WO2023039625A1 - Capteurs colorimétriques permettant la détection de contaminants chimiques et biologiques - Google Patents

Capteurs colorimétriques permettant la détection de contaminants chimiques et biologiques Download PDF

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WO2023039625A1
WO2023039625A1 PCT/AU2021/051069 AU2021051069W WO2023039625A1 WO 2023039625 A1 WO2023039625 A1 WO 2023039625A1 AU 2021051069 W AU2021051069 W AU 2021051069W WO 2023039625 A1 WO2023039625 A1 WO 2023039625A1
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colorimetric sensor
cooh
phospholipid
target analyte
formulation
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PCT/AU2021/051069
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English (en)
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Rona CHANDRAWATI
Max Weston
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Newsouth Innovations Pty Limited
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Priority to AU2021464941A priority Critical patent/AU2021464941A1/en
Priority to PCT/AU2021/051069 priority patent/WO2023039625A1/fr
Publication of WO2023039625A1 publication Critical patent/WO2023039625A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09D—COATING COMPOSITIONS, e.g. PAINTS, VARNISHES OR LACQUERS; FILLING PASTES; CHEMICAL PAINT OR INK REMOVERS; INKS; CORRECTING FLUIDS; WOODSTAINS; PASTES OR SOLIDS FOR COLOURING OR PRINTING; USE OF MATERIALS THEREFOR
    • C09D4/00—Coating compositions, e.g. paints, varnishes or lacquers, based on organic non-macromolecular compounds having at least one polymerisable carbon-to-carbon unsaturated bond ; Coating compositions, based on monomers of macromolecular compounds of groups C09D183/00 - C09D183/16
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F138/00—Homopolymers of compounds having one or more carbon-to-carbon triple bonds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/5432—Liposomes or microcapsules
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08K—Use of inorganic or non-macromolecular organic substances as compounding ingredients
    • C08K5/00—Use of organic ingredients
    • C08K5/49—Phosphorus-containing compounds
    • C08K5/51—Phosphorus bound to oxygen
    • C08K5/52—Phosphorus bound to oxygen only
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L49/00—Compositions of homopolymers or copolymers of compounds having one or more carbon-to-carbon triple bonds; Compositions of derivatives of such polymers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/02—Food
    • G01N33/025—Fruits or vegetables
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/02—Food
    • G01N33/04—Dairy products
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/02—Food
    • G01N33/12—Meat; Fish
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/02—Food
    • G01N33/14—Beverages

Definitions

  • the present disclosure generally relates to a colorimetric sensor formulation for the detection of chemical and biological contaminants both in liquid phase and vapour phase.
  • the present disclosure generally relates to a colorimetric sensor formulation comprising a diacetylene polymer composition, and methods for detection of a chemical and biological contaminant using such formulations, including various applications thereof.
  • Bacterial infections have a large impact on health. Many potentially infectious bacteria can survive on surfaces for considerable amounts of time, and they can be transmitted to humans through air, water, food, or living vectors. The ability to detect infectious bacteria in the environment easily and quickly without the need of specialised high-cost equipment is critical to improve the health being of the general public.
  • Polydiacetylene is a conjugated polymer that has been identified as a building block for the construction of colorimetric biosensors for the detection of pathogens.
  • PDA is constructed from amphiphilic diacetylene monomers that selfassemble into vesicles in aqueous solution. These vesicles can be photopolymerised with 254 nm UV light via an addition reaction between diacetylene groups on neighbouring monomers. This produces a conjugated network that is responsible for the unique optical properties of PDA.
  • PDA vesicles can be functionalised to detect an array of different analytes via incorporation of recognition elements into or onto the vesicle membrane.
  • Chemical recognition of analytes or stimulus from environmental sources such as temperature or force can induce structural perturbations in the PDA membrane. This results in a change of the effective conjugation length and a shift in the characteristic maximum absorbance from 640 nm to 540 nm which corresponds to a visible blue to red colour change.
  • the present disclosure is directed to a colorimetric sensor formulation, the process for preparing such a formulation, and use thereof, which may at least partially overcome at least one of the abovementioned disadvantages or provide the consumer with a useful or commercial choice.
  • a colorimetric sensor formulation for detection of a target analyte comprising: i) a polydiacetylene composition comprising at least one diacetylene monomer of Formula 1
  • R 1 is selected from Ci-20-alkyl or C2-20- alkenyl
  • R 2 is selected from Ci-20-alkyl-COOH or C2-20-alkenyl-COOH, in which each Ci-20-alkyl-COOH or C2-io-alkenyl-COOH are optionally substituted (for example optionally substituted with at least one NH2, SH, or a mixture thereof); n is an integer of 1-5; and
  • the at least one phospholipid molecule is incorporated into the polydiacetylene composition to form a polydiacetylene liposome comprising a recognition element;
  • the recognition element is the phospholipid molecule and has a binding affinity for the target analyte; and - the polydiacetylene liposome exhibits a colour change when contacted with the target analyte.
  • R 1 may be selected from Ci-20-alkyl.
  • R 2 may be selected from Ci-20-alkyl-COOH.
  • n may be 1 or 2.
  • R 1 may be selected from Ci-14-alkyl;
  • R 2 may be selected from Ci-s-alkyl- COOH; and
  • n may be 1 or 2.
  • R 1 may be selected from: pentadecyl, tetradecyl, tridecyl, dodecyl, undecyl, decyl, nonyl, octyl, heptyl, hexyl, pentyl, butyl or propyl.
  • R 2 may be selected from: octyl-COOH, heptyl-COOH, hexyl-COOH, pentyl-COOH, butyl-COOH, propyl-COOH, ethyl-COOH or methyl-COOH.
  • R 1 may be selected from: tetradecyl, decyl, octyl, or pentyl;
  • R 2 may be selected from octyl-COOH, hexyl-COOH, butyl-COOH, propyl-COOH or ethyl-COOH; and
  • n may be 1.
  • the phospholipid may be selected from: phosphocholines, phosphoethanolamines, phosphatidylethanolamines, phosphatidylserines, phosphatidylglycerols, and combinations thereof.
  • the phospholipid may be selected from: l,2-dimyristoyl-sn-glycero-3 -phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero- 3 -phosphocholine (DOPC), l-pahmtoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), l -pahnitoyl-2-olcoyl-.w- glycero-3-phospho-(l'-rac-glycerol) (POPG).
  • the phospholipid may be 1,2- dimyristoyl-sn-glycer
  • the polydiacetylene liposome may be unilamellar. In an embodiment, the polydiacetylene liposome may not encapsulate a probe.
  • the ratio of the diacetylene monomer : phospholipid may be in a range between about 1:1 to about 10:1.
  • the ratio of the diacetylene monomer : phospholipid may be about 3 :2 or about 4: 1.
  • the ratio of the diacetylene monomer : phospholipid may be about 4:1.
  • the colorimetric sensor formulation may further comprise cholesterol incorporated into the polydiacetylene liposome to detect the target analyte.
  • the ratio of diacetylene monomer : phospholipid : cholesterol may be in a range between about 1:1:1 to about 10:1:1.
  • the ratio of diacetylene monomer : phospholipid : cholesterol may be 5:2:3 or about 5:2:3.
  • the polydiacetylene liposome may be dispersed in an aqueous solution.
  • the aqueous solution may be selected from water or a buffer system.
  • the pH of the aqueous solution may be in a range of between about 6 and 8.
  • the polydiacetylene liposome may be coated on a substrate or incorporated into a matrix.
  • the substrate may be selected from: glass, gels, films and paper, and the matrix may be selected from: ink, gels, films and packaging material.
  • the colorimetric sensor formulation may further comprise a gel carrier for formation of a solid reservoir.
  • the polydiacetylene liposome may be in the form of a hydrogel disc.
  • the solid reservoir may encapsulate a perishable consumable product to provide a quality tag for contactless monitoring of food.
  • the gel carrier is a crosslinkable polymer.
  • the gel carrier may be selected from: agarose, alginate, poly(vinyl alcohol), pectin, carboxy methyl cellulose, hyaluronates, chitosan, cationic guar, cationic starch, or combinations thereof.
  • the gel carrier may be selected from: agarose, alginate, poly(vinyl alcohol), or combinations thereof.
  • the gel carrier may be agarose.
  • the polydiacetylene liposome may be prepared using a thin-film hydration method or a solvent injection method.
  • the colorimetric sensor formulation may have a detection limit of at least 3 pg/mL and may be capable of detecting the target analyte at concentrations of less than about 25 pg/mL.
  • the target analyte may be bacteria, directly detected or detected through the emitted toxins.
  • the target analyte may be food spoilage bacteria and/or metabolic bacteria.
  • a colorimetric sensor system for detection of a target analyte comprising:
  • a colorimetric sensor system for detection of a target analyte comprising:
  • a colorimetric sensor kit comprising one or more vessels, wherein each of the one or more vessels comprises a colorimetric sensor formulation according to any one or more embodiments or examples thereof as described herein to detect a specific target analyte.
  • the one or more vessels may be a two-part vessel system to enable the colorimetric sensor formulation to be separated from a target analyte until ready for use.
  • a colorimetric sensor tag for detection of a target analyte comprising the colorimetric sensor formulation of any one or more embodiments or examples thereof as described herein in the form of a hydrogel, wherein the hydrogel encapsulates a perishable consumable product to provide the tag for contactless monitoring of food.
  • the colorimetric sensor tag may be configured to be attached to an article.
  • a method for detection of a target analyte comprising: a) obtaining a colorimetric sensor formulation according to any one or more embodiments or examples thereof as described herein, or a colorimetric sensor system according to any one or more embodiments or examples thereof as described herein, or a colorimetric sensor kit according to any one or more embodiments or examples thereof as described herein; b) contacting the colorimetric sensor with a sample suspected of containing a target analyte; and c) observing a colour change if the target analyte is present.
  • the target analyte may be bacteria, directly detected or detected through the emitted toxins.
  • the target analyte may be a-hemolysin.
  • a method for detection of a target analyte comprising: al) obtaining a colorimetric sensor tag according to any one or more embodiments or examples thereof as described herein; and bl) observing a colour change if the target analyte is present.
  • the target analyte may be a product of bacterial lysis such as free fatty acid.
  • the observable colour change may occur in less than about 60 minutes of contacting the sensor with the sample suspected of containing an target analyte.
  • the observable colour change may occur in real-time.
  • an article comprising: a colorimetric sensor according to any one or more embodiments or examples thereof as described herein, or a colorimetric sensor tag according to any one or more embodiments or examples thereof as described herein; and a perishable consumable product.
  • Figure 2 is a TEM micrograph of PDA/DMPC particles formed via the solvent injection method.
  • Figure 4 is a series of TEM micrographs of PDA particles (a) before and (b) after the addition of 12.5 pg/mL of a-hemolysin and of PDA/DMPC/Chol particles (c) before and (d) after the addition of 12.5 pg/mL of a-hemolysin.
  • Figure 5 is a) digital colorimetric response and c), e) photographs of PDA/DMPC vesicles in refrigerated and unrefrigerated milk vs storage time, b) Digital colorimetric response and d), f) photographs of PDA/DMPC/agarose/milk gels both refrigerated and unrefrigerated vs storage time.
  • Figure 6 is a) digital colorimetric response and c) photographs of dehydrated gels in refrigerated and unrefrigerated milk vs storage time, b) Digital colorimetric response and d) photographs of rehydrated gels both refrigerated and unrefrigerated vs storage time.
  • Diacetylene monomers are the building blocks of polydiacetylenes which can be used to construct colorimetric biosensors.
  • the PDA is chromatically robust and does not exhibit chromatic responses (false positives) in the reaction to a stimulus to which it is likely to be exposed to such as changes to milk composition and temperature fluctuations. It has been shown that the sensing features of a PDA can be tuned by manipulation of the structure of the monomers. The inventors have unexpectedly shown that changing the total alkyl chain length and the positioning of the diacetylene functional group can impact thermochromism and the chromatic response to a range of analytes.
  • One or more advantages provided by the present disclosure is that relocating the diacetylene group of the diacetylene monomer towards the carboxylic acid head group may reduce its chromatic response to fluctuations in temperature and its chemical environment by increasing its association with strong hydrogen bonding on the carboxylic acid head groups of the membrane, stabilising the PDA’s conjugation. This effect may be desirable for point of care testing in complex samples.
  • alkyl refers to a straight-chained or branched saturated monovalent hydrocarbon radical, wherein the alkyl may optionally be substituted. Unless otherwise indicated, the alkyl groups typically contain from 1 to 20 carbon atoms. The term “alkyl” also encompasses both linear and branched alkyl, unless otherwise specified.
  • the alkyl is a linear saturated monovalent hydrocarbon radical that has 1 to 20 (Ci-20), 1 to 16 (Ci-ie), 1 to 14 (Ci-14), 1 to 12 (Ci-12), 1 to 10 (Ci- 10), 1 to 8 (C1-8), or 1 to 6 (C1-6) carbon atoms, or branched saturated monovalent hydrocarbon radical of 3 to 20 (C3-20), 3 to 16 (C3-16), 3 to 14 (C3-14), 3 to 12 (C3-12), 3 to 10 (C3-10), 3 to 8 (C3-8), or 3 to 6 (C3-6) carbon atoms.
  • alkyl as used herein, may include, but are not limited to optionally substituted: methyl, ethyl, propyl, (including all isomeric forms), n-propyl, isopropyl, butyl, (including all isomeric forms), n-butyl, isobutyl, sec-butyl, /-butyl, (including all isomeric forms), and hexyl (including all isomeric forms), and the like.
  • alkyl may include optionally substituted: pentadecyl, tetradecyl, tridecyl, dodecyl, undecyl, decyl, nonyl, octyl, heptyl, hexyl, pentyl, butyl or propyl.
  • alkyl groups may be mono- or polyvalent.
  • the alkyl groups may be optionally substituted and/or optionally interrupted by one or more heteroatoms.
  • the alkyl groups may be referred to as “-alkyl” in relation to use as a bivalent or polyvalent linking group.
  • alkenyl refers to a straight-chained or branched monovalent hydrocarbon radical, which contains one or more, in one embodiment, one, two, three, four, or five, in another embodiment, one, carbon-carbon double bond(s). The alkenyl may be optionally substituted.
  • alkenyl also embraces radicals having “czs” and “trans” configurations, or alternatively, “Z” and “E” configurations, as appreciated by those of ordinary skill in the art.
  • alkenyl encompasses both linear and branched alkenyl, unless otherwise specified.
  • C2-6 alkenyl refers to a linear unsaturated monovalent hydrocarbon radical of 2 to 6 carbon atoms or a branched unsaturated monovalent hydrocarbon radical of 3 to 6 carbon atoms.
  • the alkenyl is a linear monovalent hydrocarbon radical of 2 to 20 (C2-20), 2 to 15 (C2-15), 2 to 10 (C2-10), or 2 to 6 (C2-6) carbon atoms, or a branched monovalent hydrocarbon radical of 3 to 20 (C3-20), 3 to 15 (C3-15), 3 to 10 (C3- 10), or 3 to 6 (C3-6) carbon atoms.
  • alkenyl groups include, but are not limited to optionally substituted: ethenyl, propen- 1-yl, propen-2-yl, allyl, butenyl, and 4- methylbutenyl.
  • carboxyl represents a -CO2H moiety.
  • halo or halogen, whether employed alone or in compound words such as haloalkyl, means fluorine, chlorine, bromine or iodine.
  • hydroxyl represents a -OH moiety.
  • haloalkyl means an alkyl group having at least one halogen substituent, the terms “alkyl” and “halogen” being understood to have the meanings outlined above.
  • dihaloalkyl means an alkyl group having two halogen substituents
  • trihaloalkyl means an alkyl group having three halogen substituents.
  • Examples of monohaloalkyl groups include fluoromethyl, chloromethyl, bromomethyl, fluoromethyl, fluoropropyl and fluorobutyl groups; examples of dihaloalkyl groups include difluoromethyl and difluoroethyl groups; examples of trihaloalkyl groups include trifluoromethyl and trifluoroethyl groups.
  • amino represents an -NH2 moiety
  • alkylamino represents an -NHR or -NR2 group in which R is an alkyl group as defined supra. Examples include, without limitation, methylamino, ethylamino, n-propylamino, isopropylamino, and the different butylamino, pentylamino, hexylamino and higher isomers.
  • thiol refers to any organosulphur group containing a sulphurhydryl moiety -SH, which includes a R-SH group where R is a moiety containing a carbon atom for coordination to the -SH moiety, for example an alkylsulphur group as defined supra.
  • the thiol or mercapto group may be a sulphurhydryl moiety -SH.
  • optionally substituted means that a functional group is either substituted or unsubstituted, at any available position.
  • substituted as referred to above or herein may include, but is not limited to, groups or moieties such as halogen, hydroxyl, alkyl, amino, thiol, thio, mercapto, mercaptan, or haloalkyl.
  • biological and chemical contaminant(s) refers to any material that can be detected by the colorimetric sensor of the present disclosure, for example, on surfaces, in water, food (e.g. milk, juice, meat, protein powder, baby powder, etc.), and the indoor air environment.
  • materials include, but are not limited to, small molecules, pathogenic and non-pathogenic organisms, toxins, membrane receptors and fragments, volatile organic compounds, enzymes and enzyme substrates, antibodies, antigens, proteins, peptides, nucleic acids, and peptide nucleic acids.
  • Target analyte refers to the material targeted for detection by the colorimetric sensor of the present disclosure, and may include, but are not limited to, microorganisms (bacteria, directly detected or detected through the emitted toxins or through their DNA, and viruses), free fatty acids, lactic acid, antibacterial and antiviral peptides.
  • the “target analyte” is bacteria directly detected or detected through the emitted toxins.
  • the colorimetric sensor advantageously allows selective detection of a- hemolysin, a hemolytic toxin excreted by Staphylococcus aureus.
  • the colorimetric sensor advantageously allows selective detection of free fatty acids, a marker from fat rancidification by bacterial lipolysis of various perishable consumer products (e.g. milk, juice, meat, protein powder, baby powder, etc.).
  • bacteria refers to all forms of microorganisms considered to be bacteria including, but not limited to, cocci, bacilli, spirilla, spirochetes, spheroplasts, protoplasts.
  • bacteria as used herein, may refer to food spoilage bacteria and/or metabolic bacteria, and may include, but not limited to, proteolytic bacteria, lypolytic bacteria, and sucrolytic bacteria.
  • the bacteria may be Staphylococcus aureus.
  • phospholipid refers to any phospholipid molecule or assembly of phospholipid molecules with an affinity for a target contaminant and/or a probe. It will be appreciated that the phospholipid may be the recognition element.
  • the terms “assembly,” or “self-assembly,” refers to any selfordering of diacetylene molecules and phospholipids prior to polymerisation.
  • polydiacetylene liposome describes a spherical vesicle capable of turning a recognition event such as a covalent bond or a noncovalent interaction (e.g. electrostatic interaction, polar interaction, van der Waals forces) at the molecular level into an observable signal (e.g. blue to red colour transition).
  • a recognition event such as a covalent bond or a noncovalent interaction (e.g. electrostatic interaction, polar interaction, van der Waals forces) at the molecular level into an observable signal (e.g. blue to red colour transition).
  • Probe refers to a constituent that is capable of interacting with the target contaminant and/or the phospholipid. Accordingly, the probe is a type of “detectable binding reagent” i.e. an agent that specifically recognises and interacts or binds with an contaminant (i.e. the target analyte) and/or the phospholipid, wherein the probe has a property permitting detection when bound and is encapsulated by the colorimetric sensor (i.e. polydiacetylene vesicle).
  • the colorimetric sensor i.e. polydiacetylene vesicle
  • detectable binding agent physically interacts with the target contaminant or phospholipid to the substantial exclusion of other contaminants also present in the sample.
  • the binding of a detectable binding reagent useful according to the present disclosure has stability permitting the measurement of the binding.
  • covalent bond refers to the linkage of two atoms by the sharing of two electrons, one contributed by each of the atoms.
  • the term “absorption” refers, in one sense, to the absorption of light. Light is absorbed if it is not reflected from or transmitted through the colorimetric sensor, as described herein. Sensors that appear coloured have selectively absorbed all wavelengths of white light except for those corresponding to the visible colours that are seen.
  • the term “spectrum” refers to the distribution of light energies arranged in order of wavelength.
  • visible spectrum refers to light radiation that contains wavelengths from approximately 360 nm to approximately 800 nm.
  • UV irradiation refers to exposure to radiation with wavelengths less than that of visible light (i.e. less than approximately 360 nm) but greater than that of X-rays (i.e. greater than approximately 0.1 nm). UV radiation possesses greater energy than visible light and is therefore, more effective at inducing photochemical reactions.
  • chromatic transition refers to the changes of molecules or material that result in an alteration of visible light absorption.
  • chromatic transition refers to the change in light absorption of the colorimetric sensor, as described herein, whereby there is a detectable colour change associated with the transition. This detection can be accomplished through various means including, but not limited to, visual observation and spectrophotometry.
  • substrate refers to a solid object or surface upon which the colorimetric sensor is layered or attached.
  • Substrates include, but are not limited to, glass, gels, and filter paper, among others.
  • the term “formation solvent” refers to any medium, although typically a volatile organic solvent, used to solubilise and distribute material to a desired location (e.g. to a surface for producing a film or to a drying receptacle to deposit the polydiacetylene liposome, as described herein, for drying).
  • the term “tag” refers to any device that is portable and may be attached to an article (e.g. food packaging).
  • article refers to any type of food packaging.
  • the food packaging may be made of, but not limited to, paper and paperboard, rigid plastic and glass.
  • the term “device” refers to any apparatus that may or may not be portable, and used as a means of colorimetric detection.
  • the terms “positive”, “negative”, and “zwitterionic charge” refer to molecules or molecular groups that contain a net positive, negative, or neutral charge, respectively. Zwitterionic entities contain both positively and negatively charged atoms or groups whose charges cancel (i.e. whose net charge is 0).
  • zn situ refers to processes, events, objects, or information that are present or take place within the context of their natural environment.
  • aqueous refers to a liquid mixture containing water, among other components.
  • the present disclosure relates to colorimetric polydiacetylene sensor formulation for the detection of chemical and biological contaminants both in liquid phase and vapour phase.
  • the diacetylene compounds of the present disclosure can self-assemble in solution to form ordered vesicles that can be polymerised using any actinic radiation such as, for example, electromagnetic radiation in the UV or visible range of the electromagnetic spectrum.
  • the polymerisation of the diacetylene compounds result in polymerisation reaction products that have a colour in the visible spectrum less than about 540 nanometers (nm).
  • the polymerisation of the diacetylene compounds result in polymerisation reaction products that have a colour in the visible spectrum at least about 640 nm.
  • the polymerisation of the diacetylene compounds result in polymerisation reaction products that have a colour in the visible spectrum may be in a range of between about 540 nm and about 640 nm. It will be appreciated that the colour of the diacetylene compounds and their polymerisation products will depend on their conformation and exposure to external factors.
  • polymerisation of the diacetylene compounds, as disclosed herein result in meta-stable blue phase polymer networks that include a polydiacetylene backbone. These meta-stable blue phase polymer networks undergo a colour change from bluish to reddish-orange upon exposure to external factors such as heat, a change in solvent or counter ion, if available, or physical stress, for example.
  • the ability of the diacetylene compounds and their polymerisation products disclosed herein to undergo a visible colour change upon exposure to physical stress make them ideal candidates for the preparation of sensing devices for detection of a target analyte.
  • the polydiacetylene (PDA) vesicles can be functionalised to detect an array of different target analytes via incorporation of recognition elements into or onto the vesicle membrane.
  • Chemical recognition of analytes and/or stimulus from environmental sources such as temperature or force can induce structural perturbations in the PDA membrane. This results in a change of the effective conjugation length and a shift in the characteristic maximum absorbance from 640 nm to 540 nm which corresponds to a visible blue to red colour change.
  • the colorimetric sensor formulation may comprise diacetylene monomers and phospholipids that can detect bacteria using: a direct method wherein bacteria or their toxins interact with polydiacetylene/phospholipids and induce structural perturbations in polydiacetylene, leading to colorimetric changes from blue to red; and/or an indirect method wherein the by-products of bacteria metabolism, for example lactic acid or ammonia gas, react with chemically-modified polydiacetylene, and induce structural perturbations in polydiacetylene, leading to colorimetric changes from blue to red.
  • a direct method wherein bacteria or their toxins interact with polydiacetylene/phospholipids and induce structural perturbations in polydiacetylene, leading to colorimetric changes from blue to red
  • an indirect method wherein the by-products of bacteria metabolism, for example lactic acid or ammonia gas, react with chemically-modified polydiacetylene, and induce structural perturbations in polydiacetylene, leading to colorimetric
  • the present disclosure provides a colorimetric sensor formulation for detection of a target analyte comprising: i) a polydiacetylene composition comprising at least one diacetylene monomer of Formula 1
  • R 1 is selected from Ci-20-alkyl or C2-20- alkenyl
  • R 2 is selected from Ci-20-alkyl-COOH or C2-20-alkenyl-COOH, in which each Ci-20-alkyl-COOH or C2-io-alkenyl-COOH are optionally substituted; n is an integer of 1-5; and
  • the at least one phospholipid molecule is incorporated into the polydiacetylene composition to form a polydiacetylene liposome comprising a recognition element;
  • the recognition element is the phospholipid molecule and has a binding affinity for the target analyte
  • the polydiacetylene liposome exhibits a colour change when contacted with the target analyte.
  • the polydiacetylene liposome may be unilamellar. In an embodiment, the polydiacetylene liposome may not encapsulate a probe.
  • the polydiacetylene liposome may be dispersed in an aqueous solution.
  • the aqueous solution may be selected from water or a buffer system.
  • the pH of the aqueous solution may be in a range of between about 6 and 8.
  • the polydiacetylene liposome may be coated on a substrate or incorporated into a matrix.
  • the substrate may be selected from: glass, gels, films and paper, and the matrix may be selected from: ink, gels, films and packaging material.
  • the polydiacetylene liposome may be prepared using a thin-film hydration method or a solvent injection method.
  • the colorimetric sensor formulation may have a detection limit of at least 3 pg/mL and may be capable of detecting the target analyte at concentrations of less than about 25 pg/mL.
  • the target analyte may be bacteria, directly detected or detected through the emitted toxins.
  • the target analyte may be food spoilage bacteria and/or metabolic bacteria.
  • the bacteria may be hemolytic bacteria.
  • the bacteria may be a-hemolytic bacteria.
  • diacetylene monomers can be manipulated to tune the sensitivity, specificity, and chromism of PDAs.
  • a monomer that forms a PDA that is resistant to environmental stimulus is desirable.
  • R 1 may be Ci-20-alkyl or C2-20-alkenyl. In an embodiment is from C1-20- alkyl. In another embodiment Ri is C2-20-alkenyl.
  • R 1 may be a Ci-14-alkyl.
  • R 1 may be selected from: pentadecyl, tetradecyl, tridecyl, dodecyl, undecyl, decyl, nonyl, octyl, heptyl, hexyl, pentyl, butyl or propyl.
  • R 1 may be selected from: tetradecyl, decyl, octyl, or pentyl.
  • R 2 is a Ci-20-alkyl-COOH or C2-20-alkenyl-COOH, in which each C1-20- alkyl-COOH or C2-io-alkenyl-COOH are optionally substituted.
  • R 2 is an optionally substituted Ci-20-alkyl-COOH.
  • R 2 is an optionally substituted C2-20-alkenyl-COOH.
  • R 2 may be an optionally substituted Ci-s-alkyl-COOH.
  • R 2 may be selected from an optionally substituted: octyl-COOH, heptyl-COOH, hexyl-COOH, pentyl-COOH, butyl-COOH, propyl-COOH, ethyl-COOH or methyl- COOH.
  • R 2 is unsubstituted. In another embodiment, R 2 is substituted, for example R 2 is a Ci-20-alkyl-COOH or C2-20-alkenyl-COOH substituted with at least one NH2, SH, or a mixture thereof. Integer “n”
  • n may be 1, 2, 3, 4 or 5. In an embodiment, n may be 1 or 2. In an embodiment, n is 1. In another embodiment, n is 2.
  • -R 1 is Ci-i4-alkyl
  • R 2 is an optionally substituted Ci-s-alkyl-COOH
  • n is 1 or 2;
  • R 1 is: tetradecyl, decyl, octyl, or pentyl;
  • R 2 is an optionally substituted: octyl- COOH, hexyl-COOH, butyl-COOH, propyl-COOH or ethyl-COOH; and
  • n is 1.
  • the diacetylene monomer may be selected from the group consisting of 6,8-tricosadiynoic acid, 4,6-heptadecadiynoic acid, 5,7- hexadecadiynoic acid, 10,12-octadecadiynoic acid, 6,8-nonadecadiynoic acid, 8,10- henicosadiynoic acid, 10,12-tricosadiynoic acid, 10,12-pentacosadiynoic acid, or combinations thereof.
  • the PDA liposomes may comprise a recognition element.
  • the PDA can be functionalized with different recognition elements to act as a colorimetric sensor for temperature, pH, mechanical stress, proteins, bacteria, viruses, and other important target analytes.
  • the recognition element may be the phospholipid molecule and has a binding affinity for a target analyte. It has been found that incorporation of phospholipids into the PDA membranes may increase membrane flexibility and may also increase the chromatic sensitivity of PDA sensors in response to stimuli as it can allow easier conformational and structural changes around the diacetylene functional group.
  • a phospholipid can be added to the polydiacetylene composition either prior to or after polymerisation. Upon polymerisation or thereafter, the phospholipid is effectively incorporated within the polymer network such that interaction of the phospholipid with a target analyte results in a visible colour change due to the perturbation of the conjugated diacetylene polymer backbone.
  • the phospholipid may be physically mixed and dispersed among the polydiacetylene assembly.
  • the phospholipid may be covalently bonded to the polydiacetylene assembly.
  • the phospholipid may be selected from: phosphocholines, phosphoethanolamines, phosphatidylethanolamines, phosphatidylserines, phosphatidylglycerols, and combinations thereof.
  • the phospholipid may be selected from: l,2-dimyristoyl-5n-glycero-3- phosphocholine (DMPC), 1 ,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1- pahmtoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1 ,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), l-palmitoyl-2-oleoyl-5n-glycero-3-phospho-(l'-rac- glycerol) (POPG).
  • the phospholipid may be l,2-dimyristoyl-5n-glycero-3- phosphocholine (DMPC).
  • the colorimetric sensor formulation may further comprise cholesterol incorporated into the polydiacetylene liposome to detect the target analyte.
  • pathogenic bacteria produce a large variety of toxins and virulence factors.
  • Hemolytic bacteria are pathogenic bacteria that produce pore-forming toxins, ultimately resulting in cell death by necrosis or apoptosis.
  • incorporation of phospholipid and cholesterol in the PDA membrane may allow the selective detection of alpha-hemolysin, a hemolytic toxin excreted by Staphylococcus aureus, a predominant causative pathogen of bovine mastitis.
  • the ratio of diacetylene monomer : phospholipid : cholesterol may be in a range between about 1 : 1 : 1 to about 10:1:1.
  • the ratio of diacetylene monomer : phospholipid : cholesterol may be 5:2:3 or about 5:2:3. It will be appreciated that the present disclosure may mimic the composition (or ratio) of phospholipid : cholesterol in biological cells which may vary greatly.
  • the ratio of phospholipid : cholesterol may be in a range that occurs in a biological cell membranes to detect membrane active toxins (e.g. a-hemolysin). Hydrogel
  • a hydrogel may serve as a solid reservoir for the colorimetric sensor, as described herein, to advantageously transform the sensor formulation into a polydiacetylene solid sensor or colorimetric sensor tag.
  • the colorimetric sensor formulation may further comprise a gel carrier for formation of a solid reservoir.
  • the polydiacetylene liposome may be in the form of a hydrogel disc.
  • the solid reservoir may encapsulate a perishable consumable product to provide a quality tag for contactless monitoring of food.
  • the gel carrier may be a crosslinkable polymer.
  • the gel carrier may be selected from: agarose, alginate, poly(vinyl alcohol), pectin, carboxy methyl cellulose, hyaluronates, chitosan, cationic guar, cationic starch, or combinations thereof.
  • the gel carrier may be selected from: agarose, alginate, poly(vinyl alcohol), or combinations thereof.
  • the gel carrier may be agarose.
  • the inventors have advantageously developed a colorimetric sensor formulation for the selective detection of pathogenic bacteria (e.g. a-hemolysin) in complex samples (e.g. perishable consumable products such as milk).
  • pathogenic bacteria e.g. a-hemolysin
  • complex samples e.g. perishable consumable products such as milk.
  • POC point-of-care
  • a colorimetric sensor formulation for detection of a target analyte may comprise: i) a polydiacetylene composition comprising at least one diacetylene monomer of Formula 1
  • R 1 is selected from Ci-20-alkyl or C2-20- alkenyl
  • R 2 is selected from Ci-20-alkyl-COOH or C2-20-alkenyl-COOH, in which each Ci-20-alkyl-COOH or C2-io-alkenyl-COOH are optionally substituted
  • n is an integer of 1 to 5;
  • the at least one phospholipid molecule is incorporated into the polydiacetylene composition to form a polydiacetylene liposome comprising a recognition element;
  • the recognition element is the phospholipid molecule and has a binding affinity for the target analyte
  • the polydiacetylene liposome exhibits a colour change when contacted with the target analyte.
  • R 1 may be selected from Ci-20-alkyl.
  • R 2 may be selected from an optionally substituted Ci-20-alkyl-COOH.
  • n may be 1 or 2.
  • R 1 may be selected from Ci-14-alkyl
  • R 2 may be selected from optionally substituted Ci-s-alkyl-COOH
  • n may be 1 or 2.
  • R 1 may be selected from: pentadecyl, tetradecyl, tridecyl, dodecyl, undecyl, decyl, nonyl, octyl, heptyl, hexyl, pentyl, butyl or propyl.
  • R 2 may be selected from optionally substituted: octyl-COOH, heptyl-COOH, hexyl-COOH, pentyl-COOH, butyl-COOH, propyl-COOH, ethyl-COOH or methyl-COOH.
  • R 1 may be selected from: tetradecyl, decyl, octyl, or pentyl
  • R 2 may be selected from optionally substituted: octyl-COOH, hexyl-COOH, butyl-COOH, propyl-COOH or ethyl-COOH
  • n may be 1.
  • the diacetylene monomer may be selected from the group consisting of 6,8-tricosadiynoic acid, 4,6-heptadecadiynoic acid, 5,7- hexadecadiynoic acid, 10,12-octadecadiynoic acid, 6,8-nonadecadiynoic acid, 8,10- henicosadiynoic acid, 10,12-tricosadiynoic acid, 10,12-pentacosadiynoic acid, or combinations thereof.
  • the phospholipid may be selected from: phosphocholines, phosphoethanolamines, phosphatidylethanolamines, phosphatidylserines, phosphatidylglycerols, and combinations thereof.
  • the phospholipid may be selected from: l,2-dimyristoyl-sn-glycero-3 -phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero- 3 -phosphocholine (DOPC), 1 -pahmtoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), l -pahnitoyl-2-olcoyl-.w- glycero-3-phospho-(l'-rac-glycerol) (POPG).
  • the phospholipid may be l,2-dimyristoyl-sn-
  • the polydiacetylene liposome may be unilamellar. In one embodiment, the polydiacetylene liposome does not encapsulate a probe.
  • the ratio of the diacetylene monomer : phospholipid may be in a range between about 1:1 to about 10:1.
  • the ratio of the diacetylene monomer : phospholipid may be about 3 :2 or about 4: 1.
  • the ratio of the diacetylene monomer : phospholipid may be about 4:1.
  • the ratio of diacetylene monomer : phospholipid : cholesterol may be in a range between about 1:1:1 to about 10:1:1.
  • the ratio of diacetylene monomer : phospholipid : cholesterol may be about 5:2:3. It will be appreciated that the present disclosure may mimic the composition (or ratio) of phospholipid : cholesterol in biological cells which may vary greatly.
  • the ratio of phospholipid : cholesterol may be in a range that occurs in a biological cell membranes to detect membrane active toxins (e.g. a-hemolysin).
  • the polydiacetylene liposome may be dispersed in an aqueous solution.
  • the aqueous solution may be selected from water or a buffer system.
  • the buffer system may be phosphate-buffered saline (PBS) or 4-(2- hydroxyethyl)-l -piperazineethanesulfonic acid (HEPES).
  • PBS phosphate-buffered saline
  • HEPES 4-(2- hydroxyethyl)-l -piperazineethanesulfonic acid
  • the pH of the aqueous solution may be in a range of between about 6 and 8.
  • the polydiacetylene liposome may be coated on a substrate or incorporated into a matrix.
  • the substrate may be selected from: glass, gels, films and paper, and the matrix may be selected from: ink, gels, films and packaging material.
  • the polydiacetylene liposome may be prepared using a thin- film hydration method or a solvent injection method.
  • the target analyte may be bacteria, directly detected or detected through the emitted toxins.
  • the target analyte may be food spoilage bacteria and/or metabolic bacteria.
  • the target analyte is a-hemolysin. It will be appreciated that pathogenic bacteria produce a large variety of toxins and virulence factors. Hemolytic bacteria are pathogenic bacteria that produce pore-forming toxins, ultimately resulting in cell death by necrosis or apoptosis.
  • incorporation of phospholipid and cholesterol in the PDA membrane may allow the selective detection of a-hemolysin, a hemolytic toxin excreted by Staphylococcus aureus, a predominant causative pathogen of bovine mastitis.
  • the colorimetric sensor formulation may have a detection limit of at least 3 pg/mL and may be capable of detecting the target analyte at concentrations of less than about 25 pg/mL.
  • the colour change may be attributed to pore formation in the PDA membrane and lysis of the vesicles due to the action of the Staphylococcus aureus a-hemolysin. This can be evidenced by vesicle membrane destruction in transmission electron microscopy micrographs of PDA vesicles before and after incubation with the toxin.
  • the specificity of the sensor may be demonstrated by discrimination between a-hemolysin and other toxins and biomarkers for mastitis.
  • the inventors have custom-designed a PDA sensor for point-of-care application in bovine milk samples which can selectively detect a- hemolysin, a hemolytic toxin excreted by Staphylococcus aureus, a predominant causative pathogen of bovine mastitis, using the PDA-based sensor, as described herein.
  • the inventors have advantageously developed a colorimetric sensor tag for contactless monitoring of complex samples (e.g. perishable consumable products such as milk).
  • complex samples e.g. perishable consumable products such as milk.
  • the colorimetric sensor may find particular use as quality indicators in food packaging that could improve food management.
  • a colorimetric sensor formulation for detection of a target analyte comprising: i) a polydiacetylene composition comprising at least one diacetylene monomer of Formula 1
  • R 1 is selected from Ci-20-alkyl or C2-20- alkenyl
  • R 2 is selected from Ci-20-alkyl-COOH or C2-20-alkenyl-COOH, in which each Ci-20-alkyl-COOH or C2-io-alkenyl-COOH are optionally substituted;
  • n is an integer of 1 to 5;
  • the at least one phospholipid molecule is incorporated into the polydiacetylene composition to form a polydiacetylene liposome comprising a recognition element;
  • the recognition element is the phospholipid molecule and has a binding affinity for the target analyte
  • polydiacetylene liposome exhibits a colour change when contacted with the target analyte.
  • R 1 may be selected from Ci-20-alkyl.
  • R 2 may be selected from an optionally substituted Ci-20-alkyl-COOH.
  • n may be 1 or 2.
  • R 1 may be selected from Ci-14-alkyl
  • R 2 may be selected from optionally substituted Ci-s-alkyl-COOH
  • n may be 1 or 2.
  • R 1 may be selected from: pentadecyl, tetradecyl, tridecyl, dodecyl, undecyl, decyl, nonyl, octyl, heptyl, hexyl, pentyl, butyl or propyl.
  • R 2 may be selected from optionally substituted: octyl-COOH, heptyl-COOH, hexyl-COOH, pentyl-COOH, butyl-COOH, propyl-COOH, ethyl-COOH or methyl-COOH.
  • R 1 may be selected from: tetradecyl, decyl, octyl, or pentyl
  • R 2 may be selected from optionally substituted: octyl-COOH, hexyl-COOH, butyl-COOH, propyl-COOH or ethyl-COOH
  • n may be 1.
  • the diacetylene monomer may be selected from the group consisting of 6,8-tricosadiynoic acid, 4,6-heptadecadiynoic acid, 5,7- hexadecadiynoic acid, 10,12-octadecadiynoic acid, 6,8-nonadecadiynoic acid, 8,10- henicosadiynoic acid, 10,12-tricosadiynoic acid, 10,12-pentacosadiynoic acid, or combinations thereof.
  • the phospholipid may be selected from: phosphocholines, phosphoethanolamines, phosphatidylethanolamines, phosphatidylserines, phosphatidylglycerols, and combinations thereof.
  • the phospholipid may be selected from: l,2-dimyristoyl-sn-glycero-3 -phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero- 3 -phosphocholine (DOPC), 1 -pahmtoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), l -pahnitoyl-2-olcoyl-.w- glycero-3-phospho-(l'-rac-glycerol) (POPG).
  • the phospholipid may be l,2-dimyristoyl-sn-
  • the polydiacetylene liposome may be unilamellar. In one embodiment, the polydiacetylene liposome does not encapsulate a probe.
  • the ratio of the diacetylene monomer : phospholipid may be in a range between about 1:1 to about 10:1.
  • the ratio of the diacetylene monomer : phospholipid may be about 3 :2 or about 4: 1.
  • the ratio of the diacetylene monomer : phospholipid may be about 4:1.
  • the polydiacetylene liposome may be in the form of a hydrogel disc.
  • the gel carrier is a crosslinkable polymer.
  • the gel carrier may be selected from: agarose, alginate, poly(vinyl alcohol), pectin, carboxy methyl cellulose, hyaluronates, chitosan, cationic guar, cationic starch, or combinations thereof.
  • the gel carrier may be selected from: agarose, alginate, poly(vinyl alcohol), or combinations thereof.
  • the gel carrier may be agarose.
  • the polydiacetylene liposome may be prepared using a thin-film hydration method or a solvent injection method.
  • the solid reservoir may encapsulate the perishable consumable product to provide a tag for contactless monitoring of the perishable consumable.
  • the perishable consumable may be a food or drink product, optionally selected from: milk, juice, meat, coffee, sauce, broth, protein powder, and the like.
  • the colorimetric sensor tag may be used to monitor the quality of fruit, vegetable, and animal products, and products that contain sugar, protein and fats (e.g. baby formula).
  • the senor may be provided in a dehydrated form when not in use, optionally for storage and/or transportation.
  • the colorimetric sensor tag can be attached to the external surface of an article (e.g. food packaging) to provide an indirect indication of food quality without the need for contact with the food product.
  • the quality tags can be “switched” on and off using dehydration and rehydration methods, advantageously removing the need for in situ manufacturing and allowing storage before use.
  • the target analyte may be a free fatty acid. It will be appreciated that free fatty acids are a product of microbial spoilage of perishable consumable products.
  • the polydiacetylene liposome may prepared using a thin-film hydration method or a solvent injection method.
  • a method to obtain polydiacetylene liposomes may involve dissolving diacetylene monomers and phospholipids in an organic solvent (e.g. chloroform) which is then evaporated to dryness (e.g. by nitrogen) leaving a thin lipid film.
  • the dry lipid film is then hydrated in an appropriate amount of aqueous phase and the mixture is heated to above the phase transition temperature (T m ) of the lipids/lipid mixture and lipid film is allowed to “swell”.
  • T m phase transition temperature
  • the resulting liposomes which typically consist of multilamellar vesicles (MLV's) are dispersed by shaking the test tube.
  • This preparation provides the basis for producing small unilamellar vesicles by methods such as sonication (Papahadjopoulos et al., 1967), extrusion as described by Cullis et al. in U.S. Pat. No.5, 008, 050, or high-pressure homogenisation.
  • This process is the conventional method for synthesis of polydiacetylene liposomes and is known as the thin film hydration method (“Bangham method”) which was developed in 1965 for the production of liposomes.
  • Another method which may be used to prepare the polydiacetylene liposomes, as described herein, may be the solvent injection method and may be a suitable alternative for the synthesis of the polydiacetylene liposomes.
  • the diacetylene monomers may be dissolved in a polar solvent (e.g. ethanol).
  • a polar solvent e.g. ethanol
  • the diacetylene solvent solution is then slowly added to an aqueous medium at above the phase transition temperature of the monomer under vigorous stirring.
  • the ethanol is evaporated, and the amphiphilic diacetylenes self-assemble into vesicles.
  • Preparation of liposomes of phospholipids by the double emulsion method for encapsulating a water soluble compound, e.g. a protein, which does not have strong affinity to the liposome is described by Schneider in US 4,008,801 and US 4,224,179.
  • the emulsion with liposomes containing the compound is prepared by the double emulsion method: A small volume of an aqueous solution with a water soluble compound e.g. a protein antigen and/or an immunostimulator is mixed with a larger volume organic solvent with the phospholipid dissolved. The formed emulsion is mixed with a larger volume aqueous solution and the organic solvents are removed with an airstream.
  • a method described by Carmona-Ribeiro and Chaimovich involves injecting an organic e.g. chloroform, methanol, ethanol, solution of the desired lipids into an aqueous buffer where the lipids spontaneously forms liposomes as the solvent evaporates.
  • the ethanol injection method for large scale manufacturing of liposomes has been evaluated in recent years (Wagner 2006, Justo 2010), and this manufacturing method was judged more feasible to large scale production than for example the lipid film method.
  • the activity of the toxin results in pore formation in the lipid membrane and the dye is released from the liposome. This leads to an increase in fluorescent signal which provides a quantitative indication of the toxin concentration.
  • this biosensor is faster and more portable than blood agar tests, encapsulation of the dye in the liposome for a signal transducer and the requirement of fluorometer adds complexity to the particle synthesis and signal reading.
  • Electrochemical sensors have also been previously developed that convert the action of the toxin on endothelial cells coating an electrode into an electrical signal. Whilst, this sensor may exhibit good sensitivity (0.1 ng/mL), electrochemical transduction requires a digital signal display for use by untrained operators.
  • the present disclosure provides for the incorporation of PDA into a membrane wherein the signal generated from a chemical recognition event is a colour change that is visible to the human eye. This advantageously removes the necessity for signal reading equipment such as fluorometers or voltammeters.
  • the PDA systems as described herein, can offer new opportunities for mobilisation of alpha-toxin diagnostics and use by unskilled operators due to its convenient colour change.
  • a method for detection of a target analyte may comprise: a) obtaining a colorimetric sensor formulation according to any one or more embodiments or examples thereof as described herein, or a colorimetric sensor system according to any one or more embodiments or examples thereof as described herein, or a colorimetric sensor kit according to any one or more embodiments or examples thereof as described herein; b) contacting the colorimetric sensor with a sample suspected of containing a target analyte; and c) observing a colour change if the target analyte is present.
  • the target analyte may be bacteria, directly detected or detected through the emitted toxins.
  • the target analyte may be a-hemolysin.
  • a-hemolysin is a hemolytic toxin excreted by Staphylococcus aureus, a predominant causative pathogen of bovine mastitis.
  • a method for detection of a target analyte may comprise: al) obtaining a colorimetric sensor tag according to any one or more embodiments or examples thereof as described herein; and bl) observing a colour change if the target analyte is present.
  • the target analyte may be a product of bacterial lysis such as free fatty acid. It will be appreciated that free fatty acids are a product of microbial spoilage of perishable consumable products.
  • the observable colour change may occur in less than about 60 minutes of contacting the sensor with the sample suspected of containing an target analyte. In an embodiment, the observable colour change may occur in less than about 60 minutes, about 45 minutes, about 30 minutes, about 15 minutes, about 10 minutes, about 5 minutes, or about 1 minute. For example, the observable colour change may occur in less than about 30 minutes. In a preferred example, the observable colour change may occur in less than about 15 minutes.
  • the observable colour change may occur in realtime. It will be appreciated that the term “real-time” may refer to the actual time for which the observable colour change occurs, i.e. at the time the recognition event occurs.
  • the present disclosure provides a colorimetric sensor formulation that can be amenable to a variety of applications that demand cost-effective, stable, accurate, consistent and quick diagnostics outside the laboratory setting. Applications include point-of-care (POC) testing, home testing diagnostics, and food processing.
  • POC point-of-care
  • a colorimetric sensor system for detection of a target analyte may comprise: (i) the colorimetric sensor formulation of any one or more embodiments or examples thereof as described herein; and
  • a colorimetric sensor system for detection of a target analyte may comprise:
  • the colorimetric sensor tag may comprise opaque materials such as food ingredients.
  • software for the analysis of images of colorimetric sensors may include Python programming language and Digital Colorimetric Response (DCR) to observe the colour change.
  • DCR Digital Colorimetric Response
  • the colorimetric sensor formulation comprising the PDA vesicles can be obtained without the need to form a film by the conventional LB (Langmuir-Blodgett) process before transferring it onto an appropriate support.
  • the polydiacetylene vesicles can be formed on a substrate using the known LB process as described in A. Ulman, An Introduction to Ultrathin Organic Films, Academic Press, New York (1991), pp. 101-219.
  • the present disclosure can provide bio sensing capabilities in a matrix, such as ink.
  • the sensors are self-contained and do not require additional instrumentation to convey a measurable result.
  • use with other analytical instrumentation is possible to further enhance sensitivity, such as fluorescence with the fluorescent “red” phase developed after detection of the target analyte.
  • the colorimetric sensor formulation as described herein may be in the form of a solution.
  • the solution can be provided in a simple vial system, the targe analyte may be directly added to a vial containing a solution of the colorimetric sensor, as described herein, designed specifically to the target analyte of interest.
  • the colorimetric sensor kit could comprise multiple vials in the kit, with each vial containing a colorimetric sensor formulation, as described herein, particular to different target analytes. For those applications in which the target analyte cannot be added directly to the colorimetric sensor formulation, a two-part vial system could be used.
  • one compartment of the vial could contain reagents for sample preparation of the target analyte physically separated from the second compartment containing the colorimetric sensor formulation. Once sample preparation is complete, the physical barrier separating the compartments would be removed to allow the analyte to mix with the transducer for detection.
  • a colorimetric sensor kit may comprise one or more vessels, wherein each of the one or more vessels comprises a colorimetric sensor formulation according to any one or more embodiments or examples thereof as described herein to detect a specific target analyte.
  • the one or more vessels may be a two-part vessel system to enable the colorimetric sensor formulation to be separated from a target analyte until ready for use.
  • bovine mastitis is a common herd disease typically caused by bacterial infection and is characterised by an inflammation of the mammary gland. It represents one of the most difficult veterinary diseases to control and is the most costly to the dairy industry. Animal welfare concerns, treatment costs, extreme antibiotic use, and the reduced milk yield associated with mastitis incur significant economic and environmental expenses. Early and more specific diagnosis has been identified as an opportunity to better direct herd management strategy and reduce costs.
  • Standard bovine mastitis diagnostic techniques include a range of laboratory and farm-based approaches. Some methods evaluate milk characteristics such as somatic cell count, enzymatic activity, electrical conductivity, and pH. These can indicate mastitis quickly and at low cost, however they do not specify the causative pathogen. Viable pathogen-specific methods include cell culturing and DNA-based techniques (e.g. polymerase chain reaction). These are restricted to laboratory settings, require specialised equipment, and are time-consuming. It has been identified that PDA sensors may be a useful alternative for pathogen- specific diagnosis that exhibit the sensitivity and specificity of laboratory-based techniques with the rapidity, mobility, and price of existing non-specific farm-based methods. Previous efforts have aimed to develop sensors for pathogen-specific diagnosis tools for mastitis.
  • Staphylococcus aureus is a gram-positive bacterium and is a predominant causative pathogen of mastitis
  • a-hemolysin a-toxin
  • the toxin is secreted by the bacteria as a water soluble monomer which binds to the membrane of a host cell and forms an oligomer containing seven subunits.
  • the oligomerisation process forms a pore in the lipid bilayer of the host cell between 1-3 nm which allows the flow of cations, ATP, and small molecules ( ⁇ 4 kDa) to either side of the cell membrane.
  • date marking tools such as use-by and sell-by dates
  • shelf life estimates are static and do not adapt to variables such as storage conditions, leading to frequently invalid indications of food quality.
  • shelf life estimates are overly conservative as they pre-empt poor storage conditions.
  • Cautious date marking is understandable as poisoning from spoiled food is a serious public health concern that can precipitate economic and legal ramifications for food producers.
  • increasing concerns over sustainable food production now means that food loss due to conservative date marking is unacceptable and an attractive avenue to address the global food waste crisis.
  • Traditional sensors directly monitor the chemical constitution of a sample, deducing qualitative or quantitative information by specific detection of targeted markers. Although this approach works well in controlled laboratory settings, in out-of- lab applications it can be difficult to establish and maintain a reliable interface between a sample and the sensor. This is particularly true for sensors that are intended for incorporation into food packaging to indicate quality.
  • the present inventors have advantageously prepared colorimetric sensor tags which can encapsulate a variety of perishable consumable products (e.g. milk).
  • the colorimetric sensor tags as described herein, may be incorporated into, or retrofitted onto an article (e.g. food packaging) as quality tags for contactless monitoring of food to be consumed.
  • an article e.g. packaged food product
  • attaching the colorimetric sensor tags to an article may subject both the sensor and the consumable product to the same storage conditions and therefore the sensor tag may “spoil” or change in composition with the same kinetics as the food product being emulated.
  • the colorimetric sensor tag can advantageously indirectly indicate the quality of the food by measuring the degree of the colour change of the quality tag. This removes the challenge of maintaining an interface between the food and sensor as it is no longer required.
  • a colorimetric sensor tag for detection of a target analyte may comprise the colorimetric sensor formulation of any one or more embodiments or examples thereof as described herein in the form of a hydrogel, wherein the hydrogel encapsulates a perishable consumable product to provide the tag for contactless monitoring of food.
  • the colorimetric sensor tag may be configured to be attached to an article.
  • the article may be food packaging prepared from cardboard, aluminium, plastic, glass, and the like.
  • DA diacetylene
  • a solvent e.g. chloroform
  • the DA monomer formed a thin film on the substrate.
  • the thin film of DA monomer was hydrated with 10 mL of deionised water at 80 °C under probe sonication for 10 minutes utilising a Branson Digital Sonifier (250 W, 20 kHz) with a 1/8” diameter tapered circular tip (Branson 101-148-062). Hydration produced liposomes at a concentration of 1 mg/mL.
  • the liposomes were left to anneal for 16 hours at 4 °C.
  • the DA liposomes were then subjected to photopolymerisation to form polydiacetylene (PDA) liposomes by being exposed to 254 nm UV light to induce an addition reaction between diacetylene groups on neighbouring monomers.
  • PDA vesicle size distribution and ( ⁇ -potential were measured using dynamic light scattering (DLS) using a Malvern Zetasizer Nano ZS.
  • DLS dynamic light scattering
  • the PDA sample was diluted in water by a factor of 10 (i.e. 0.1 mg/ml).
  • the z-average size of the PDA liposomes ranged from about 100 nm to about 400 nm.
  • the average ( ⁇ -potential of the PDA liposomes ranged between about - 30 mV to about -40 mV. Examples of the liposome sizes and zeta potentials are shown in Figure 1.
  • Polydiacetylene liposome 1A may be prepared according to the general process prepared in Example 1, wherein the diacetylene monomer is 6,8-tricosadiynoic acid and the solvent is chloroform.
  • the z-average size of PDA liposome 1A was 140.14 ⁇ 38.97 nm.
  • Polydiacetylene liposome IB may be prepared according to the general process prepared in Example 1, wherein the diacetylene monomer is 10,12-tricosadiynoic acid and the solvent is chloroform.
  • the z-average size of PDA liposome IB was 117.18 ⁇ 7.28 nm.
  • Polydiacetylene liposome 1C may be prepared according to the general process prepared in Example 1, wherein the diacetylene monomer is 6,8-nonadecadiynoic acid and the solvent is chloroform.
  • the z-average size of PDA liposome C was 325.50 ⁇ 42.80 nm.
  • Polydiacetylene liposome ID may be prepared according to the general process prepared in Example 1, wherein the diacetylene monomer is 10,12-pentacosadiynoic acid and the solvent is chloroform.
  • Polydiacetylene liposome IE may be prepared according to the general process prepared in Example 1, wherein the diacetylene monomer is 8,10-henicosadiynoic acid and the solvent is chloroform.
  • Polydiacetylene liposome IF may be prepared according to the general process prepared in Example 1, wherein the diacetylene monomer is 10,12-octadecadiynoic acid and the solvent is chloroform.
  • Polydiacetylene liposome 1G may be prepared according to the general process prepared in Example 1, wherein the diacetylene monomer is 4,6-heptadecadiynoic acid and the solvent is chloroform.
  • Polydiacetylene liposome IH may be prepared according to the general process prepared in Example 1, wherein the diacetylene monomer is 5,7-hexadecadiynoic acid and the solvent is chloroform.
  • DA monomer and a phospholipid e.g. l,2-dimyristoyl-5n-glycero-3- phosphocholine (DMPC)
  • a solvent e.g. chloroform
  • the solvent was evaporated by nitrogen for 60 minutes at room temperature (20 °C).
  • the DA monomer and phospholipid formed a thin film on the substrate.
  • the thin film of DA monomer and phospholipid was hydrated with 10 mL of deionised water at 80 °C under probe sonication for 10 minutes utilising a Branson Digital Sonifier (250 W, 20 kHz) with a 1/8” diameter tapered circular tip (Branson 101-148-062). Hydration produced liposomes at a concentration of 1 mg/mL. The liposomes were left to anneal for 16 hours at 4 °C. The DA/phospholipid liposomes were then subjected to photopolymerisation to form PDA/phospholipid liposomes by being exposed to 254 nm UV light to induce an addition reaction between diacetylene groups on neighbouring monomers.
  • Polydiacetylene/phospholipid liposome 2A may be prepared according to the general process prepared in Example 2, wherein the diacetylene monomer is 6,8- tricosadiynoic acid, the phospholipid is DMPC, and the solvent is chloroform.
  • Example 2b Synthesis of polydiacetylene/phospholipid liposome 2B
  • Polydiacetylene/phospholipid liposome 2B may be prepared according to the general process prepared in Example 2, wherein the diacetylene monomer is 10,12- tricosadiynoic acid, the phospholipid is DMPC, and the solvent is chloroform.
  • Polydiacetylene/phospholipid liposome 2C may be prepared according to the general process prepared in Example 2, wherein the diacetylene monomer is 6,8- nonadecadiynoic acid, the phospholipid is DMPC, and the solvent is chloroform.
  • Polydiacetylene/phospholipid liposome 2D may be prepared according to the general process prepared in Example 2, wherein the diacetylene monomer is 10,12- pentacosadiynoic acid, the phospholipid is DMPC, and the solvent is chloroform.
  • Polydiacetylene/phospholipid liposome 2E may be prepared according to the general process prepared in Example 2, wherein the diacetylene monomer is 8,10- henicosadiynoic acid, the phospholipid is DMPC, and the solvent is chloroform.
  • Polydiacetylene/phospholipid liposome 2F may be prepared according to the general process prepared in Example 2, wherein the diacetylene monomer is 10,12- octadecadiynoic acid, the phospholipid is DMPC, and the solvent is chloroform.
  • Polydiacetylene/phospholipid liposome 2H may be prepared according to the general process prepared in Example 2, wherein the diacetylene monomer is 4,6- heptadecadiynoic acid, the phospholipid is DMPC, and the solvent is chloroform.
  • Polydiacetylene/phospholipid liposome 2H may be prepared according to the general process prepared in Example li, wherein the diacetylene monomer is 5,7- hexadecadiynoic acid, the phospholipid is DMPC, and the solvent is chloroform.
  • Example 3 General process for the preparation of PDA/phospholipid/cholesterol liposomes via thin film hydration method
  • DA monomer, a phospholipid (e.g. DMPC), and cholesterol were dissolved in 500 pL of a solvent (e.g. chloroform) at 25 °C, to produce a solution of DA monomer, phospholipid, and cholesterol in the solvent.
  • a solvent e.g. chloroform
  • the solvent was evaporated by nitrogen for 60 minutes at room temperature (20 °C).
  • the DA monomer, phospholipid, and cholesterol formed a thin film on the substrate.
  • the thin film of DA monomer, phospholipid, and cholesterol was hydrated with 10 mL of deionised water at 80 °C under probe sonication for 10 minutes utilising a Branson Digital Sonifier (250 W, 20 kHz) with a 1/8” diameter tapered circular tip (Branson 101- 148-062). Hydration produced liposomes at a concentration of 1 mg/mL. The liposomes were left to anneal for 16 hours at 4 °C. The DA/phospholipid/cholesterol liposomes were then subjected to photopolymerisation to form PDA/phospholipid/cholesterol liposomes by being exposed to 254 nm UV light to induce an addition reaction between diacetylene groups on neighbouring monomers.
  • Polydiacetylene/phospholipid/cholesterol liposome 3A may be prepared according to the general process prepared in Example 3, wherein the diacetylene monomer is 6,8-tricosadiynoic acid, the phospholipid is DMPC, and the solvent is chloroform.
  • Polydiacetylene/phospholipid/cholesterol liposome 3B may be prepared according to the general process prepared in Example 3, wherein the diacetylene monomer is 10,12-tricosadiynoic acid, the phospholipid is DMPC, and the solvent is chloroform.
  • Polydiacetylene/phospholipid/cholesterol liposome 3C may be prepared according to the general process prepared in Example 3 wherein the diacetylene monomer is 6,8-nonadecadiynoic acid, the phospholipid is DMPC, and the solvent is chloroform.
  • Polydiacetylene/phospholipid/cholesterol liposome 3D may be prepared according to the general process prepared in Example 3, wherein the diacetylene monomer is 10,12-pentacosadiynoic acid, the phospholipid is DMPC, and the solvent is chloroform.
  • Polydiacetylene/phospholipid/cholesterol liposome 3E may be prepared according to the general process prepared in Example 3, wherein the diacetylene monomer is 8,10-henicosadiynoic acid, the phospholipid is DMPC, and the solvent is chloroform.
  • Polydiacetylene/phospholipid/cholesterol liposome 3F may be prepared according to the general process prepared in Example 3, wherein the diacetylene monomer is 10,12-octadecadiynoic acid, the phospholipid is DMPC, and the solvent is chloroform.
  • Polydiacetylene/phospholipid/cholesterol liposome 3G may be prepared according to the general process prepared in Example 3, wherein the diacetylene monomer is 4,6-heptadecadiynoic acid, the phospholipid is DMPC, and the solvent is chloroform.
  • Polydiacetylene/phospholipid/cholesterol liposome 3H may be prepared according to the general process prepared in Example 3, wherein the diacetylene monomer is 5,7-hexadecadiynoic acid, the phospholipid is DMPC, and the solvent is chloroform.
  • DA monomer (8 mg) was dissolved in 250 pl of polar organic solvent (e.g. ethanol), to produce a solution of DA monomer in solvent at a concentration of 32 mg/ml).
  • the solution was injected into a vial containing 10 ml of water at flow rate of 100 pl/min. During the solution injection process, the solution was maintained at 80 °C under constant stirring at 300 rpm. After the injection step was complete, the DA/ethanol/water mixture was incubated for further 30 minutes at 80 °C to allow for ethanol evaporation. The self-assembled DA liposomes were allowed to cool to room temperature and then stored at 4 °C for 24 hours. Photo-induced polymerisation was carried out under 254 nm UV irradiation (UV lamp LF-206.LS, 6W, UVfTEC, UK) for 10 minutes to obtain blue-phase PDA liposomes.
  • UV irradiation UV lamp LF-206.LS, 6W, UVfTEC, UK
  • the data were analysed using the general purpose non-negative least squares (NNLS) fitting algorithm, with a size range analysis of 0.4-10000 nm.
  • the liposomes’ size and standard deviation were calculated from the intensity distributions.
  • Zeta potential measurements were collected using a disposable folded capillary cell (DTS1070), containing 0.6 ml of the sample without dilution.
  • DTS1070 disposable folded capillary cell
  • Different amphiphilic DA monomer building blocks self-assembled to form vesicles of a characteristic size with their diameters ranging from about 130 nm to about 800 nm and zeta-potentials ranging from about -25 mV to about -30 mV.
  • Polydiacetylene liposome 4A may be prepared according to the general process prepared in Example 4, wherein the diacetylene monomer 10,12-pentacosadiynoic acid and the solvent is ethanol.
  • the z-average size of PDA liposome 4A was 127.2 ⁇ 58.3 nm and the zeta potential was -32.2 ⁇ 8.6 mV.
  • Polydiacetylene liposome 4B may be prepared according to the general process prepared in Example 4, wherein the diacetylene monomer is 10,12-tricosadiynoic acid and the solvent is ethanol.
  • the z-average size of PDA liposome 4B was 184.1 ⁇ 59.9 nm and the zeta potential was -29.0 ⁇ 6.8 mV.
  • Polydiacetylene liposome 4C may be prepared according to the general process prepared in Example 4, wherein the diacetylene monomer is 10,12-octadecadiynoic acid and the solvent is ethanol.
  • the z-average size of PDA liposome 4C was 800.3 ⁇ 237.1 nm and the zeta potential was -26.9 ⁇ 7.5 mV.
  • Example 4d Synthesis of polydiacetylene liposome 4D
  • Polydiacetylene liposome 4D may be prepared according to the general process prepared in Example 4, wherein the diacetylene monomer is 8,10-henicosadiynoic acid and the solvent is ethanol.
  • the z-average size of PDA liposome 2D was 281.9 + 91.3 nm and the zeta potential was -26.7 ⁇ 6.7 mV.
  • Polydiacetylene liposome 4E may be prepared according to the general process prepared in Example 4, wherein the diacetylene monomer is 6,8-nonadecadiynoic acid and the solvent is ethanol.
  • the z-average size of PDA liposome 4E was 456.1 + 130.7 and the zeta potential was -32.0 ⁇ 7.1 mV.
  • Polydiacetylene liposome 4F may be prepared according to the general process prepared in Example 4, wherein the diacetylene monomer is 6,8-tricosadiynoic acid and the solvent is ethanol.
  • Polydiacetylene liposome 4G may be prepared according to the general process prepared in Example 4, wherein the diacetylene monomer is 4,6-heptadecadiynoic acid and the solvent is ethanol.
  • Polydiacetylene liposome 4H may be prepared according to the general process prepared in Example 4, wherein the diacetylene monomer is 5,7-hexadecadiynoic acid and the solvent is ethanol.
  • DA monomer was dissolved in 250 pl of polar organic solvent (e.g. ethanol).
  • 2 mg of phospholipid e.g DMPC
  • 250 pl of polar organic solvent e.g. ethanol
  • the mixed solution was then injected (100 pl/min for 5 min) via pipette into 10 ml of water which was being heated at 80 °C and mixed at 300 rpm in a thermomixer. After the injection step was complete, the solution containing self-assembled diacetylene/phospholipid vesicles was left at 80 °C for a further 30 minutes to allow evaporation of residual ethanol.
  • the vial was then wrapped in foil and placed in the refrigerator at 4 °C for 24 hours to allow the vesicles to anneal.
  • Photo-induced polymerisation was carried out under 254 nm UV irradiation (UV lamp LF-206.LS, 6W, UVITEC, UK) for 10 minutes at a distance of 15 cm from lamp base to solution surface was then performed to produce PDA/phospholipid liposomes.
  • Polydiacetylene/phospholipid liposome 5A may be prepared according to the general process prepared in Example 5, wherein the diacetylene monomer is 6,8- tricosadiynoic acid, the phospholipid is DMPC, and the solvent is ethanol.
  • Polydiacetylene/phospholipid liposome 5B may be prepared according to the general process prepared in Example 5, wherein the diacetylene monomer is 10,12- tricosadiynoic acid, the phospholipid is DMPC, and the solvent is chloroform.
  • TEM micrographs were obtained using a JEOL 1400 TEM (JEOL, Akishima, Japan) operating at an accelerating voltage of 100 keV. Samples were ultra- sonicated, and then drop cast onto formv ar- supported copper grids (ProSciTech) without staining. The size and morphology of the nanoparticles were recorded using a Phurona CCD Camera (Emsis) and Radius software (Emsis). TEM analysis (Fig.2) confirmed that the particles were of the same morphology as PDA/DMPC vesicles constructed via the thin film hydration method as described in Example 1.
  • Polydiacetylene/phospholipid liposome 5C may be prepared according to the general process prepared in Example 5, wherein the diacetylene monomer is 6,8- nonadecadiynoic acid, the phospholipid is DMPC, and the solvent is ethanol.
  • Polydiacetylene/phospholipid liposome 2M may be prepared according to the general process prepared in Example 5, wherein the diacetylene monomer is 10,12- pentacosadiynoic acid, the phospholipid is DMPC, and the solvent is ethanol.
  • Example 5e Synthesis of polydiacetylene/phospholipid liposome 5E
  • Polydiacetylene/phospholipid liposome 5E may be prepared according to the general process prepared in Example 5, wherein the diacetylene monomer is 8,10- henicosadiynoic acid, the phospholipid is DMPC, and the solvent is ethanol.
  • Polydiacetylene/phospholipid liposome 5F may be prepared according to the general process prepared in Example 5, wherein the diacetylene monomer is 10,12- octadecadiynoic acid, the phospholipid is DMPC, and the solvent is ethanol.
  • Polydiacetylene/phospholipid liposome 5G may be prepared according to the general process prepared in Example 5, wherein the diacetylene monomer is 4,6- heptadecadiynoic acid, the phospholipid is DMPC, and the solvent is ethanol.
  • Polydiacetylene/phospholipid liposome 5H may be prepared according to the general process prepared in Example 5, wherein the diacetylene monomer is 5,7- hexadecadiynoic acid, the phospholipid is DMPC, and the solvent is ethanol.
  • Example 6 Detection of q-Hemolysin a-hemolysin powder was hydrated with 0.01 M phosphate buffered- saline (PBS) to make a 0.5 mg/mL stock solution. 100 pL of the polydiacetylene liposome 1A synthesised according to Example la was mixed with the stock a-hemolysin solution to produce a solution with final a-hemolysin concentration of 12.5 pg/mL.
  • PBS phosphate buffered- saline
  • Example 2a 100 pL of the polydiacetylene/phospholipid liposome 2A synthesised according to Example 2a was mixed with the stock a-hemolysin solution to produce a solution with final a-hemolysin concentration of 12.5 pg/mL.
  • Example 3a 100 pL of the polydiacetylene/phospholipid/cholesterol liposome 3A synthesised according to Example 3a was mixed with the stock a-hemolysin solution to produce a solution with final a-hemolysin concentration of 12.5 pg/mL.
  • Example 6a Liposomes synthesised according to Example 3a selectivity
  • LPS lipopolysaccharide
  • Liposomes synthesised according to Example 3a were incubated with 12.5 Listeriolysin O toxin (LLO) (100 pg/mL) and no colour change in the sensor was detected. This was surprising, however the action of pore-forming toxins is known to require specific lipid compositions or binding sites.
  • LLO Listeriolysin O toxin
  • Liposomes synthesised according to Example 3a were exposed to other known biomarkers for mastitis in bovine milk including FFA (octanoic acid, 0.5 pg/mL) and lactic acid (3 mM) and no colour change in the liposomes was observed.
  • FFA octanoic acid, 0.5 pg/mL
  • lactic acid 3 mM
  • Example 6b Liposomes synthesised according to Example 3a morphology pre and post a-hemolysin incubation
  • TEM micrographs were taken of liposomes synthesised according to Example la and Example 3a before and after the addition of alpha-hemolysin (12.5 pg/mL).
  • polydiacetylene/phospholipid liposomes 5A-5H prepared according to examples 5a-5h are referred to as PDA based liposomes.
  • a 6 wt% agarose solution was prepared by dissolving agarose (600 mg) in deionised water (10 ml) under constant stirring at 60 °C for 1 hour. 2 ml of the 6 wt% agarose solution was mixed with 2 ml of a perishable consumable product (e.g milk) and 2 ml of PDA based liposomes (1 mg/ml). The resultant mixture had a constitution of 0.33 mg/ml PDA based liposome, 33 vol% milk, and 2 wt% agarose. 400 pl of the mixture was cast into the cap of a glass vial and placed in the fridge for 1 hour to allow the gel to set forming the PDA based liposome hydrogels.
  • a perishable consumable product e.g milk
  • PDA based liposomes 1 mg/ml
  • PDA based liposome hydrogel 7 A may be prepared according to the general process prepared in Example 7, wherein the PDA based liposome is prepared according to Example 5a.
  • PDA based liposome hydrogel 7B may be prepared according to the general process prepared in Example 7, wherein the PDA based liposome is prepared according to Example 5b.
  • PDA based liposome hydrogel 7C may be prepared according to the general process prepared in Example 7, wherein the PDA based liposome is prepared according to Example 5c.
  • PDA based liposome hydrogel 7D may be prepared according to the general process prepared in Example 7, wherein the PDA based liposome is prepared according to Example 5d.
  • PDA based liposome hydrogel 7E may be prepared according to the general process prepared in Example 7, wherein the PDA based liposome is prepared according to Example 5e.
  • PDA based liposome hydrogel 7F may be prepared according to the general process prepared in Example 7, wherein the PDA based liposome is prepared according to Example 5f.
  • PDA based liposome hydrogel 7G may be prepared according to the general process prepared in Example 7, wherein the PDA based liposome is prepared according to Example 5g.
  • PDA based liposome hydrogel 7H may be prepared according to the general process prepared in Example 7, wherein the PDA based liposome is prepared according to Example 5h.
  • Example 7b To make quality tags that mimic the spoilage kinetics of milk, two PDA based liposome hydrogel were generated according to Example 7b. To study the colour change behaviour of the hydrogels, one sample set was refrigerated at 4°C and the other was stored at 20 °C for a period of 48 hours.
  • the blue to red colour change of the samples stored at 20 °C is due to the insertion of free fatty acids into the PDA membrane.
  • Free fatty acids are a product of fat rancidification by bacterial lipolysis.
  • the hydrogel acts as a quasi-liquid and does not impede or alter the spoilage of milk fats.
  • the tags were first dehydrated in a vacuum to 13.8 ⁇ 1.4% of their original weight under refrigeration. The dehydrated tags were then subject to the same experimental conditions used to compare the spoilage kinetics of the PDA based liposome/milk solutions and PDA based liposome hydrogels (Fig. 5). One sample set of the dehydrated gels was refrigerated keeping the milk ingredients fresh. In parallel with this experiment, another sample set was stored at 20 °C, allowing the milk ingredients to spoil. Both the refrigerated and unrefrigerated dehydrated gels did not change colour over 48 hours (Fig. 6a, c).
  • a set of dehydrated gels were then rehydrated to see if increasing the water content could “switch” the gels back “on”.
  • the dehydrated gels were submerged in a small Petri dish containing 5 ml of water and left to soak for 6 hours under refrigeration.
  • the gels were then weighed and found to have returned to 74.4 ⁇ 3.9% of their original weight before dehydration.
  • the gels were then subject to refrigerated and unrefrigerated conditions for 48 hours.
  • the rehydrated gels that were refrigerated remained blue for the 48 hour period. It was found that the encapsulated milk was not expected to spoil while refrigerated.

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Abstract

La présente divulgation se rapporte, de manière générale, à une formulation de capteur colorimétrique permettant à la détection de contaminants chimiques et biologiques à la fois en phase liquide et en phase vapeur. En particulier, la présente divulgation se rapporte, de manière générale, à une formulation de capteur colorimétrique comprenant une composition de polymère de diacétylène, et à des procédés de détection d'un contaminant chimique et biologique à l'aide de telles formulations, y compris diverses applications de ces dernières.
PCT/AU2021/051069 2021-09-16 2021-09-16 Capteurs colorimétriques permettant la détection de contaminants chimiques et biologiques WO2023039625A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060172371A1 (en) * 2003-07-10 2006-08-03 Ben-Gurion University Of The Negev Research And Development Authority Polydiacetylene-containing solid colorimetric and/or fluorescent detector, method for its preparation and uses thereof
WO2013049525A1 (fr) * 2011-09-30 2013-04-04 Cirle Emballages ou autres matériaux comportant un biocapteur et leurs procédés d'utilisation

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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