WO2023101609A2

WO2023101609A2 - A highly sensitive colourimetric sensor composed of multifunctional copolymers

Info

Publication number: WO2023101609A2
Application number: PCT/SG2022/050873
Authority: WO
Inventors: Mingfeng WANG; Hang Zhang; Bee Eng Mary Chan
Original assignee: Nanyang Technological University
Priority date: 2021-12-01
Filing date: 2022-12-01
Publication date: 2023-06-08
Also published as: WO2023101609A9; WO2023101609A3

Abstract

Disclosed herein is a copolymeric colourimetric sensor material formed from the crosslinking of: a random copolymer of formula Ia: x yzIar r with a random copolymer of formula Ib: x' y'z'Ibr r where R1, x, y, z, R2, x', y' and z' are as defined in the description.

Description

A Highly Sensitive Colourimetric Sensor Composed of Multifunctional Copolymers

Field of Invention

The current invention relates to a colourimetric sensor material that can be used to detect whether a foodstuff is in a state fit to eat. The invention also relates to the manufacture of the sensor material and its integration into food packaging.

Background

The listing or discussion of a prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

Issues associated with food quality and safety are increasingly becoming significant public concerns. Particularly, concerns on perishable foods, for example, meat, seafood, and milk, are more outstanding. This comes from the growth of pathogens and spoilage microorganisms which are easier to occur in these foods when exposed to elevated temperatures (5-60 °C), and therefore lead to food poisoning. Ammonia and biogenic amines are the main metabolites during food spoilage, which are generally produced by protein decomposition. Through precise monitoring of these compounds in food, one can accurately evaluate freshness and quality of perishable foods. Furthermore, based on this principle, many analytical methods such as spectroscopy, chromatography, and electrochemistry have been used to directly measure the amount of these compounds in food. However, these existing technologies require expensive and non-portable instruments as well as trained personnel to precisely determine the amount of food-spoilage associated compounds to assess the freshness and the quality of food products.

Colorimetric sensor is a promising solution for precise, portable, and low-cost evaluation of the food quality. Generally, this type of sensor is an optochemical sensing device based on the analyte-binding induced color change of chromophores. Such relatively simple principle could minimize the need for expensive instruments and tedious analytical procedures. However, the development of such colorimetric sensors is limited by factors such as analyte detection sensitivity and selectivity, safety of the chromophore itself, as well as manufacturing feasibility of the sensing devices. The most common seafood freshness indicator is based on pH-sensitive dyes which respond to Total Volatile Basic nitrogen (TVB-N) molecules, that are basic (or alkaline) in nature. The TVB-N molecules caused by degradation of protein and other nitrogen-containing compounds include TMA, DMA, ammonia and etc. For pH sensing labels, the commonly used dyes include anthocyanin, bromocresol purple, bromocresol green, bromophenol blue, methyl red, and cresol red, which are impregnated into various films such as polypropylene, nylon or cellulose. Many previous publications on different gas sensing systems are available but commercial freshness indicators available today are limited. Products such as SensorQ™, an anthocyaninbased pH sensing technology that measures volatile amines, did not take off. A common challenge in the commercial application of these dye-labels is the lack of sensitivity and stability of the pH indicator. The colorants generally do not have enough sensitivity in the small pH range around neutral pH, which is most relevant for food spoilage (though they have pronounced color change at very low or very high pH values). The dyes usually do not have more sensitivity than unaided human vision for slightly spoiled food. Further, leaching of the dye results in inaccurate responses or false positive indications of spoilage. The stable fixation of the colorant in suitable matrix to avoid leaching in actual use is another challenge. Also, spoilage does not necessarily produce basic pH as some spoilage caused by Lactobacillus spp., for example, does not cause basicity.

Multifunctional polymers, due to their multiple reactive sites, superior processor ability, and tunable mechanical properties, represent an interesting material choice for development of advanced colorimetric sensors. In particular, polymers with covalently bound chromophores have been developed for simple fabrication of sensor (ca. smart coating, surface modification). Recently, several dye-functionalized cellulose derivatives, conjugated polymers, and polymers functionalized with natural anthocyanins have been reported for sensing of food quality.

Despite recent advances, the majority of previously reported colorimetric sensors for monitoring food spoilage is based on the change of the optical density or fluorescence intensity of a monochromic channel. Such change in monochromic density is difficult for both qualitative assessment of food quality by naked eyes and quantitative measurement using instruments in which careful and reliable calibration is required. Colorimetric sensors with multicolor evolution provide opportunities to address this issue, in which the multicolor change could generate strong colorimetric contrast to precisely monitor the extent of food spoilage. Although some colorimetric sensors based on small molecular metal complexes, inorganic nanoparticles, and physically blended composites consisting of dyes and inorganic/polymeric matrices have been reported, they still face challenges such as potential leaching of the sensing components which could cause food contamination. Therefore, there exists a need for new colorimetric sensors for highly sensitive detection of food spoilage. Summary of Invention

Aspects and embodiments of the invention will now be discussed be reference to the following numbered clauses. 1. A copolymeric colourimetric sensor material formed from the crosslinking of: a random copolymer of formula la:

the point of attachment to the rest of the molecule; and x, y and z represent the repeating units of the copolymer, with a random copolymer of formula lb:

where:

R2 is selected from

, where the dotted line represents the point of attachment to the rest of the molecule; and x’, y’ and z’ represent the repeating units of the copolymer, wherein: the crosslinks between the random copolymer of formula la and the random copolymer of formula lb are formed via repeating units y and y’ as depicted below:

where the dotted lines represent the points of attachment to the rest of the molecule. 2. The copolymeric colourimetric sensor material according to Clause 1, wherein R2 is

3. The copolymeric colourimetric sensor material according to Clause 1 or Clause 2, wherein the molar ratio of the repeating units x:y:z in the random copolymer of formula la and the molar ratio of the repeating units x’:y’:z’ in the random copolymer of formula lb is from 150:1 :1 to 100:10:20, such as about 100:6:8.

4. The copolymeric colourimetric sensor material according to any one of the preceding clauses, wherein the molar ratio of the random copolymer of formula la to the random copolymer of formula lb is from 1:10 to 10:1 , such as from 2:5 to 5:2, such as 1 :1.

5. The copolymeric colourimetric sensor material according to any one of the preceding clauses, wherein the copolymeric colourimetric sensor material is in a sensitized state where the repeating unit z has the following structure:

6. The copolymeric colourimetric sensor material according to any one of the preceding clauses, wherein the copolymeric colourimetric sensor material is provided in a sensitized state where the copolymeric colourimetric sensor material in the sensitized state has a pink colour. 7. The copolymeric colourimetric sensor material according to any one of the preceding clauses, wherein the copolymeric colourimetric sensor material is suitable for the detection of rotting food, optionally wherein the copolymeric colourimetric sensor material is suitable for the detection of a rotting protein-rich food (e.g. a fish or a meat).

8. A food packaging material comprising: a substrate polymeric material having a surface, the substrate polymeric material comprising polymeric strands; and a copolymeric colourimetric sensor material in a sensitized state as described in Clause 5 or Clause 6 anchored to the surface of the substrate polymeric material.

9. The food packaging material according to Clause 8, wherein the crosslinking of the random copolymer of formula la to the random copolymer of formula lb results in entanglement between the copolymeric colourimetric sensor material and the polymeric strands of the substrate polymeric material, thereby anchoring the copolymeric colourimetric sensor material to the surface of the substrate polymeric material.

10. The food packaging material according to Clause 8 or Clause 9, wherein the substrate polymeric material is polyethylene terephthalate.

11. The food packaging material according to any one of Clauses 8 to 10, wherein the substrate polymeric material is provided in the form of a film.

12. A method of forming a copolymeric colourimetric sensor material as described in any one of Clauses 1 to 7, the method comprising the steps of:

(a) providing a mixture comprising a random copolymer of formula la, a random copolymer of formula lb, and, optionally, a solvent; and

(b) subjecting the mixture to UV light for a period of time to provide the copolymeric colourimetric sensor material.

13. The method according to Clause 12, wherein the molar ratio of the random copolymer of formula la to the random copolymer of formula lb is from 1 :10 to 10:1 , such as from 2:5 to 5:2, such as 1 :1.

14. A method of forming a food packaging material as described in any one of Clauses 8 to 11 , the method comprising the steps of: (i) providing a mixture comprising a random copolymer of formula la, a random copolymer of formula lb, and a solvent, where the random copolymer of formula la and the random copolymer of formula lb are as described in any one of Clauses 1 to 4;

(ii) applying the mixture to a surface of a substrate polymeric material comprising polymeric strands to provide a precursor food packaging material; and

(ii) subjecting the precursor food packaging material to UV light for a period of time to provide the food packaging material.

15. A method of detecting rotting food, the method comprising the steps of:

(aa) wrapping an article of food susceptible of rotting in a food packaging material as described in any one of Clauses 8 to 11 to provide a wrapped food article, where the copolymeric colourimetric sensor material has an initial pink colour;

(ab) monitoring the wrapped food article over a period of time and detecting rotting through one or more of the following: a reduction in an intensity of the initial pink colour; loss of the initial pink colour, such that the copolymeric colourimetric sensor material appears colourless; or the appearance of a yellow colour.

16. The method according to Clause 15, wherein the food susceptible of rotting is a protein-rich food, optionally wherein the food susceptible of rotting is a fish or a meat.

Drawings

FIG. 1 depicts the (a) ultraviolet (UV) reactor surrounded by 365 nm light-emitting diodes (LEDs), and (b) preparation of multifunctional polymer.

FIG. 2 depicts (a) molecular structure transformation of rhodamine and fluorescein after exposure to the different stimuli (including heat, base, acid, and UV), and dimerization of Coumarin derivatives after irradiation of UV light, (b) illustration on the preparation of the sensor material, and (c) rhodamine activation and cross-linking of the practical sensor before and after UV irradiation, in which the dye-polymer adducts (as the active sensing component) were patterned on polyethylene terephthalate (PET) substrates.

FIG. 3 depicts the synthesis of functional monomer and multifunctional polymers via reversible addition-fragmentation transfer (RAFT) polymerization. FIG. 4 depicts the gel permeating chromatography (GPC) curves of four types of multifunctional polymers including POCoRh, POCoFI, PHCoRh (acylated), and PHCoFI (acylated), using THF as eluent and polystyrene (PS) as standard.

FIG. 5 depicts (a) the comparative differential scanning calorimetry (DSC) heating thermograms (scan 2) for the dried multifunctional polymers PHCoRh and PHCoFI, and (b) ring opening/closing of rhodamine spirocycle/open form with UV and pH, respectively, resulting in pink and colorless forms.

FIG. 6 depicts (a, b) UV absorbance spectrum of POCoRh and PHCoRh before and after UV irradiation, followed by ammonia, and (c) microscopy photographs of POCoRh and PHCoRh coated on the glass substrate (2.2 x 2.5 cm²) at different stages (original, after UV irradiation, and then treated with ammonia), under daylight and UV light, and (d) steady-state fluorescence emission spectra of POCoRh and PHCoRh coating after UV irradiation (A_ex = 365 nm).

FIG. 7 depicts (a, b) UV absorbance spectra of POCoFI and PHCoFI before and after interaction with ammonia hydroxide (27 wt%), (c) colorless sensors separately prepared with POCoFI and PHCoFI, and (d) after interaction with ammonium hydroxide (27 wt%), the sensor’s color changed to the brilliant yellow.

FIG. 8 depicts (a) ultraviolet-visible (UV/vis) absorption spectra of blended PHCoRh/POCoFI before and after UV irradiation followed by ammonia exposure. Shown in the inset are the digital photographs of blended PHCoRh/POCoFI coated on the glass substrate, (b) UV absorbance spectrum of blended PHCoRh/POCoFI after treatment with ammonia at different concentrations, (c) relative absorbance (at A₅ei, A500 nm) of blended PHCoRh/POCoFI after exposure to ammonia at different concentrations, and (d) relative absorbance (at A561 nm) of blended PHCoRh/POCoFI after incubation with different volatile organic chemicals (VOCs, hexane, dichloromethane, ethyl acetate, methanol, chloroform, 3.5 pL in a 4.3 cm³ of cuvette) and alkaline organic amine (morpholine, triethylamine, trimethylamine, ammonia, 1000 ppm).

FIG. 9 depicts the UV absorbance spectra of (a) PHCoRh in THF and (b) blended POCoFI and PHCoRh coated on the glass substrate, after different irradiation times.

FIG. 10 depicts (left) the blended POCoFI/PHCoRh sensor patterned on the PET film before and after UV irradiation, and (right) both sensors were immersed in PBS buffer (pH = 9), in which the sensor without UV-crosslinking is above the line, and the sensor after UV- crosslinking is below the line.

FIG. 11 depicts the UV-activated POCoFI/PHCoRh sensor stored with ammonium hydroxide at different concentrations (10-10⁶ ppm). The pictures were collected at 0 minute, 30 minutes, and 2 hours.

FIG. 12 depicts the UV absorbance spectra of blended POCoFI and PHCoRh coated on the glass substrate before and after interaction with volatile organic chemicals: (a) hexane; (b) ethyl acetate; (c) methanol; and (d) dichloromethane.

FIG. 13 depicts the UV absorbance spectra of blended POCoFI and PHCoRh coated on the glass substrate before and after interaction with chloroform and organic amines: (a) chloroform; (b) morpholine; (c) triethylamine; and (d) trimethylamine.

FIG. 14 depicts that the UV-activated POCoFI/PHCoRh sensor pattern was stored in a desiccator at 23 °C without any special protection from the light, and the pictures at different times (original, 2, 4, 6, 10, 20, and 30 days) were collected.

FIG. 15 depicts (a) sensor (marked with a box in the upper right corner) was stored with the fish in a tray at 23 °C, and the reference sensor (marked with a box in the upper left corner) was fixed outside. Meanwhile, the change in color (red and yellow) was highlighted by the triangle with a gradient color, (b) sensor (marked with a box in the upper right corner) was stored with the fish in a tray at 3 °C, and (c) sensor (marked with a box in the upper left corner) was stored with the fish in a tray at -20 °C. For each storage condition, pictures are collected under the daylight (top row) and 365 nm UV light (bottom row).

FIG. 16 depicts that the sensor was stored with salmon in a closed environment at different temperatures: 23 °C; 3 °C; and -20 °C. For the different storage conditions, the samples in the top row was exposed to room light, while the samples in the bottom row were exposed to UV (365 nm) light.

FIG. 17 depicts that the sensor was stored with shrimp in a closed box at different temperatures: (a) 23 °C; and (b) 3 °C. For each storage condition, pictures are collected under the daylight (top row) and 365 nm UV light (bottom row).

Description It has surprisingly been found that a multi-functional polymer system comprising of hydrophilic, sensing and anchoring components can be used to provide a highly sensitive colourimetric sensor that can be used for the detection of food spoilage. Such multifunctional polymers can be directly patterned on the surface of commercial packaging films (e.g. food packaging films), such as PP or PET to provide a colourimetric sensor in situ. The sensors disclosed herein exhibit a prominent colorimetric response (from deep pink, to colourless and through to a brilliant yellow), are ultra-sensitive and selective. One application of the disclosed sensors is in monitoring the freshness of a foodstuff, such as seafood (e.g. shrimp, salmon, etc.). The related results further evidenced its promising practical application, in which the sensors can accurately indicate the quality of seafood in real-time in a sensitive way.

In a first aspect of the invention, there is provided a copolymeric colourimetric sensor material formed from the crosslinking of: a random copolymer of formula la:

where:

Ri is selected from

, where the dotted line represents the point of attachment to the rest of the molecule; and x, y and z represent the repeating units of the copolymer, with a random copolymer of formula lb:

where:

R2 is selected from

where the dotted lines represent the points of attachment to the rest of the molecule. In embodiments herein, the word “comprising” may be interpreted as requiring the features mentioned, but not limiting the presence of other features. Alternatively, the word “comprising” may also relate to the situation where only the components/features listed are intended to be present (e.g. the word “comprising” may be replaced by the phrases “consists of” or “consists essentially of”). It is explicitly contemplated that both the broader and narrower interpretations can be applied to all aspects and embodiments of the present invention. In other words, the word “comprising” and synonyms thereof may be replaced by the phrase “consisting of” or the phrase “consists essentially of’ or synonyms thereof and vice versa.

The phrase, “consists essentially of” and its pseudonyms may be interpreted herein to refer to a material where minor impurities may be present. For example, the material may be greater than or equal to 90% pure, such as greater than 95% pure, such as greater than 97% pure, such as greater than 99% pure, such as greater than 99.9% pure, such as greater than 99.99% pure, such as greater than 99.999% pure, such as 100% pure.

As will be appreciated, the copolymeric colourimetric sensor material is generated from two separate copolymeric materials that are crosslinked together via the coumarin groups. As noted above, this results in the following crosslinked structure:

In the event that the crosslinking is 100% efficient, so all coumarin groups are crosslinked, then the copolymeric colourimetric sensor material could be represented by the structure of formula Ic.

However, as will be appreciated, crosslinking is generally not 100% efficient, so there will remain some coumarin groups in a non-crosslinked state in the random copolymers of formula la and lb. The degree of crosslinking achieved in the copolymeric colourimetric sensor materials disclosed herein is not known precisely. However, any suitable degree of crosslinking may be used herein. For example, a suitable degree of crosslinking may be an amount sufficient to form a crosslinked network that is interlocked and non-leachable (e.g. when applied to a polymeric food packaging film). As will be appreciated, the random copolymers of formula la and lb are each constructed from three monomers. Broadly speaking, a first monomeric unit in each random copolymer provides a hydrophilic functionality, a second monomeric unit provides a sensing functionality and a third monomeric unit provides an anchoring functionality. This is depicted in FIG. 1 b. The hydrophilic functionality may be provided by oligo(ethylene glycol) methyl ether methacrylate (OEGMA) or 2-hydroxyethyl methacrylate (HEMA) in either the random copolymer of formula la or lb. This hydrophilic monomer may form the main backbone of the copolymers, with the other monomeric components being provided in relatively small amounts compared to the hydrophilic component.

The anchoring functionality refers to the coumarin repeating units within each of the random copolymers of formula la and lb. For example, the coumarin monomer may be 7-(2-methacryloyloxyethoxy)-4-methylcoumarin. In the context of the current invention, when the random copolymers of formula la and formula lb are subjected to UV light, the coumarin dimerizes to form a crosslinked network so that the polymer chains are interlocked and non- leachable. For example, if it is desired to form a food (spoilage) sensor on a polymeric film suitable for food packaging, then the random copolymers of formula la and lb may be applied to a surface of the polymeric film and subjected to UV light, resulting in the dimerization of some of the coumarin units to provide polymer chains that are interlocked and non-leachable. This also increases the adhesion of the resulting copolymeric colourimetric sensor material to the polymeric film (i.e. substrate) on which the copolymeric colourimetric sensor material is deposited.

The sensing functionality is formed of two components - one on the random copolymer of formula la and one on the random copolymer of formula lb.

In the random copolymer of formula la, the sensing monomer is one that contains a rhodamine spirocycle (e.g. a rhodamine methacrylate monomer). While rhodamine is depicted in the random copolymer of formula la in a spirocycle form, it is believed that the conditions used for crosslinking of the coumarin anchoring monomers (e.g. UV light treatment) may also result in the opening of the lactam ring to provide a stabilised cationic material that has a pink colour, as depicted below. It will be appreciated that if the crosslinking is achieved by means other than UV light, then the rhodamine may remain in a colourless ring-closed state. If this is the case, then UV light may then be applied in a separate step to ring-open the rhodamine to provide a material that is pink in colour.

This ring-opening may be reversed by changing the ambient pH conditions to which the rhodamine moiety is exposed to. For example, the pink colour of the ring-opened rhodamine species may be maintained when the pH of the ambient environment is less than about 7. However, if the pH is allowed to rise, then the pink colour obtained by the ring-opened rhodamine species will fade away as the ring-opened rhodamine species is converted back to the ring-closed species. This results in the loss of the pink colour and a return to a colourless state (this may occur close to neutral pH).

In the random copolymer of formula lb, the sensing monomer is one that contains a fluorescein (e.g. a fluorescein methacrylate monomer). The fluorescein may be provided in a colourless state and it may change to a yellow-coloured state upon raising pH levels.

It has been surprisingly found that the combination of the components provided herein provides an especially effective sensing system for use in food packaging. In the first instance, the rhodamine sensing moiety changes colour from pink to colourless over an ammonia concentration range of from 10 to 1000 ppm, and it remains colourless above 1000 ppm. Fluorescein changes colour from colourless to yellow at a higher ammonia concentration range (e.g. from 1000 to 10⁶ ppm). This allows for the early detection of the degradation of a foodstuff and it may also provide a way for an end user to determine if the foodstuff is in a state that is fit to eat depending on the colour of the sensor. For example, if the sensor has a pink colour, then the foodstuff may be in a state that is fit to eat, while if the sensor is colourless some caution may be advised and if the sensor is yellow, then the foodstuff may be in a state that is no longer safe to eat.

As will be appreciated, there are two possible random copolymers of formula la and two possible random copolymers of formula lb. These are summarized in short-hand below, where “P” refers to “poly”, “O” refers to OEGMA (i.e. OEG₅MA), “H” refers to HEMA, “Rh” refers to rhodamine, “Fl” refers to fluorencin, and “Co” refers to coumarin:

POCoRh;

POCoFI;

PHCoRh; and

PHCoFI.

These copolymers may be obtained by random copolymerization. For example, the copolymers may be obtained using RAFT copolymerization. The above copolymers can be combined to provide four different possible final products: POCoRh - POCoFI;

POCoRh - PHCoFI;

PHCoRh - POCoFI; and

PHCoRh - PHCoFI.

As noted hereinbefore, the hydrophilic functionality may be provided by oligo(ethylene glycol) methyl ether methacrylate (OEGMA) or 2-hydroxyethyl methacrylate (HEMA) in either the random copolymer of formula la or lb. In particular embodiments of the invention that may be

mentioned herein, R2 may be 5 and R1 may be . In other words, the final product may be PHCoRh - POCoFI.

As noted hereinbefore, the amount of the hydrophilic functionality may be significantly greater than that of the other functionalities present in the random copolymers of formula la and lb. In particular embodiments that may be mentioned herein, the molar ratio of the repeating units x:y:z in the random copolymer of formula la and the molar ratio of the repeating units x’:y’:z’ in the random copolymer of formula lb may be from 150:1 :1 to 100:10:20, such as about 100:6:8.

Any suitable amount of the random copolymer of formula la to the random copolymer of formula lb may be used herein, provided that a suitable degree of crosslinking between these copolymers can be obtained and that the resulting sensor can change colour from pink to yellow when faced with an appropriate change in the ambient pH environment. For example, the molar ratio of the random copolymer of formula la to the random copolymer of formula lb may be from 1 :10 to 10:1 , such as from 2:5 to 5:2, such as 1 :1.

As noted above, in embodiments of the invention that may be mentioned herein, the copolymeric colourimetric sensor material may be in a sensitized state where the repeating unit z has the following structure:

In embodiments of the invention that may be mentioned herein, the copolymeric colourimetric sensor material may be provided in a sensitized state where the copolymeric colourimetric sensor material in the sensitized state has a pink colour.

As noted hereinbefore, the copolymeric colourimetric sensor material may be suitable for the detection of rotting food. For example, the copolymeric colourimetric sensor material may be suitable for the detection of a rotting protein-rich food (e.g. a fish or a meat).

As will be appreciated, the copolymeric colourimetric sensor material may be particularly suited to use in combination with food packaging materials. As such, in a further aspect of the invention, there is provided a food packaging material comprising: a substrate polymeric material having a surface, the substrate polymeric material comprising polymeric strands; and a copolymeric colourimetric sensor material in a sensitized state as described hereinbefore, anchored to the surface of the substrate polymeric material.

As will be appreciated, this anchoring may be achieved by the crosslinking of the coumarin moieties on the random copolymers of formula la and lb disclosed herein. For example, the crosslinking of the random copolymer of formula la to the random copolymer of formula lb results in entanglement between the copolymeric colourimetric sensor material and the polymeric strands of the substrate polymeric material, thereby anchoring the copolymeric colourimetric sensor material to the surface of the substrate polymeric material. Any suitable substrate polymeric material may be used herein. For example, the substrate polymeric material may be polyethylene terephthalate.

The substrate polymeric material may be provided in any suitable form. For example, the substrate polymeric material may be provided in the form of a film.

In a further aspect of the invention, there is provided a method of forming a copolymeric colourimetric sensor material as described herein, the method comprising the steps of:

In embodiments of this aspect, the molar ratio of the random copolymer of formula la to the random copolymer of formula lb may be from 1:10 to 10:1, such as from 2:5 to 5:2, such as 1:1.

In a further aspect of the invention, there is also provided a method of forming a food packaging material as described herein, the method comprising the steps of:

(i) providing a mixture comprising a random copolymer of formula la, a random copolymer of formula lb, and a solvent, where the random copolymer of formula la and the random copolymer of formula lb are as described herein;

In yet a further aspect of the invention, there is provided a method of detecting rotting food, the method comprising the steps of:

(aa) wrapping an article of food susceptible of rotting in a food packaging material as described herein to provide a wrapped food article, where the copolymeric colourimetric sensor material has an initial pink colour;

In embodiments of this aspect, the food susceptible of rotting may be a protein-rich food. For example, the food susceptible of rotting may be a fish or a meat.

Further aspects and embodiments of the invention will now be described by reference to the following non-limiting examples.

Examples

Materials

All chemicals and reagents were purchased from Aldrich and used as-received, without further purification, unless specified. RAFT agent, 2-cyano-2-propyl benzodithioate (CPBD), was purchased from Aldrich and stored in the refrigerator. Radical initiator azobisisobutyronitrile (AIBN) was recrystallized from ethanol three times, and then stored in the refrigerator. Polyethylene glycol) methyl ether methacrylate (OEG5MA, M_n = 300 g/mol) was passed through a column of basic alumina to remove inhibitors before using. Hydroxyethyl methacrylate (HEMA) was purified through the distillation under the nitrogen protection to remove inhibitors and ethylene glycol dimethacrylate (EGDMA) and methacrylic acid. LED strips (365 nm, power 3W) were bought from Guangsheng led through NTU e-procurement platform. Silica gel 60 (0.04-0.063 mm) was used for column chromatography.

Nuclear magnetic resonance (NMR) spectroscopy

The monomers and polymers were dissolved in chloroform-d (or DMSO-ck) for ¹H NMR measurements on a Bruker AV300 MHz NMR spectrometer.

GPC

Molecular weights of polymers were measured using GPC (Agilent 1260). The eluent was tetrahydrofuran (THF) at a flow rate of 1.0 mL/min. A series of low polydispersity polystyrene standards were employed for the GPC calibration.

Fluorescence spectrophotometry

Emission spectra of solutions were measured by fluorescence spectrophotometry on a PerkinElmer LS-55 at 25 °C. UV/vis spectroscopy

UV/vis spectra of the polymers were measured on a Shimadzu UV-2450 spectrophotometer.

General set-up for the UV reactor

LED strips (365 nm, power 3W) were used as shown in FIG. 1a. The photoactivation of sensor and rhodamine containing polymer proceeded on the bottom of UV reactor.

Example 1. Molecular design and synthesis of multifunctional polymers

We report a series of multifunctional covalently bound polymer-dye adducts and their applications as colorimetric sensors for sensitive detection of seafood spoilage. The polymer- dye adducts consist of matrix blocks (poly(2-hydroxyethyl methacrylate)/poly(oligo(ethylene glycol) methyl ether methacrylate, PHEMA/POEG5MA), UV-crosslinking moiety (coumarin), and sensing motifs (rhodamine/fluorescein) (FIG. 2a).

The multifunctional polymers were directly synthesized through RAFT polymerization as a controllable radical polymerization method. RAFT polymerization has advantages such as robust regulation over multiple monomers engaged copolymerization, metal-free, and compatibility of diverse functionalities. Five derivatives of different methacrylates were selected to prepare the multifunctional polymers, including coumarinMA/rhodamineMA/fluoresceinMA/HEMA/OEGsMA (FIG. 3).

FIG. 2a shows the detailed molecular structure transformations of the functionalities including rhodamine and coumarin triggered by UV light irradiation. Fluorescein is well-known for its base-responsive property, where base-involved deprotonation can promote its conjugated molecular structure transformation and provide a respective change in optical property. Thus, the commercially available fluorescein-MA was utilized as the functional sensing modules to impart the synthesized polymers with base-responsive behavior. Rhodamine spirolactam has been well studied for its prominent responsive behavior against multiple stimuli (acid, UV light, stress, mechanical strength, and electricity) and rhodamine-MA was prepared and incorporated into the functional polymers. Rhodamine-MA was synthesized by two steps according to previous work (K.-K. Yu et al., Polym. Chem. 2014, 5, 5804): (1) the preparation of spiro-lactam intermediate (Rhodamine-OH); and (2) subsequent methacrylation through reaction with methacryloyl chloride (FIG. 3a). of Rhodamine-OH com et al., J. Am. Chem. Soc. 2009, 131

Rhodamine B (0.5 g, 1.04 mmol) was dissolved in ethanol (40 mL), and then 2-aminoethanol (252 pL, 4.16 mmol) was added under N2 protection. The mixture was stirred at 120 °C for 2 days. After evaporation of solvent under vacuum, the residue was dissolved in ethyl acetate (EA, 30 mL), washed with H2O and brine, and dried over anhydrous MgSC . The crude product was purified by column chromatography using EA/dichloromethane (DCM) (1/10, v/v) as the eluent, affording Rhodamine-OH as a pale-yellow solid (200 mg, yield: 40%).

¹H NMR (CDCh, 300 MHz): 7.89 (m, 1 H), 7.44 (m, 2H), 7.07 (m, 1 H), 6.48 (d, 2H), 6.37 (s, 2H), 6.27 (d, 2H), 4.19 (m, 1 H), 3.46 (q, 2H), 3.32 (m, 10H), 1.17 (t, 12H).

Synthesis of Rhodamine-MA compounds (K.-K. Yu et al., Polym. Chem. 2014, 5, 5804) Rhoamine-OH (200 mg, 0.41 mmol) and triethyl amine (172.4 pL, 1.24 mmol) were dissolved in anhydrous DCM (10 ml), and the mixture was cooled down in an ice bath. Metharyloyl chloride (47pL, 0.5 mmol) was dissolved in anhydrous DCM (1 ml), and the solution was added dropwise to the former mixture, under N₂ protection. The reaction was stirred overnight under room temperature. After removal of the solvent under vacuum, the residue was dissolved by DCM (20 mL) and washed with H₂O and brine, and dried over anhydrous MgSCU. The crude product was purified by column chromatography using EA/DCM (1/20, v/v) as the eluent, affording Rhodamine-MA as a white solid (196 mg, yield: 86%).

¹H NMR (CDCh, 300 MHz): 7.91 (m, 1 H), 7.42 (m, 2H), 7.07 (m, 1 H), 6.48 (d, 2H), 6.37 (d, 2H), 6.27 (d, 2H), 6.01 (d, 1 H), 5.48 (t, 1 H), 3.78 (t, 2H), 3.49 (t, 2H), 3.32 (m, 8H), 1.84 (s, 3H), 1.17 (t, 12H).

The remarkable mechanical stability of sensor is important for its practical application, especially considering the potential food contamination caused by unexpected mechanical breakage. Therefore, mechanically durable materials would be an ideal matrix material for the development of novel polymeric sensors. Based on the above consideration, we hypothesized that a multifunctional polymer capable of covalent cross-linking is highly suitable for sensor application. Cross-linking of polymeric matrices would be achieved by 365 nm UV light irradiation which triggers the dimerization reaction between coumarin units that are covalently appended from the polymeric backbones to form a crosslinked network so that the polymer chains are interlocked and non-leachable; this also increases dye adhesion to the plastic substrates on which the coating would be deposited. The target methacrylate containing coumarin (CouMA) was prepared according to previously reported methods (FIG. 3b) (C. P.

Kabb et al., ACS Appl. Mater, interfaces 2018, 10, 16793).

Synthesis of 7-(2-hydroxvethoxv)-4-methylcoumarin (coumarin-OH) (C. P. Kabb et al., ACS Appl. Mater, interfaces 2018, 10, 16793)

4-Methylumbelliferone (2.0 g, 11.4 mmol) and potassium carbonate (3.1 g, 22.7 mmol) were added into anhydrous DMF (20 mL) under N2 atmosphere. 2-Bromoethanol (1.2 mL, 17.0 mmol) was added, and then the mixture was stirred at 90 °C for 18 h. The reaction mixture was cooled to room temperature and poured into ice-cold water to precipitate the product. The white product was collected by filtration and dried in vacuum. Yield: 2.3 g (92.0%).

¹H NMR (300 MHz, DMSO-d₆): 5 (ppm) 7.68-7.64 (d, 1 H), 6.98-6.94 (m, 2H), 6.20 (s, 1 H), 4.95 (s, 1 H), 4.09 (t, 2H), 3.75 (t, 2H), 2.37 (s, 3H).

Synthesis of 7-(2-methacryloyloxyethoxy)-4-methylcoumarin (CouMA) (C. P. Kabb et al., ACS Appl. Mater, interfaces 2018, 10, 16793)

Briefly, 7-(2-hydroxyethoxy)-4-methylcoumarin (2.0 g, 9.1 mmol) and triethylamine (2.6 ml, 18.2 mmol) were added into anhydrous chloroform (40 mL) under N₂ atmosphere. The mixture was stirred at O °C for 15 minutes. Then, methacryloyl chloride (1.8 mL, 18.2 mmol) was added slowly via the syringe at 0 °C. The solution was reacted for 12 h at room temperature. Dichloromethane (60 mL) was added, then the solution was washed with the brine (80 x 2 mL), dried over sodium sulfate, filtered, and concentrated in vacuum to obtain the crude product. The product was further recrystallized from ethanol to afford white powdery solid. Yield: 1.8g (69%).

¹H NMR (300 MHz, CDCI3): 6 (ppm) 7.50 (d, 1 H), 6.91-6.82 (m, 2H), 6.15 (m, 2H), 5.61 (m, 1 H), 4.52 (t, 2H), 4.28 (t, 2H), 2.40 (s, 3H), 1.96 (s, 3H).

Meanwhile, direct fabrication of food sensors on surface of packaging materials is highly desirable, which will significantly simplify food monitoring and enable real-time detection of food freshness. Surface coating provides a relatively convenient and effective way to achieve this, but it remains challenging to balance the mechanical stability of the coating sensor and its adhesion property to the surface of packaging materials. To address this issue, we selected two different hydrophilic and polar polymers, POEG5MA and PH EMA, with different glasstransition temperatures (T_g) and adhesion ability as the main matrix for the resulting polymeric sensor. As such, herein, we developed four types of multifunctional polymers composed of hydrophilic blocks, anchoring segments, and biogenic amine-responsive sensing modules by combining 2 synergistic dyes which are rhodamine and fluorescein.

RAFT agent (CPBD) was employed to regulate the RAFT copolymerization, as shown in FIG. 3c. Four sets of multifunctional polymers were prepared in such a way, including OEGsMA- CouMA-RhoMA (POCoRh), OEG₅MA-CouMA-FluMA (POCoFI), HEMA-CouMA-RhoMA (PHCoRh), and HEMA-CouMA-FluMA (PHCoFI), respectively. The related polymer synthesis is depicted in FIG. 1b.

Synthesis of multifunctional polymers including POCoRh, POCoFI, PHCoRh, and PHCoFI Four polymers were prepared by the same method. The following is for the synthesis of POCoRh.

RAFT agent CPDB (4.42 mg, 20 pmol, 1eq.), OEG5MA (600 mg, 2 mmol, 100 eq.), CouMA (35 mg, 120 pmol, 6 eq.), Rhodamine-MA (89 mg, 160 pmol, 8 eq.) and AIBN (0.033 mg, 2 pmol, 0.1 eq.) were dissolved in 1.5 ml of 1 ,4-dioxane in a Schlenk tube. After three freeze- and-thaw cycles for removal of oxygen, the tube was then placed in a preheated oil bath at 70 °C and reacted for 24 h. Then, the solution was poured into excess cold diethyl ether, precipitating the polymer product, which was collected by centrifugation and reprecipitated 3 times using THF and diethyl ether. The resultant polymer was dried in vacuum at room temperature for 24 hours and obtained as a sticky solid (650 mg, yield 79%).

For POCoFI, PHCoRh, and PHCoFI, the corresponding yields were 75%, 86%, and 93%, respectively. Notably, DMF was used as solvent for the synthesis of PHCoRh and PHCoFI.

Protection of PHEMA containing multifunctional polymers, including PHCoRh and PHCoFI Attempts to directly determine the M_n and dispersity (£>) of PHCoRh and PHCoFI using the GPC running by THF failed due to the strong interaction between the polymer and the column. Hence, the hydroxyl groups of the two polymers were further protected to reduce their polarity.

The two polymers were protected by the same method. Taking the protection of PHCoRh as an example, 18 mg of polymer was dissolved in pyridine (0.5 mL), followed by the addition of acetic anhydride (0.1 mL). The mixture was reacted for 3 hours at room temperature, and subsequently precipitated in cold diethyl ether, in which precipitation was repeated twice using THF and diethyl ether. After drying in vacuum at room temperature for 12 hours, the polymer was dissolved in THF (HPLC grade) for GPC analysis. The characterization data for POCoRh, POCoFI, PHCoRh, and PHCoFI are provided in Example 2.

Example 2. Characterization of multifunctional polymers

The multifunctional polymers prepared in Example 1 were characterized.

Differential scanning calorimetry (DSC) analysis

Glass transition (T_g) temperatures were measured by DSC on an PerkinElmer DSC 4000. All T_g values were obtained from the second scan. Both heating rate and cooling rate were 10 °C/min unless indicated otherwise.

Results and discussion

Successful preparation of designed polymers was confirmed by ¹H NMR spectroscopy and GPC (Table 1). The characteristic proton signal of rhodamine (at 7.89 ppm), coumarin (at 6.15 ppm), and OEG (at 3.63 ppm) can be observed and assigned in the representative ¹H NMR spectrum of POCoRh. Through the integral ratios of the proton peaks in the ¹H NMR spectrum, the molar fractions of the different components in the polymer product were easily calculated, which are consistent with the feed ratio of the monomers. Similarly, through ¹H NMR spectra of the other three polymers, the corresponding characteristic signal of each functional block can also be assigned.

All GPC curves of the polymers showed a unimodal distribution and relatively narrow dispersity (1.2-1.5) (FIG. 4). These results suggest that the three different monomers engaged in the RAFT copolymerization in a relatively controllable manner. Notably, two polymers (PHCoRh and PHCoFI) only showed a single glass-transition temperature (T_g = 107.3 °C for PHCoRh and 117.3 °C for PHCoFI) through DSC analysis, which further indicates the successful synthesis of the statistical random copolymers (FIG. 5a).

Table 1. Characterization of multifunctional polymers.³

P(OEGsMAi6o-co-

POCoRh [105/6/8J/1/0.1 20.0 1.29 <-30

CoUyCO-RhOs)

P(OEGsMAi22-co-

POCoFI [105/6/8J/1/0.1 18.4 1.57 <-30

COUT-CO-FIUS)

P(HEMAioo-co-Coii7-

PHCoRh [100/6/8J/1/0.2 14.7 1.20 107.3 co- Rh Os)

P(HEMAioo-co-Coii6-

PHCoFI [100/6/8J/1/0.2 15.0 1.28 117.3 co- Flu?) ^aFour types of multifunctional polymers were prepared by the RAFT copolymerization, using CPDB as RAFT agent and Al BN as radical initiator. ^ftThe degree of polymerization (DP) of the repeating units was calculated by the corresponding ¹H NMR spectrum. ^cGPC data were determined in THF, using PS standard. ^dDSC analysis.

Example 3. Optical properties of the four multifunctional polymers before and after different treatments

The optical properties of the four multifunctional polymers prepared in Example 1 were studied.

Study of the optical properties of the four multifunctional polymers before and after different treatments

PHCoRh (20 mg) and PHCoFI (20 mg) were separately dissolved in the mixed solvent (ethanol/water/1 ,4-butanediol, 200/4/2 pL). POCoRh (20 mg) and POCoFI (20 mg) were separately dissolved in ethanol (200 pL). Then, each solution (10 pL) was uniformly coated on the corresponding same size glass substrate in an area of 0.9 x 0.9 cm². The samples separately coated with PHCoRh or POCoRh were placed in the UV reactor for 1 hour to finish the photoactivation.

For the testing of biogenic amine, the four samples were separately stored with 5 pL of ammonia hydroxide (27 wt%) in a cuvette (4.3 cm³) for 5 minutes. The UV absorption spectra of every sample before and after reaction were recorded. Results and discussion

Rhodamine spirolactams are an intriguing series of organic dyes due to their multiple responsive behaviors. The underlying mechanism is that ring-closed spirolactam of rhodamine could undertake a molecular isomerization from a twisted form to a planar zwitterionic structure in the ring-opening state (FIG. 5b), which significantly red-shifts the absorption band and turns on the fluorescence emission. As the open-form is red, it is easily visible to the naked eye at neutral pH. Importantly, this transformation is reversible and could return to the pristine state upon exposure to stimuli such as a base or thermal treatment. In the presence of a base such as ammonia, the open-form is closed again to form the closed-form which is colorless (FIG. 5b).

Here, we propose to use UV light-irradiated rhodamine as an indicator to monitor the presence of biogenic amines. Specifically, two polymer-dye adducts, POCoRh and PHCoRh, were separately treated with UV light at 365 nm to activate rhodamine spirolactam, leading to its color change from colorless to deep pink (A_abs = 561 nm), as shown in FIGS. 6a-c, so as to be easily visible to the naked eye at neutral pH. As we expected, both UV-activated POCoRh/PHCoRh exhibited a color change from pink red to colorless upon exposure to ammonia solution. As such, the polymers need to be UV-irradiated to change color from colorless to pink, which are then sensitive to a base. Therefore, the Rh-containing polymers need to be UV-irradiated to change from colorless to pink before being used as a sensor.

Surprisingly, we found that PHCoRh was much more sensitive to UV light than POCoRh, evidenced by the deeper color change (FIG. 6c), broader absorption band of activated PHCoRh (FIGS. 6a-b) and a slight red-shift (~10 nm) of the fluorescence emission peak (FIG. 6d). Such interesting phenomenon could be attributed to the fact that hydroxyl groups of PHEMA could stabilize the activated rhodamine’s zwitterion through hydrogen bonding. Hence, PHCoRh seems to be a more suitable candidate for the sensor design based on the more visible color change. However, this response dominated by the photo-activated rhodamine only showed a monochromic change from the deep pink to colorless, which may be insufficient for the development of food sensor with superior performance.

To identify a polymer with complementary color response and contrast to the rhodaminecontaining polymer as described above, we synthesized two fluorescein-containing copolymers and characterized their optical response to exposure of ammonia as a base. The synthesized polymers, denoted as POCoFI and PHCoFI, showed a color change from colorless to brilliant yellow after being exposed to the ammonia. Such a color change could be attributed to the emergence of a remarkable absorption band at 500 nm as determined through UV-vis absorption spectroscopy (FIG. 7). Nevertheless, such color change from the colorless to pale yellow is still not easy to be detected by naked eyes, particularly at a relatively low concentration of amines.

Example 4. Optical property of blended POCoRh/ PHCoFI sensor

To enhance the color contrast of the polymer-dye films in response to food spoilage, we adopted a bicomponent strategy to construct the food sensor with multicolor responsive behavior through the UV irradiation protocol. The constructing route of sensor using two different polymers and simultaneous transformation of relevant molecular structure are depicted in FIG. 2b.

Study of the optical property of blended POCoRh/ PHCoFI sensor

100 mg of POCoFI and 20 mg of PHCoRh were blended and dissolved in 1 ml of ethanol, and the mixture was stirred for 5 hours at room temperature to obtain a homogeneous solution. The aforementioned blended sensor solution (10 pL) was uniformly coated on the substrate of glass in an area of 0.9 x 0.9 cm². Then, the sample was placed in the UV reactor and irradiated for 1 hour. The activated sensor was directly stored with 5 pL of ammonium hydroxide (27 wt%) in a cuvette (4.3 cm³) for 5 minutes to verify the related sensing behavior. The UV absorbance spectra of the sample at every step before and after interaction were recorded.

Swelling test

The patterned sensor before and after UV treatment were immersed into a PBS buffer (pH = 9), respectively. The apparent state of each sensor array was recorded by the camera over the different storage times (20 min, 24 h and 48 h).

Results and discussion

FIG. 8a shows the color evolution of the sensor film, when activated by UV light and later responded to ammonia, from colorless to deep pink to yellow. Thus, the resultant sensor materials did exhibit a broader color transition from deep pink to colorless to yellow after interaction with ammonia, which indicate the rational design of sensor (FIG. 8a). Specifically, a characteristic absorption peak at 561 nm appeared in the UV/vis absorption spectrum after exposure to UV light. Such spectral change, as reflected from the color transition from colorless to deep pink, indicates the successful activation of the “dormant” rhodamine by the UV light irradiation. When the UV-activated sensor film was subsequently exposed to ammonia and the basicity increased, the absorption peak at 561 nm disappeared and a new peak at 500 nm emerged, corresponding to a color change from deep pink to dark yellow. Such spectral and color change could be attributed to the deactivation of the activated rhodamine and deprotonation of the fluorescein upon exposure to ammonia as a volatile base. Notably, activated rhodamine displayed stronger sensitivity to ammonia than fluorescein, which can be seen from the gradient color transition and sensitivity testing (FIG. 8b).

Therefore, via a strategy of activating “dormant states” by UV light irradiation, activated rhodamine derivative could be obtained, which could further act as a detector of biogenic amines. Further, its combination with fluorescein imparted the polymeric sensor a multicolor evolution when interacting with analytes. Moreover, such a UV treatment can be integrated into the fabrication of sensor, analogous to a UV curing of polymer-dye films.

To maximize the activation of rhodamine in the polymeric sensor, the irradiation time of UV light was optimized and finally fixed as 1 hour. It was determined through the characteristic absorption of activated rhodamine at 561 nm in UV/vis absorption spectrum, in which the absorption tends to be constant after 1 hour of irradiation (FIG. 9). Nevertheless, it remains difficult to rely on spectroscopy techniques including UV/vis, Fourier-transform infrared (FT- IR) and Raman to quantify the degree of associated crosslinking in this mixture after the UV treatment, because of the overlap of the coumarin signal with other chromophores with similar aromatic structure. As a result, the stability of the photo-crosslinked sensor was tested through a swelling test, in which the patterned sensor before and after UV treatment were immersed into a phosphate-buffered saline (PBS) buffer (pH = 9), respectively. It was found that UV- treated sensor was just swollen, whereas the untreated sensor quickly dissolved into the buffer, indicating the formation of desirable covalent crosslinking in the former (FIG. 10). However, after two days storage of cross-linked sensor in PBS buffer, peeling off of the sensor from the substrate occurred, indicating insufficient surface adhesion between the sensor and the polymeric substrate without pretreatment.

Example 5. Sensitivity and selectivity of blended POCoRh/ PHCoFI sensor

Sensitivity and selectivity are two important criteria in the evaluation of a new type of sensor for food spoilage.

Sensitivity and selectivity of blended POCoRh/ PHCoFI sensor

UV-irradiated POCoRh/ PHCoFI blended sensor (prepared in Example 4) was treated with different concentration of ammonia from 10-10⁶ ppm to verify its sensitivity. The interacted concentration of ammonia was calculated based on ideal gas law, through the addition of different concentrations of ammonium hydroxide into the air-tight cuvette (4.3 cm³). After the addition of ammonium hydroxide, the closed system needed 2 hours to make a sufficient interaction between ammonia and sensor. Then, the corresponding cuvette was directly used to test the UV absorbance to record the sensor’s response to ammonia. The ideal gas law is provided below.

PV= nRT where P is the pressure (1 atm), V is the volume of cuvette (4.3*1 O'³ L), n is the amount of ammonia (mol), R is the ideal gas constant (0.08206 L atm (mol K)^-1), and T is the absolute temperature of testing (297.15 K).

For the characterization of the selectivity of the POCoRh/PHCoFI sensor, the sensor was interacted separately with different volatile organic solvent (3.5 pL) including hexane, dichloromethane, chloroform, ethyl acetate, and methanol for 1 hour in an air-tight cuvette (4.3 cm³). Then, the optical responses were determined via the absorbance change at 561 nm before and after interaction with the different analytes. Alkaline chemicals including morpholine, trimethylamine, and triethylamine were separately stored with the sensor in an air-tight cuvette for 2 hours. Their concentrations were also calculated by the ideal gas law. The related UV absorbance spectra recorded the responsive change.

Results and discussion

Using ammonia solution as the model analyte, we first examined the sensitivity of the sensor prepared in Example 4. FIGS. 8b, c show the sensitivity upon exposure to ammonia at different concentrations. When the concentration is below 1000 ppm (10 to 1000 ppm), the activated sensor mainly exhibited a color change from deep pink to colorless, thus demonstrating great sensitivity from 10 to 1000 ppm which is near neutral pH. The UV-irradiated Rh dye changes color from pink to colorless over the ammonia concentration range 10 to 1000 ppm, which is near neutral pH; it remains colorless above 1000 ppm.

When the concentration of ammonia is in the range of 1000 to 10⁶ ppm, a color change from colorless to brilliant yellow was observed on the polymeric film as the second dye, fluorescein, changes color from colorless to yellow at a higher range (1000 to 10⁶ ppm) of ammonia concentration (FIG. 8). The sensitivity against ammonia could reach as low as 10 ppm, as determined, ca. 6% of decrease of the maximum absorbance at 561 nm (FIG. 8b). FIG. 11 shows the color change of the sensor at different concentrations of ammonia atmosphere, in which an obvious evolution of pink to colorless to yellow is presented. The ultra-high sensitivity of our system in the near neutral pH regime, which is very unique, is derived from the UV- irradiated form of Rh and the high range, 5 orders of magnitude, is achieved through the use of two complementary dyes.

To confirm the selectivity our polymer-dye adduct sensor, it was exposed to different vapors of VOCs (including dichloromethane, hexane, ethyl acetate, methanol, and chloroform) and alkaline organic amines (including triethylamine, trimethylamine, ammonia, and morphine). FIGS. 12-13 show the optical responses as determined via the absorbance change at 561 nm before and after interaction with the different analytes. Specifically, the absorbance at 561 nm of the sensor was dramatically decreased upon exposure to the aforementioned alkaline organic amines (at 1000 ppm of concentration) including triethylamine, trimethylamine, ammonia, and morphine, while little change in the optical absorbance was observed in the sensor films exposed to neutral VOCs (3.5 L in a 4.3 cm³ of cuvette) such as dichloromethane, hexane, ethyl acetate, methanol, and chloroform, even at a higher concentration. Trends in these selective responses and the accompanied changes in optical absorbance are further depicted in FIG. 8d.

Example 6. Optical stability of blended POCoRh/ PHCoFI sensor

The optical stability of the blended POCoRh/ PHCoFI sensor prepared in Example 4 was investigated.

Optical stability

The optical stability of UV-activated sensor array (prepared in Example 4) during long-term storage is evaluated in a top-closed desiccator under room light irradiation. Sensor’s color was recorded qualitatively by camera under room light over different periods of storage (2, 4, 6, 10, 20 and 30 days).

Results and discussion

FIG. 14 shows negligible change of the color after up to 30 days of preservation, indicating UV-activated rhodamine can be trapped safely in this cross-linked polymer system.

As demonstrated in the experiments in Examples 4-6, the activated sensors exhibited a great potential for monitoring biogenic amine in terms of selectivity and sensitivity. In addition, its simple fabrication procedure and excellent stability further make these blended functional polymer systems suitable for applications in detection of food spoilage.

Example 7. Fabrication of colorimetric polymeric sensors Importantly, the preparation of blended sensing materials via the UV irradiation can be directly applied to the formation of surface pattern sensor on commercial packaging film such as PET or polypropylene (PP) (PET film was used here as the substrate to fabricate pattern sensor) because the dye-activation and sensor-formation could be simply integrated in one step.

Specifically, two polymer-dye adducts, PHCoRh and POCoFI, were co-dissolved in a common good solvent (ethanol) and patterned by drop-casting on the commercial polymer substrates such as PET films, followed by 1 hour of UV light (365 nm) irradiation, according to the absorption (A_abs= 561 nm) of blended PHCoRh/POCoFI as a function of UV irradiation time. The UV light plays two roles during this process: (1) photo-activation of rhodamine; and (2) crosslinking of coumarin. A sensor with activated rhodamine and stable cross-linked network structure can be fabricated (FIGS. 2b, c). Importantly, the rational combination of segments of PHEMA and POEG5MA resulting from the above multifunctional polymers not only enables the sensor to have a good mechanical property, but it also provides a reliable interface interaction with the substrate. Thus, based on the above principle, a pattern sensor was directly fabricated on the surface of a commercial package film (PET) (FIGS. 2b, c).

Sensors fabrication through blended POCoRh and PHCoFI

100 mg of POCoFI and 20 mg of PHCoRh were blended and dissolved in 1 mL of ethanol. The reaction mixture was stirred for 5 hours at room temperature to obtain a red-color homogeneous solution. Then, through the drop casting method, each 3 pL of the aforementioned solution was continuously transferred onto the surface of a PET film to construct the sensor array (3 pL for every independent module), in which a plastic template could be used if the desired pattern is in a square-shape. The coated PET film was carefully placed in a homemade UV reactor (FIG. 1a) and exposed to UV light (365 nm) for 1 hour to maximize the activation of rhodamine and desirable cross-linking.

Thus, using the commercially available PET film as the substrate without any special surface treatment, the solution of PHCoRh/POCoFI mixed in ethanol was directly dropcast on PET. Through UV light irradiation for 1 hour, an array of cured surface pattern sensor with deep pink color was successfully prepared.

Example 8. Sensing fish and shrimp using the activated surface pattern sensor

The pattern sensor prepared in Example 7 was taken to sense the freshness of fish and shrimp. Test of food monitoring

The fresh fish (kambong and salmon) and shrimp were bought from a local supermarket. The pattern sensors fabricated on the PET film in Example 7 were placed with different foods in a closed package but without physical contact and stored at different temperatures (23 °C, 3 °C and -20 °C). Specifically, kambong (around 0.6 kg) was placed in a tray with the sensor, and the whole system was further wrapped by plastic wrap to ensure a closed storage environment. For the sample stored at 23 °C, a control sensor was taped on the outside surface of the tray to verify that the sensor changes are caused only by food spoilage. For the sensing of salmon and shrimp, the target foods were placed in a closed box or tray with the sensor. During the storage, the related color and fluorescence evolution of sensors along time were acquired by camera under room light and UV light (from a hand-held UV lamp, 365 nm, 6 W), respectively.

Results and discussion

Given the influence of temperature on the spoilage of food, three different storage temperatures (23 °C, 3 °C and -20 °C) were selected to examine the relationship between the color evolution of the sensor and the food freshness. As shown in FIG. 15, with the spoilage of kambong stored at 23 °C, deep pink sensor gradually faded to colorless (0-9 hours) and later turned to pale yellow (9-24 hours). Notably, the reference sensor fixed outside of the food tray did not exhibit any color fading, demonstrating that the color transition of the inner sensor was solely triggered by the presence of biogenic amines from the fish spoilage. By contrast, lower temperature storage of food can greatly extend itself freshness, especially for meat, which was also indicated by the accompanied sensor. For the food preserved at 3 °C, the sensor experienced a slow color change from original pink to colorless and finally changed to pale yellow after 6 days. Such a visible color change did not occur in the sensor film stored at -20 °C and the sensor array remained deep pink during storage for as long as 30 days.

Our sensor array exhibited a very similar tendency in the monitoring of salmon freshness (FIG. 16). It took 8 hours for the color change from deep pink to almost colorless and later to brilliant yellow (8 hours-2 days) at 23 °C, and 8 days for the same color change when the salmon was stored at 3 °C. However, negligible color change was observed after 15-day storage at -20 °C.

Interestingly, this sensor exhibited a superior sensitivity to shrimp spoilage, as shown in FIG. 17, in which the color of the sensor quickly faded within 3 hours and then evolved into a brilliant yellow during 3-24 hours storage at 23 °C. Even at a lower temperature of 3 °C, the sensor also experienced a quicker color change during exposure to shrimp compared to fish under the same condition. After only 24 hours, the sensor’s pink color disappeared at 3 °C. Such a different response of the sensor to the spoilage of shrimp should be attributed to the different protein composition in the organism and the microbial involved in the decomposition of these proteins.

The experimental results described in the above examples demonstrate that our colorimetric and fluorometric sensor is highly sensitive to the freshness of fish and shrimp stored at different temperatures, and exhibited a superior performance in monitoring the spoilage of seafood including fish and shrimp. In addition, the presented sensor exhibited different sensing behaviors to the same food stored at different temperatures and to different food stored at the same storage condition, which further indicates its promising practical application for monitoring spoilage of seafood products.

Therefore, we have presented a type of colorimetric sensor based on multifunctional polymers through the polymerization of monomers with different functionalities containing biogenic amine-responsive rhodamine and fluorescein, UV-crosslinking coumarin, and hydrophilic PEG-oligomer, and hydroxyl moieties. Taking advantage of the UV-responsive features of coumarin and rhodamine spirolactam, the isomerized zwitterionic rhodamine was combined with fluorescein and further used as sensing components to analytes such as biogenic amines, while UV-induced dimerization of coumarin enabled facile deposition and curing of the sensing polymers on solid substrates. To tune the physical properties of surface adhesion and mechanical stability of the sensor films, two different polymers including POCoFI/ POCoRh were further blended to coat the polymeric sensor on the surface of commercialized packaging film such as PET. Such blended POCoFI/ POCoRh sensor exhibited high sensitivity and good specificity against biogenic amines, and are promising for facile and real-time monitoring of food spoilage such as fish, shrimp and other seafood products. The combination of different functionalities with processability of polymer materials provides a promising solution for the development of high performance food sensors.

Claims

1. A copolymeric colourimetric sensor material formed from the crosslinking of: a random copolymer of formula la:

where:

R2 is selected from

where the dotted lines represent the points of attachment to the rest of the molecule.

2. The copolymeric colourimetric sensor material according to Claim 1 , wherein R2 is

3. The copolymeric colourimetric sensor material according to Claim 1 or Claim 2, wherein the molar ratio of the repeating units x:y:z in the random copolymer of formula la and the molar ratio of the repeating units x’:y’:z’ in the random copolymer of formula lb is from 150:1 :1 to 100:10:20, such as about 100:6:8.

4. The copolymeric colourimetric sensor material according to any one of the preceding claims, wherein the molar ratio of the random copolymer of formula la to the random copolymer of formula lb is from 1 :10 to 10:1 , such as from 2:5 to 5:2, such as 1 :1.

5. The copolymeric colourimetric sensor material according to any one of the preceding claims, wherein the copolymeric colourimetric sensor material is in a sensitized state where the repeating unit z has the following structure:

6. The copolymeric colourimetric sensor material according to any one of the preceding claims, wherein the copolymeric colourimetric sensor material is provided in a sensitized state where the copolymeric colourimetric sensor material in the sensitized state has a pink colour.

7. The copolymeric colourimetric sensor material according to any one of the preceding claims, wherein the copolymeric colourimetric sensor material is suitable for the detection of rotting food, optionally wherein the copolymeric colourimetric sensor material is suitable for the detection of a rotting protein-rich food (e.g. a fish or a meat).

8. A food packaging material comprising: a substrate polymeric material having a surface, the substrate polymeric material comprising polymeric strands; and a copolymeric colourimetric sensor material in a sensitized state as described in Claim 5 or Claim 6 anchored to the surface of the substrate polymeric material.

9. The food packaging material according to Claim 8, wherein the crosslinking of the random copolymer of formula la to the random copolymer of formula lb results in entanglement between the copolymeric colourimetric sensor material and the polymeric strands of the substrate polymeric material, thereby anchoring the copolymeric colourimetric sensor material to the surface of the substrate polymeric material.

10. The food packaging material according to Claim 8 or Claim 9, wherein the substrate polymeric material is polyethylene terephthalate.

11. The food packaging material according to any one of Claims 8 to 10, wherein the substrate polymeric material is provided in the form of a film.

12. A method of forming a copolymeric colourimetric sensor material as described in any one of Claims 1 to 7, the method comprising the steps of:

13. The method according to Claim 12, wherein the molar ratio of the random copolymer of formula la to the random copolymer of formula lb is from 1 :10 to 10:1 , such as from 2:5 to 5:2, such as 1 :1.

14. A method of forming a food packaging material as described in any one of Claims 8 to

11 , the method comprising the steps of: (i) providing a mixture comprising a random copolymer of formula la, a random copolymer of formula lb, and a solvent, where the random copolymer of formula la and the random copolymer of formula lb are as described in any one of Claims 1 to 4;

15. A method of detecting rotting food, the method comprising the steps of:

(aa) wrapping an article of food susceptible of rotting in a food packaging material as described in any one of Claims 8 to 11 to provide a wrapped food article, where the copolymeric colourimetric sensor material has an initial pink colour;

16. The method according to Claim 15, wherein the food susceptible of rotting is a proteinrich food, optionally wherein the food susceptible of rotting is a fish or a meat.