+: Fisetin (1:2.8 molar ratio) in ethyl cellulose (EC)

[0116] 28.8 g of dry ethyl cellulose polymer (4cP) was added to a 69:31 (v / v) mixture of ethanokethyl acetate (140.9 ml ethanol; 63.9 ml ethyl acetate) and stirred overnight to ensure full dissolution of the polymer. 146.6 mg (1.088xl0-4mol) of ruthenium (II) 4,7-diphenyl-l,10'- phenanthroline ([Ru(dpp)s]2+, Lumophore 1) and 88.4 mg (3.089xl0-4mol) of 2-(3,4- dihydroxyphenyl)-3,7-dihydroxychromen-4-one (Fisetin, Lumophore 2), were then added sequentially to the solution in powder form and the solution stirred until complete homogenisation was achieved. The formulation was then printed using the same methodology as described in Example 1.1. The film incorporating the printed sensor squares was measured and the colour change response was assessed as outlined in Example 2 below. 1.4 Platinum(ll) 5,10,15,20-tetrakis(2,3,4,5,6-pentafluorophenyl)porphyrin-22,24-diide in ethyl cellulose (EC) and sodium [(2S,3R,4R)-5-(7,8-dimethyl-2,4-dioxobenzo[g]pteridine- 10-yl)-2,3,4-trihydroxypentyl] hydrogen phosphate in hydroxypropyl cellulose (HPC) PtTFPP:Flavin mononucleotide sodium salt (FMN) (1:6.0 molar ratio) in ethyl cellulose / hydroxypropyl cellulose (EC / HPC)

[0117] 2.88 g of dry ethyl cellulose polymer (4 cP) was added to a 69:31 (v / v) mixture of ethanokethyl acetate (14.1 ml ethanol; 6.4 ml ethyl acetate) and stirred to ensure full dissolution of the polymer. 12.7 mg (3.1 ml, 1.087xl0-5mol) of platinum(ll) 5,10,15,20-tetrakis(2,3,4,5,6- pentafluorophenyl)porphyrin-22,24-diide (PtTFPP, Lumophore 1) was first dissolved in THF before being added to the EC polymer solution. Separately, 2.00 g of dry hydroxypropyl cellulose (HPC) was solubilised in 34.6 ml ethanol and stirred until full dissolution. 33.3 mg (0.74 ml, 6.539xl0-5mol) of flavin mononucleotide (FMN) sodium salt was then added to the HPC polymer solution and the solution stirred until complete homogenisation was achieved. The FMN formulation was printed onto a self-adhesive PE substrate (EZ-Pierce™) using a 20 pm spiral coating bar before the printed substrate was dried in the oven at 40 °C for 2 hrs. The printed substrate was then coated with the PtTFPP / EC formulation using the same 20 pm coating bar. The printed substrate was cut into 1 cm x 1 cm squares and stored in the dark at room temperature until used. The colour change response of the printed sensor formulation was measured as outlined in Example 2 below.

[0118] 1.5 Further Formulations

[0119] Further formulations comprising [Ru(dpp)3]2+:Riboflavin (1:2.8 molar ratio) in HPMC; PtTFPP:Curcumin (1:3.0 molar ratio) in polystyrene (PS); PtOEP:Paeonol (1:10.0 molar ratio) in CAP / EC; PtTFPP:Ellagic acid (1:6.0 molar ratio) in EC / HPC; PtTPP:Curcumin (1:3.0 molar ratio) in EC; PtTPP:Morin (1:3.0 molar ratio) in CAB; PtTPP:Safflower extract (1:1.9 molar ratio) in EC / HPMC; PtOEP:Curcumin (1:3.0 molar ratio) in CAP; PtOEP:Riboflavin (1:6.0 molar ratio) in CAB; PtTFPP:Bixin (1:3.0 molar ratio) in EC / HPC; PtOEP:Fisetin (1:3.0 molar ratio) in EC, [Ru(dpp)3]2+:Quercetin (1:2.8 molar ratio) in EC;PtTPP:FMN (1:6.0 molar ratio) in EC / HPC) and PtTFPP:3-hydroxyflavone (1:4.0 molar ratio) in EC / CAB were prepared using the methodology described in Example 1.1. Forthe [Ru(dpp)3]2+:Riboflavin (1:2.8 molar ratio) in HPMC example, a 10 pm spiral coating bar was used.

[0120] The formulations that were prepared in Examples 1.1 to 1.5 are summarised in Table 1 below:

[0121] Table 1: preparation of oxygen sensors Example 2: Measurement of colour response

[0122] The colour response of the sensor formulation to changes in oxygen concentration was determined as follows:

[0123] For Examples 1.1 and 1.3 to 1.5, the non-adhesive side of commercially available self-adhesive PE and PET substrates (EZ-Pierce™ and SealPlate™ films from Excel Scientific) was printed using spiral coating bars from Elcometer™ allowing for the deposition of wet ink films with a 10 or 20 pm thickness (see Table 1). The coated substrates were then dried, cut into 1 cm x 1 cm squares, and stored in the dark at room temperature until used (up to 6 months). For Example 1.2, the sensor was printed using a rotogravure printing press as outlined in that example.

[0124] Spectrophotometric assessment of the sensor response (i.e. measurement of the spectral response) was performed by sticking a single sensor onto a pre-cut glass slide and placing it in a disposable poly(methyl methacrylate) (PMMA) cuvette or cell under a controlled gas atmosphere. A Teflon-based sample holder was used to hold the sensor-in the LED excitation light path (at 385 nm for PtOEP and PtTPP or at 405 nm for [Ru(dpp)s]2+and PtTFPP) at a fixed angle of 45°, thus ensuring that the sample position was the same for each sensor measured. The modification of the atmosphere within the cuvette was achieved by mixing O2 and N2 gases at the required ratios (0-100% O2, which corresponds to 100-0% N2) using a computer- controlled gas blender. Two holes on the top of the sample holder allowed for the gas mix IN flow and OUT flow.

[0125] A vision system equipped with a CMOS camera and 2 LED bar lights was used to assess the sensor response (i.e. colour response). 1 cm2square sensors were adhered to the underside (i.e. the food contact side) of a commercial multilayer lidding film before the film was placed in a measuring chamber. Typically, six sensors were used to prevent against sensor failure and provide an average sensor response. [Although not done here, a printed reel of film could alternatively be dispensed over the measuring chamber using a reel-to-reel feeder]. The gas atmosphere in the chamber can be continuously modified by flowing IN the desired gas mixture using a computer-controlled gas blender and confirming the modified atmosphere within the chamber by analysing the OUT flow using a gas analyser. Once the required oxygen within the chamber is reached, a manual trigger strobes the LED bar lights, and the camera captures an RGB image of the sensors during the illumination pulse. The gas is then modified within the chamber and the image capture is repeated once the gas atmosphere set on the gas blender matches the gas reading from the gas analyser. For low O2 formulations (i.e. operating at oxygen levels < 21%), images were captured at 0, 1, 3, 5, 10, 21% O2. For high O2 formulations (i.e. operating at oxygen levels >35%), images were recorded at 0, 20, 35, 50, 60, 70 and 80% O2. The RGB and HSI outputs from the camera are then analysed to obtain the colour response of the various formulations tested. To standardise the reporting of the colour response, the relative percentage changes are presented as follows: For formulations having their application for high O2 packaging, the difference in hue is indicated using the 80% O2 hue value as reference (AHue = Hueso-Huei, where Huei, is the value measured at i% O2), so that the relative percentage change is obtained by dividing the hue difference by the hue at 80% O2, i.e. AHue / Hueso. Similar reporting is performed for formulations working at low O2, but in that case, the 0% O2 Hue value is used as reference, so that the relative percentage change is given by (Huei-Hueo) / Hueo .

[0126] The results of these measurements for PtOEP: Curcumin (1:3) in CAP (Example 1.5), for [Ru(dpp)s]2+: Curcumin (1:2.8) in EC (Example 1.2) and for [Ru(dpp)s]2+: Curcumin (1:7.5) in EC (Example 1.1) are shown in Figure 1.

[0127] Figure 1(a) shows that a discernible colour change is exhibited for the PtOEP: Curcumin [(1:3) in CAP, Example 1.5] sensor from an initial bright orange to a final green colour between 0- 21% O2. The three-colour transition from orange to yellow to green in this particular oxygen range is relevant to vacuum-packed products, oxygen-sensitive MAP foodstuffs (cooked meat, nuts), as well as MAP of fresh produce. The average hue and % change was measured, and the results are included in Table 2 below. These show that the change of colour resulted in a relative change in Hue between 0 and 5% O2 (Hues-Hueo / Hueo) of 210% for this lumophore combination.

[0128] Table 2: Hue change for PtOEP: Curcumin sensor [(1:3) in CAP, (Example 1.5)]

[0129] Figure 1(b) shows that for the [Ru(dpp)s]2+: Curcumin [(1:2.8) in EC (Example 1.2)] sensor, an orange to green colour change was evident between 0-80% O2. The average hue and % change were measured, and the results are included in Table 3 below. The relative change in Hue between 80 and 50% O2 (Hueso-Hueso / Hueso) reached -19.3%. The oxygen range where the colour change takes place is relevant to MAP gas mixtures used for products such as raw meats which are packed under a Ch-rich atmosphere (70-80% O2). Table 3: Hue change for [Ru(dpp)s]2+: Curcumin sensor [(1:2.8) in EC (Example 1.2)] Figure 1(c) shows that for [Ru(dpp)3]2+:Curcumin [(1:7.5) in EC (Example 1.1)] a yellow to green colour change is produced in a modified atmosphere containing 0-21% O2. The average hue and % change were measured, and the results are included in Table 4 below. The change in colour resulted in a Hue difference between 0 and 10% O2 of > 20 or a relative change (Hueio-Hueo / Hueo) of 46%. The colour transition occurs in a range of oxygen suitable for MAP packaging of oxygen-sensitive foodstuffs, such as bagged lettuce.

[0130] Table 4: Hue change for [Ru(dpp)s]2+: Curcumin sensor [(1:7.5) in EC, (Example 1.1)]

[0131] Example 3: Effect of Antioxidant on Sensor Performance

[0132] The effect of the addition of an antioxidant on the photostability of the sensor formulations was investigated as follows.

[0133] The formulations of Examples 1.1 to 1.5 were prepared as described above. After homogenisation, the formulation was then split in two vials. The first vial was used as a control, while an antioxidant was added directly to the formulation in the second vial to give the molar ratio of lumophore 2: antioxidant indicated in Table 5 below. The control and the antioxidant-containing formulation were then each coated onto PE self-adhesive substrates using spiral coating bars as described in Example 2 above. The photostability of the printed sensor squares from the formulations was then assessed by continuously illuminating the sensors with the 405 nm LED bar lights (FWHM = 20 nm ) at full power for 5 min under (i) a 80% O2 atmosphere for formulations relevant to products packed in 02-rich MAP packaging or (ii) a 21% O2 (air) atmosphere for formulations developed for oxygen-sensitive products (low O2). An image was captured every minute and changes in RGB and HSI were compared. The results are shown in Table 5 below.

[0134] Table 5: Effect of antioxidant on photostability of sensor formulations

[0135] The results demonstrate that the incorporation of an antioxidant into the formulation leads to surprisingly significant enhanced photostability, i.e. > 30% (hue) for the [Ru(dpp)s]2+formulations, when compared with the formulations in which no antioxidant was included.

[0136] For instance, for the [Ru(dpp)3]2+:curcumin lumophore combination of Example 1.2 (1:2.8 molar ratio), the formulation displayed a significant decrease in both RGB and HSI over 5 minutes. Specifically, the green (G), red (R) and hue (H) values decreased by 12.3, 4.0 and 7.5%, respectively, over the 5-minute illumination period. Conversely, when the antioxidant was included in the formulation (in this case tocopherol at L2:AO of 1:2), the green (G), red (R) and hue (H) values decreased by 7.2, 2.2 and 3.8%, respectively, over the same 5-minute illumination period. This suggests an improvement in photostability in the region of 50% for hue, 45% for red and 41% for green when an antioxidant is included in the formulation.

[0137] Similarly, when bixin, which can act both as antioxidant and second lumophore, is added (1:1 L2:AO molar ratio) to a formulation comprising the [Ru(dpp)3]2+:fisetin lumophore combination of Example 1.3 (1:2.8 molar ratio), the photostability improvement reaches 48% for hue, 108% for red and 61% for green.

[0138] When quercetin, which can act both as antioxidant and second lumophore is added to a formulation comprising the PtTPP:FMN lumophore combination of Example 1.5 (1:6 molar ratio), the photostability is significantly improved by 87% and 57% for red (R) and green (G), respectively, while a smaller 5% stabilisation is observed for hue. The lower stabilisation observed in hue is a direct result of the larger stability improvement for the red compared to the green.

[0139] Smaller and simpler compounds such as vanillin was also shown to contribute in the surprising stabilisation of the PtTPP: FM N lumophore pair, with a photostability improvement of 8% for hue, 11% for red, and 8% for green observed.

[0140] The surprising efficacy of the antioxidant addition means that the naturally-occurring lumophores and their synthetic analogues can be effectively exploited for use in the luminescence-based sensors of the invention.

[0141] Example 4: Effect of Metal Coordination to Lumophore 2 on Sensor Response

[0142] The effect of the coordination of a metal ion to Lumophore 2 on sensor performance was investigated as follows. The formulations listed in Table 1 were prepared following a similar protocol to that described in Example 1, with the addition of a metal to yield a coordination complex with lumophore 2 carried out as described below in Examples 4.1-4.3.

[0143] In summary, the formation of the metal complex was achieved either by (i) direct addition of an aliquot of the metal stock solution to the formulation or (ii) pre-formation of the metal complex by mixing a solution of lumophore 2, deprotonated by the addition of base if required, with the metal ion to give the molar ratio of lumophore 2: M indicated in Table 6 below, and subsequent addition to the formulation. After the addition of lumophore 1 and homogenisation, the control and metal-containing formulations were each coated onto self- adhesive PE substrates (EZ-Pierce™ from Excel Scientific) using spiral coating bars as described in Example 2 above.

[0144] 4.1 Ruthenium (II) 4,7-diphenyl-l,10'-phenanthroline and 2-(3,4-dihydroxyphenyl)- 3,5,7-trihydroxychromen-4-one with Al (II I)

[0145] [Ru(dpp)3]2+:Quercetin-AI(lll) (1:2.8 molar ratio) in ethyl cellulose (EC)

[0146] 28.8 g of dry ethyl cellulose polymer (4 cP) was added to a 69:31 (v / v) mixture of ethanokethyl acetate (140.9 ml ethanol; 63.9 ml ethyl acetate) and stirred overnight to ensure full dissolution of the polymer. 146.6 mg (1.088xl0-4mol) of ruthenium (II) 4,7-diphenyl-l,10'- phenanthroline ([Ru(dpp)s]2+, Lumophore 1) and 105.9 mg (3.089xl0-4mol) of 2-(3,4- dihydroxyphenyl)-3,5,7-trihydroxychromen-4-one (Quercetin hydrate, Lumophore 2), were then added sequentially to the solution in powder form and the solution stirred until complete homogenisation was achieved. The metal salt, in this case aluminium(lll) chloride hexahydrate (AICI3.6H20, 77.9 mg, 3.195xl0-4mol or 1.05 molar equivalent), was added to the formulation, which was further stirred. Sufficient time (l-2hrs) between the addition of Al(lll) and the printing was allowed to ensure the complete formation of the coordination complex L2-M. Printing and preparation of the sensor squares was performed as described in Example 1.1. The colour change response of the sensor was then assessed as outlined in Example 2 above. 4.2 Platinum(ll) 5,10,15,20-tetrakis(2,3,4,5,6-pentafluorophenyl)porphyrin-22,24-diide in ethyl cellulose (EC) and 6,7,13,14-tetrahydroxy-2,9-dioxatetracyclo [6.6.2.04’16.01:l’15]hexadeca-l(15),4,6,8(16),ll,13-hexaene-3,10-dione with B(lll) in hydroxypropyl cellulose (HPC)

[0147] PtTFPP:Ellagic acid - B(lll) (1:6.0 molar ratio) in ethyl cellulose / hydroxypropyl cellulose (EC / HPC)

[0148] 2.88 g of dry ethyl cellulose polymer (4 cP) was added to a 69:31 (v / v) mixture of ethanokethyl acetate (14.1 ml ethanol; 6.4 ml ethyl acetate) and stirred to ensure full dissolution of the polymer. 12.7 mg (1.087xl0-5mol) of platinum(ll) 5,10,15,20-tetrakis(2,3,4,5,6- pentafluorophenyl)porphyrin-22,24-diide (PtTFPP, Lumophore 1) was first dissolved in THF before being added to the EC polymer solution. 2.00g of dry hydroxypropyl cellulose (HPC) polymer was dissolved in a 75:25 (v / v) mixture of ethanokwater (54.2 ml ethanol; 18.0 ml deionised water) and stirred overnight. The coordination of B(lll) to ellagic acid was performed prior to the addition to the HPC solution. A 4g / l stock solution of ellagic acid in basic water (pH 11) and a 0.067 M stock solution of Borax (sodium tetraborate) in deionised water were prepared. 10.0 mg (2.5 ml, 3.31xl0-5mol) of the ellagic acid stock solution was then added to 0.994 ml (25.3 mg, 6.62xl0-5mol) of the Borax solution to obtain a L2:M molar ratio of 1:2.

[0149] The ellagic acid:B(lll) coordination compound was then added to the HPC polymer solution and stirred until homogenisation was achieved.

[0150] The formulations, i.e. PtTFPP in EC, and ellagic acid-B(lll) in HPC, were printed onto a self- adhesive PE substrate (EZ-Pierce™ from Excel Scientific) as described in Example 2. To ensure a rapid response to oxygen, the oxygen sensitive layer, PtTFPP in EC, was printed after the ellagic acid-HPC layer. The printed substrate was dried in the oven at 40 °C for 2 hrs. The printed PE substrate was then cut into 1 cm x 1 cm squares and stored in the dark at room temperature until needed. The sensor squares were then placed on the inside of a MAP lidding film (i.e. a commercial multilayer film from Plastopil (Toplex HB44)). The colour change response of the multilayer print was measured as outlined in Example 2. 4.3 Ruthenium (II) 4,7-diphenyl-l,10'-phenanthroline and (lE,6E)-l,7-bis(4-hydroxy-3- methoxyphenyl)hepta-l,6-diene-3, 5-dione with Al(lll)

[0151] [Ru(dpp)3]2+:Curcumin-AI(lll) (1:2.8 molar ratio) in ethyl cellulose (EC)

[0152] 28.8 g of dry ethyl cellulose polymer (4 cP) was added to a 69:31 (v / v) mixture of ethanokethyl acetate (140.9 ml ethanol; 63.9 ml ethyl acetate) and stirred overnight. While 146.6 mg (1.088xl0-4mol) of ruthenium (II) 4,7-diphenyl-l,10'-phenanthroline ([Ru(dpp)s]2+, Lumophore 1) was added to the EC solution from a freshly prepared stock solution, (1E,6E)- l,7-bis(4-hydroxy-3-methoxyphenyl)hepta-l,6-diene-3, 5-dione (Curcumin, Lumophore 2) was first coordinated to Al(lll) before being added to the polymer. In brief, a 1 ml stock solution of curcumin (20.4 g / l in ethyl acetate:ethanol 2:1 (v / v), 5.543xl0-5mol) was mixed with 1 equivalent of NaOH IM (0.055 ml, 5.543xl0-5mol) to deprotonate curcumin's enol functional group. This step was followed by the addition of 13.4mg of AICI3.6H2O (1 eq., 5.543xl0-5mol) to allow for the coordination complex to be formed in solution. The solution was stirred vigorously to ensure full coordination of Al ( 111 ). Finally, 0.549 ml (3.043xl0-5mol curcumin) of the curcumin-AI(lll) solution was added to the EC polymer containing the first lumophore in order to obtain a L1:L2 molar ratio of 1:2.8. The formulation was then stirred until complete homogenisation was achieved and no sedimentation of the lumophores was observed. After printing the formulation onto a self-adhesive PE substrate, the backing paper was removed, and the sensor squares were placed on the inside of a MAP lidding film (i.e. a commercial multilayer film from Plastopil (Toplex HB44)) for testing. The colour change response of the printed sensor formulation was measured as outlined in Example 2 below.

[0153] 4.4 Platinum(ll) 5,10,15,20-tetrakis(2,3,4,5,6-pentafluorophenyl)porphyrin-22,24-diide in ethyl cellulose (EC) and 3-hydroxy-2-phenylchromen-4-one with Zn(ll) in cellulose acetate butyrate (CAB)

[0154] PtTFPP:3-hydroxyflavone - Zn(ll) (1:4.0 molar ratio) in ethyl cellulose / cellulose acetate butyrate (EC / CAB)

[0155] 2.88 g of dry ethyl cellulose polymer (4 cP) was added to a 69:31 (v / v) mixture of ethanokethyl acetate (14.1 ml ethanol; 6.4 ml ethyl acetate) and stirred to ensure full dissolution of the polymer. 12.7 mg (1.087xl0-5mol) of platinum(ll) 5,10,15,20-tetrakis(2,3,4,5,6- pentafluorophenyl)porphyrin-22,24-diide (PtTFPP, Lumophore 1) was first dissolved in THF before being added to the EC polymer solution. 4.20g of dry cellulose acetate butyrate (CAB) polymer was dissolved in a 69:31 (v / v) mixture of ethanokethyl acetate (14.1 ml ethanol; 6.4 ml ethyl acetate) and stirred overnight. The coordination of Zn(ll) to 3-hydroxyflavone was performed prior to the addition to the CAB solution. A 13 g / l stock solution of 3- hydroxyflavone in ethanokTHF 75:25 (v / v) and a 0.076 M stock solution of zinc(ll) chloride (ZnCb) in ethanol were prepared. 10.4 mg (0.804 ml, 4.35xl0-5mol) of the 3-hydroxyflavone stock solution was then mixed with 0.570 ml (5.9 mg, 4.35xl0-5mol) of the ZnCb solution to obtain a L2:M molar ratio of 1:1.

[0156] The 3-hydroxyflavone:Zn(ll) coordination compound was then added to the CAB polymer solution and stirred until homogenisation was achieved.

[0157] The formulations, i.e. PtTFPP in EC, and 3-hydroxyflavone-Zn(ll) in CAB, were printed onto a self-adhesive PE substrate (EZ-Pierce™ from Excel Scientific) as described in Example 2. To ensure a rapid response to oxygen, the oxygen sensitive layer, PtTFPP in EC, was printed after the 3-hydroxyflavone-containing CAB layer. The printed substrate was dried in the oven at 40 °C for 2 hrs. The printed PE substrate was then cut into 1 cm x 1 cm squares and stored in the dark at room temperature until needed. The sensor squares were then placed on the inside of a MAP lidding film (i.e. a commercial multilayer film from Plastopil (Toplex HB44)). The colour change response of the multilayer print was measured as outlined in Example 2.

[0158] 4.5 Assessment of Colour Response and Photostability

[0159] The colour response of the control (Table 1) and metal-containing formulations (Examples 4.1 to 4.4) were assessed as described in Example 2 and relative increase / decrease in sensitivity (colour response) is reported in Table 6. The photostability of the printed sensor squares from the formulations was then assessed by continuously illuminating the sensors with the 405 nm LED bar lights (FWHM = 20 nm ) at full power for 5 min under (i) an 80% O2 atmosphere for formulations relevant to products packed in C -rich MAP packaging or (ii) a 21% O2 (air) atmosphere for formulations developed for oxygen-sensitive products (low O2). An image was captured every minute and changes in RGB and HSI were compared. The results are shown in Table 6 below and represent the stability improvement for the same illumination exposure (i.e. taking into consideration the absorption changes induced by metal coordination).

[0160] Table 6: Effect of metal coordination on the fluorescence, colour response and photostability of sensor formulations.

[0161] As can be seen from the results in Table 6, the sensor comprising [Ru(dpp)3]2+:Quercetin-AI(l 11 ) prepared in Example 4.1 showed significantly increased brightness compared to the same formulation without the addition of Al(lll), with a significant colour change from red to green clearly discernible to the naked eye. The O2 sensitivity of the complexed-Quercetin formulation was also improved significantly compared with its free metal analogue, with a 27% increase in sensitivity observed. The change in Hue observed at oxygen concentrations between 80% and 50% was 15.4% for the control (non-complexed sensor) and 19.3% for the metal-containing formulation. A significant 33% improvement in photostability (based on Hue values) was also observed for the Al(lll) complexed Quercetin sensor compared with its uncomplexed counterpart.

[0162] As for Example 4.1, the lumophore pair of Example 4.2 (i.e. PtTFPP: El lagic acid-B(lll)) showed improved brightness compared to the same formulation without the addition of B(lll). Similarly, to what was observed in Example 4.1 with Al(lll), the addition of sodium tetraborate (as source of B(lll)) also contributed in improving the O2 sensitivity, with a 24% increase in sensitivity recorded. The photostability was tested and compared with the metal-free control formulation. A similar photostability improvement was observed across the hue, red and green outputs (23-25%).

[0163] For the complexed formulation of Example 4.3, (i.e. [Ru(dpp)3]2+:Curcumin (1:2.8, in EC), the formulation exhibited an increase in brightness, which (as also seen in Examples 4.1 and 4.2.) enhanced the colour response, resulting in a 11% improvement in O2 sensitivity. In addition, the metal-containing formulation showed a much greater photostability compared to its corresponding metal-free formulation, with a photostability improvement of 42% in Hue, 34% red and 38% green achieved. The decrease in Hue went from 4.3% in the absence of Al(lll) to 2.5% after coordination to Al(lll), while the red and green went from 5.3% and 12.6%, respectively for the metal-free formulation to 3.5% and 7.8% decrease in the presence of Al(lll).

[0164] For the complexed formulation of Example 4.4, (i.e. PtTFPP:3-hydroxyflavone (1:4.0, in EC / CAB), the formulation exhibited an increase in brightness, which (as also seen in Examples 4.1 and 4.2.) enhanced the colour response, resulting in a 61% improvement in O2 sensitivity. In addition, the metal-containing formulation showed a much greater photostability compared to its corresponding metal-free formulation, with a photostability improvement of 31% in red and 62% in green achieved. Compared to previous examples, 3-hydroxyflavone as well as its Zn(ll) coordinated counterpart show sufficient blue emission to be able to identify a significant 58% improvement in photostability in blue. The decrease in red went from 0.9% in the absence of Zn(ll) to 0.6% after coordination to Zn(ll), while the green and blue went from 4.2% and 2.8%, respectively for the metal-free formulation to 2.6% and 1.7% decrease in the presence of Zn(ll). It is important to note that in some cases, as for the lumophore pair [Ru(dpp)3]2+:fisetin (1:2.8 molar ratio, in EC), the photostabilisation was disproportionately observed in favour of one of the lumophores. In this instance, the red output from the sensor originating from [Ru(dpp)s]2+displays a 3-fold greater improvement in photostability than the green output, from fisetin. The colour of the sensor, represented by the hue value, changes towards a redder colour (i.e. lower hue), which results in an apparent decrease in stability.

[0165] Example 5: Combined Effect of Antioxidant and Metal Coordination on Sensor Response

[0166] [Ru(dpp)3]2+:Curcumin (1:2.8 molar ratio) in ethyl cellulose (EC) (4 cP) was prepared as outlined in Example 1.1, but at a molar ratio of 1:2.8. Briefly, 14.4 g of dry ethyl cellulose polymer was added to a 69:31 (v / v) mixture of ethanokn-propyl acetate (70.5 ml ethanol; 32.0 ml ethyl acetate) and stirred overnight. 73.3 mg (5.438xl0-5mol) of [Ru(dpp)s]2+, lumophore 1, and 56.1 mg (1.523xl0-4mol) of Curcumin, lumophore 2, were dissolved in a 69:31 (v / v) mixture of ethanol :ethyl acetate before being added to the polymer solution. After complete homogenisation, the formulation was split equally into 2 different flasks. The first flask was kept as prepared , while DL-a-tocopherol (2 eq., 1.523xl0-4mol ) was added to the second flask.

[0167] Concomitantly, the metal-containing formulation described in Example 4.3 was prepared and also separated equally into 2 different flasks; flask 3 contained the Al(lll) based formulation, [Ru(dpp)3]2+:Curcumin-AI(lll) (1:2.8 molar ratio), as prepared. DL-a-tocopherol was added to flask 4 to obtain a formulation where both antioxidants and metal coordination were used.

[0168] The following formulations were therefore obtained:

[0169] Flask 1: [Ru(dpp)3]2+:Curcumin;

[0170] Flask 2: [Ru(dpp)3]2+:Curcumin with TP;

[0171] Flask 3: [Ru(dpp)3]2+:Curcumin-AI(lll);

[0172] Flask 4: [Ru(dpp)3]2+:Curcumin-AI(lll) with TP. After printing each of the formulations of Flasks 1-4 onto a self-adhesive PE substrate (EZ- Pierce™ from Excel Scientific), the sensors were cut and placed onto the inner layer MAP lidding film from Plastopil (Toplex HB44) for testing. The photostability of curcumin within each of the 4 formulations was then assessed and the stabilisation effect was measured as described in Example 4.5.

[0173] While both tocopherol and Al(lll) coordination individually led to a significant increase in the photostability of curcumin (see Tables 5 and 6), combining both additives in the same formulation led to a further improvement. When comparing the reference, [Ru(dpp)3]2+:Curcumin only, with the metal- and antioxidant-containing formulation, a 72% 66% and 63% stabilisation in Hue, red and green, respectively, was observed. The reference exhibited a Hue, red and green decrease of 4.3%, 5.3%and 12.6%, respectively, while these same values were 1.2%, 1.8%and 4.6% for [Ru(dpp)3]2+:Curcumin-AI(lll) + tocopherol. The results suggest that the combination of metal co-ordination of the naturally-occurring lumophore, along with the addition of an antioxidant to the formulation comprising the second lumophore, leads to very significant increases in sensor performance.

[0174] Example 6: Application of colorimetric sensor to low O2 MAP packs - MAP bagged lettuce

[0175] The two formulations [Ru(dpp)3]2+:Curcumin (1:7.5 molar ratio) and PtTFPP-Kaemperol-AI(lll) (1:3.0 molar ratio) were prepared as outlined in Example 1.1 and Example 4.1, respectively. Once printed on a PE self-adhesive sheet (EZ-Pierce™ from Excel Scientific) and cut into 1cm2square sensors, 4 sensors were placed on the inside of a commercial film used to pack bagged shredded lettuce. The lettuce was placed inside the bag, before sealing was performed using a heat-sealing device. To create the modified atmosphere, the air was evacuated from the bags with a needle going through a rubber septum and connected to the vacuum. Once most of the air had been removed (close to vacuum), vacuum was stopped, and the needle was connected to the N2 gas line. The bagged lettuce was then flushed with N2. The cycle was repeated three times to achieve an O2 level of 1% O2 or lower within the headspace of the bagged lettuce. Following this protocol, 30-40 shredded lettuce bags were produced and imaged using the vision system described in Example 2 above. The O2 level within the headspace of the bagged lettuce was first calculated using the vision system and the Hue value obtained from the captured images. The bags were then destructively tested using a gas analyser and the readings from the gas analyser were compared with the ones provided by the vision system. A clear orange-yellow to green colour change was observed for the [Ru(dpp)3]2+:Curcumin and a clear colour change from orange to green colour was observed for PtTFPP-Kaemperol-AI(lll). For both sensors, the O2 levels determined using the vision system were within ±1% from the gas analyser readings for most of the bags. Lettuce bags with poor sealing were detected immediately as the O2 increased significantly between measurements, indicating air ingress through a leak or crease in the seal.

[0176] Example 7: Application of colorimetric sensor to high O2 MAP packs - MAP mince beef

[0177] A 5kg ink batch of [Ru(dpp)3]2+:Curcumin (1:2.8 molar ratio) was prepared as described in Example 1.2. An antioxidant, DL-a-tocopherol (2eq., 1.523xl0-4mol) was subsequently added to the sensor formulation, before the viscosity of the formulation was adjusted for printing on a rotogravure printing press by adding solvent mix until required viscosity was obtained as measured using a Zahn Cup #2. The ink formulation was printed on a 285 mm wide, 1,000 m long co-extruded film reel, which had been pre-treated using corona discharge to increase the adhesion of the ink to the substrate film.

[0178] The printed film was used to seal the headspace of modified atmosphere packaged raw mince beef, at MAP of 80% 02 / 20% CO2 on a commercial food packaging line. The MAP mince beef packages were stored in retailer display cabinets at 4 °C over 12 days. The two shelf-life trials (36 packs per trial) involved measuring each pack daily to monitor sensor colour outputs using the vision system described in Example 2. The purpose of the trial was to monitor non- invasively the O2 level inside the MAP mince beef packs throughout the course of the product shelf life. The changes in the O2 levels observed within the headspace of the packages were grouped into 3 categories:

[0179] Group I: Leak / Damage: Significant drop in O2 level attributed to leak in or damage to the packaging tray or lidding film;

[0180] Group II: Stable / no significant drop in O2 level observed during the shelf life;

[0181] Group III: Drop in O2 level observed during the shelf life. Packs from Group I exhibited a visible colour change from green to orange, due to a significant decrease in oxygen level, which is typical behaviour for a pack that has lost its integrity due to leakage, poor sealing, or other structural damage.

[0182] The Group 2 packs showed stable O2 readings and thus displayed no colour change for the duration of the shelf life of the packaged product.

[0183] The Group 3 packs showed no, or only slight colour change for first few days, but the sensor readings dropped several days before the product "use by" date (i.e. typically 3 days before the "use by" date), indicating that an oxygen level decrease within those packs. The decrease resulted in a small but measurable change in colour over the last three days of the shelf-life.

[0184] As a control, 6 packs of MAP minced beef were destructively tested using a Mocon Dansensor O2 and CO2 gas analyser (Model: Checkpoint 3) and the results compared with the sensor outputs. Gas analyser readings correlated with the non-destructive testing within ~ ±5%.

[0185] As the inventors have demonstrated, the non-toxic luminescent-based colorimetric sensors of the invention combine high sensitivity towards oxygen and tuneability to specific oxygen concentrations, such as those relevant for the MAP of particular types of foodstuffs, via the colour change induced by luminescence quenching. Due to the non-toxic nature of the formulation, the sensor can be embedded within food packaging atmospheres, for instance where it can come into direct contact with foodstuffs or other ingestible items, with legislative approval for the sensors streamlined by the non-toxic and naturally-occurring nature of the second lumophore. The sensors enable semi-quantitative analysis to be performed based solely on the colorimetric changes induced, while ratiometric measurements can be used if required to perform fully quantitative measurements. The sensors can be used alongside digital image analysis, performed via software or an app pre-programmed with an internal sensor calibration, to provide high throughput visualisation and assessment of the sensor response. The non-destructive nature of the sensor allows continuous quality control, for example with every packaged product on a manufacturing line being analysed in-situ, and throughout the supply chain. This means that any defective or spoiled items can be removed from the supply chain when identified, without affecting the remainder of the batch, leading to significant environmental benefit due to the reduction in food waste and associated packaging. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. Each feature disclosed in this specification (including any accompanying claims, abstract and drawings) may be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features. The invention is not restricted to the details of the foregoing embodiment(s). The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

[0186] With respect to the use of substantially any plural and / or singular terms herein, those having skill in the art can translate from the plural to the singular and / or from the singular to the plural as is appropriate to the context and / or application. The various singular / plural permutations may be expressly set forth herein for sake of clarity.

[0187] It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims are generally intended as "open" terms (e.g., the term "including" should be interpreted as "including but not limited to," the term "having" should be interpreted as "having at least," the term "includes" should be interpreted as "includes but is not limited to," etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases "at least one" and "one or more" to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles "a" or "an" limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases "one or more" or "at least one" and indefinite articles such as "a" or "an" (e.g., "a" and / or "an" should be interpreted to mean "at least one" or "one or more"); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of "two recitations," without other modifiers, means at least two recitations, or two or more recitations).

[0188] It will be appreciated that various embodiments of the present disclosure have been described herein for purposes of illustration, and that various modifications may be made without departing from the scope of the present disclosure. Accordingly, the various embodiments disclosed herein are not intended to be limiting, with the true scope being indicated by the following claims.

Claims

Claims1. A colorimetric oxygen sensor comprising a first lumophore and a second lumophore, each lumophore being dispersed in a polymer matrix, wherein the first lumophore is selected from ruthenium (II) 4,7-diphenyl-l,10'-phenanthroline [Ru(dpp)s]2+; platinum octaethylporphyrin (PtOEP); platinum 5,10,15,20-tetrakis(2,3,4,5,6- pentafluorophenyl)porphyrin (PtTFPP) and platinum meso-tetraphenylporphyrin (PtTPP); and the second lumophore is a naturally-occurring compound or a synthetic analogue thereof selected from a diarylheptanoid; a flavonol; a carotenoid selected from bixin, capsanthin and capsorubin; a co-enzyme; a minor flavonoid; paeonol, or ellagic acid.

2. A colorimetric oxygen sensor as claimed in claim 1, wherein the diarylheptanoid is selected from curcumin; the flavonol is selected from quercetin, fisetin, kaempferol, myricetin, 3-hydroxyflavone and morin; the co-enzyme is selected from riboflavin, NADH, FAD and FMN; the minor flavonoid is safflower extract; or the second lumophore is paeonol, ellagic acid, bixin, capsanthin or capsorubin.

3. A colorimetric oxygen sensor as claimed in claim 1 or claim 2, wherein the naturally- occurring compound or synthetic analogue thereof is selected from a diarylheptanoid or a flavonol.

4. A colorimetric oxygen sensor as claimed in any preceding claim, further comprising an antioxidant.

5. A colorimetric oxygen sensor as claimed in claim 4, wherein the antioxidant is a carotenoid, a phenolic compound, or a vitamin.

6. A colorimetric oxygen sensor as claimed in claim 5, wherein the antioxidant is bixin, quercetin, or DL-a-tocopherol.

7. A colorimetric oxygen sensor as claimed in any preceding claim, wherein the second lumophore is complexed with Aluminium, Boron or Zinc.

8. A colorimetric oxygen sensor as claimed in any preceding claim, wherein the polymer is selected from polystyrenes, polyvinyls, polyamides, polyurethanes, acrylates, shellac, rosin, rosin esters, celluloses and cellulose-derivatives; and mixtures thereof.

9. A colorimetric oxygen sensor as claimed in any preceding claim, wherein the first and second lumophores are dispersed in the same polymer.

10. A colorimetric oxygen sensor as claimed in any of claims 1 to 8, wherein the first and second lumophores are dispersed in different polymers.

11. A colorimetric oxygen sensor as claimed in any preceding claim, wherein the second lumophore is curcumin or fisetin.

12. A colorimetric oxygen sensor as claimed in any preceding claim, wherein the first lumophore is ruthenium (II) 4,7-diphenyl-l,10'-phenanthroline [Ru(dpp)s]2+or platinum octaethylporphyrin (PtOEP).

13. A colorimetric oxygen sensor as claimed in claim 12, wherein the first lumophore is ruthenium (II) 4,7-diphenyl-l,10'-phenanthroline [Ru(dpp)s]2+and the second lumophore is curcumin.

14. A colorimetric oxygen sensor as claimed in claim 12, wherein the first lumophore is ruthenium (II) 4,7-diphenyl-l,10'-phenanthroline [Ru(dpp)s]2+and the second lumophore is fisetin.

15. A colorimetric oxygen sensor as claimed in claim 13 or claim 14, further comprising an antioxidant.

16. A colorimetric oxygen sensor as claimed in claim 15, wherein the antioxidant is DL-a- tocopherol.

17. A colorimetric oxygen sensor as claimed in any of claims 13 to 16, wherein the polymer is ethyl cellulose.

18. A colorimetric oxygen sensor as claimed in any preceding claim, wherein the molar ratio of lumophore 1: lumophore 2 is from 1:1.9 to 1:10.

19. A colorimetric oxygen sensor as claimed in any preceding claim, wherein the sensor is in the form of one or more inks.

20. A method of determining the oxygen content of an atmosphere, the method comprising exposing the colorimetric sensor of any of claims 1 to 19 to the atmosphere; applying a source of UV or UV-visible excitation to the colorimetric sensor, and observing the colour of the sensor.

21. A method of determining the oxygen content of an atmosphere as claimed in claim 20, wherein the method further comprises comparing the colour of the sensor with a reference colour or calibration curve to make an assessment of the oxygen content.

22. A method of determining the oxygen content of an atmosphere as claimed in claim 20 or claim 21, where observing the colour of the formulation is performed via the naked eye, via the use of spectrometry, or via the use of a digital imaging apparatus.

23. A method as claimed in any of claims 20 to 22, wherein the atmosphere is a food packaging atmosphere.

24. A method as claimed in claim 23, wherein the sensor is positioned within the headspace of a modified atmosphere packaged foodstuff.