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WO2025022116A1 - Hydrogels pour la liaison à des protéines, leurs formulations et utilisations - Google Patents

Hydrogels pour la liaison à des protéines, leurs formulations et utilisations Download PDF

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Publication number
WO2025022116A1
WO2025022116A1 PCT/GB2024/051945 GB2024051945W WO2025022116A1 WO 2025022116 A1 WO2025022116 A1 WO 2025022116A1 GB 2024051945 W GB2024051945 W GB 2024051945W WO 2025022116 A1 WO2025022116 A1 WO 2025022116A1
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Prior art keywords
hydrogel
suitably
proteins
protein
monomers
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Ruchi Gupta
Nicholas John Goddard
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Cancer Research Technology Ltd
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Cancer Research Technology Ltd
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    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • 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
    • C09D133/00—Coating compositions based on homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by only one carboxyl radical, or of salts, anhydrides, esters, amides, imides, or nitriles thereof; Coating compositions based on derivatives of such polymers
    • C09D133/24—Homopolymers or copolymers of amides or imides
    • C09D133/26—Homopolymers or copolymers of acrylamide or methacrylamide

Definitions

  • Novel hydrogels that are capable of binding to proteins, and additionally concentrating, labelling and releasing said labelled and concentrated proteins, are provided.
  • Corresponding formulations, oral formulations, biomaterials, uses, methods and kits of parts are also provided.
  • ELISA enzyme-linked immunosorbent assay
  • ELISA uses two antibodies where a capture antibody allows a target protein to be pulled out of samples and a detection antibody is labelled with an enzyme. A substrate is then added, which is acted upon by the enzyme to produce a fluorescent or coloured product. The fluorescence intensity or optical absorbance is proportional to the concentration of the target protein. Furthermore, as each enzyme can act upon multiple substrate molecules, the fluorescent or absorbance signal is amplified, allowing measurement of low abundance proteins.
  • ELISA requires multiple adding and washing steps, and hence is laborious.
  • ELISA has been implemented on microfluidic paper-based analytical devices by controlling the flow rate and hence arrival times of reagents to the detection zone 3 .
  • the flow control requires patterning of paper and multiple layers which must be precisely aligned.
  • label-free biosensors have been used to measure low abundance proteins.
  • label-free biosensors 4 require only capture antibodies and rely on changes in refractive index 5 ' 7 or impedance 8,9 or mass 10,11 to determine the concentration of target proteins.
  • the present invention aims to address one or more of the above-mentioned problems in the art.
  • the inventors have surprisingly found a novel hydrogel capable of pre-concentration, fluorescent labelling, and light-triggered release of proteins which can be used in assays to detect proteins with far fewer steps than ELISA based assays.
  • the pre-concentration is achieved by covalent capture of proteins in the hydrogels with a much smaller volume of sample than is required for currently available assays, enabling either direct use on patients, for example in the mouth to absorb a saliva sample, or in vitro, for example in a urine sample.
  • the covalent capture of proteins occurs as a result of the reaction between primary amines in proteins with a fluorescent group such as the isothiocyanate group in fluorescein isothiocyanate (FITC)17, 18 present in the hydrogels.
  • FITC fluorescein isothiocyanate
  • FITC is fluorescent
  • proteins are pre-concentrated and labelled in a single step.
  • the FITC is attached to the hydrogel’s backbone via a photolabile group, o-nitrobenzyl 19 ' 25 , which exhibits controllable photoreactions with tunable absorption for wavelengths >300 nm 23,25 .
  • the release of the fluorogenic labelled proteins can be triggered through the irradiation of the hydrogels with UV light (365 nm).
  • the released proteins can then be captured and measured using standard fluorescence detection techniques.
  • streptavidin was initially used as an exemplar protein.
  • the streptavidin used in the examples was tagged with rhodamine so that fluorescence of rhodamine can be used to determine the protein concentration before and after incubation with the hydrogels. This in turn provided the pre-concentration factor.
  • the designed hydrogels offered a preconcentration factor of up to 295. Equally, the rhodamine label was used to study the light- triggered release kinetics of streptavidin from the hydrogels.
  • the inventors showed that 50% of streptavidin was released from the hydrogels in -100 s, in general at least 50% of all proteins are released very quickly from the hydrogels in under 10 minutes. Finally, the inventors showed that released streptavidin was labelled with 85 fluorescein molecules per one molecule of the protein.
  • the designed hydrogel when combined with capture of released streptavidin using biotin and fluorescence detection, allowed detection of very low concentrations of at least 0.0033 ppm (or ⁇ 60 pM) of the protein in a sample.
  • hydrogel of the present invention can be used to achieve pre-concentration, labelling, and controlled release of many different types of proteins, including proteins which may be indicative of disease.
  • the designed hydrogel when combined with capture of released CRP and fluorescence detection, allowed detection of very low concentrations of at least 0.0022 ppm (or -19 pM) of the protein in a sample.
  • the designed hydrogel when combined with capture of released IL6 and fluorescence detection, allowed detection of very low concentrations of at least 0.005 ppm (or -240 pM) of the protein in a sample.
  • the hydrogels do not require secondary antibodies, enzyme labels, and substrates nor do they require multiple adding/washing steps which are required for traditional ELISA assays.
  • a hydrogel comprising a polymer formed of a plurality of inactive monomers and a plurality of active monomers, wherein each active monomer comprises at least one fluorophore capable of covalently binding to a protein, wherein each fluorophore is attached to the active monomer by a cleavable bond.
  • the plurality of inactive monomers is selected from the group consisting of polyethylene glycol (PEG), acrylamide, N-isopropylacrylamide, methacrylamide, methacrylate, and PEG bis-azide.
  • PEG polyethylene glycol
  • acrylamide acrylamide
  • N-isopropylacrylamide methacrylamide
  • methacrylate methacrylate
  • PEG bis-azide PEG bis-azide
  • the plurality of active monomers is selected from the group consisting of polyethylene glycol acrylamide, polyethylene glycol N-isopropylacrylamide, polyethylene glycol methacrylamide, polyethylene glycol methacrylate, allylamide, and PEGwoo-azide.
  • the at least one fluorophore is selected from the group consisting of fluorescein, fluorescein isothiocyanate (FITC), eosin Y, eosin B, tetrachlorofluorescein, carbofluoresceins, naphthofluoresceins, and (semi)naphthofluoresceins with suitable protein reactive groups.
  • the cleavable bond is o-nitrobenzyl.
  • the plurality of inactive monomers are acrylamide
  • the plurality of active monomers are polyethylene glycol methacrylamide
  • each polyethylene glycol methacrylamide monomer comprises at least one fluorescein isothiocyanate capable of covalently binding to a protein, wherein the (molar) ratio of inactive monomers to active monomers is between 100 to 1 and 5 to 1, and wherein each fluorescein isothiocyanate is attached to said polyethylene glycol methacrylamide monomer by a cleavable bond which is suitably o-nitrobenzyl.
  • a formulation comprising a core and a shell
  • the core comprises the hydrogel of any one of the preceding aspects and embodiments
  • the shell comprises a hydrogel having a pore size of less than 30 nm.
  • an oral formulation comprising the hydrogel according to any of the preceding aspects and embodiments is provided, or the formulation according to the preceding aspect is provided.
  • the oral formulation is a pill, tablet, capsule, granule, troch, lozenge, a lollipop, or sampling material.
  • the oral formulation is a disc.
  • a disc comprising the hydrogel of the invention.
  • a sampling device comprising the hydrogel according to any one of the preceding aspects and embodiments, the formulation of the preceding aspects and embodiments, or the oral formulation according of the preceding aspects and embodiments, wherein the sampling device comprises a test tube.
  • an inert substrate comprising a coating thereon, wherein the coating comprises the hydrogel of the invention.
  • the coating is a film.
  • a container comprising an inner surface operable to be contacted with a reaction mixture, wherein the inner surface is coated with a base layer upon which is coated a reactive layer, the reactive layer comprising a mixture of a binding molecule and a blocking agent, wherein the blocking agent comprises a compound which does not contain amine groups.
  • the base layer is a polymer comprising free amine groups.
  • such polymers are, for example acrylamide/bisacrylamide optionally copolymerised with aminopropyl methacrylamide, or 4 arm PEG succinimidyl ester (NHS) copolymerised with PEG-bis amine, or chitosan.
  • the base layer is chitosan.
  • the binding molecule is defined hereinbelow, and is a protein which recognises and binds to a protein of interest, for example biotin or an antibody, suitably an antibody or a binding fragment thereof which specifically binds to the protein of interest.
  • the blocking agent comprises PEG-methyl.
  • the capture protein and the blocking agent bind to the base layer.
  • the reactive layer comprises a majority of binding molecules, and a minority of blocking agent.
  • the blocking agent is bound to the base layer only where the binding molecule is not bound to the base layer.
  • the blocking agent binds to any free amine groups in the base layer.
  • the blocking agent prevents free FITC binding to the base layer of the container, suitably when in use.
  • the container may be any container suitable for carrying out a method of the invention therein.
  • the container is a microwell or microtitre plate.
  • kit-of-parts comprising: a. the hydrogel according to any one of the preceding aspects and embodiments, the formulation according to any one of the preceding aspects and embodiments, the oral formulation (optionally which is a disc) according to any one of the preceding aspects and embodiments, the sampling device according to any one of the preceding aspects and embodiments, or the substrate according to any one of the preceding aspects and embodiments,; and b. instructions for use.
  • kit may further comprise the container above.
  • a biomaterial comprising the hydrogel according to any one of the preceding aspects and embodiments or comprising the formulation according to any previous aspect and embodiment is provided.
  • hydrogel according to any one of the preceding aspects and embodiments, or a formulation or an oral formulation (optionally which is a disc) according to any one of the preceding aspects and embodiments, or the sampling device according to any one of the preceding aspects and embodiments, or the substrate according to any one of the preceding aspects and embodiments, or the container according to any one of the preceding aspects and embodiments, for concentrating and labelling proteins in a sample.
  • the hydrogel according to any one of the preceding aspects and embodiments, or a formulation or oral formulation (optionally which is a disc) according to any one of the preceding aspects and embodiments, or the sampling device according to any one of the preceding aspects and embodiments, or the substrate according to any one of the preceding aspects and embodiments, or the container according to any one of the preceding aspects and embodiments, for use in a method of diagnosing a disease or disorder is provided.
  • a method of concentrating and labelling proteins in a sample is provided, the method comprising: a.
  • a method of detecting proteins in a sample comprising: a. Contacting a sample with the hydrogel of any one of the preceding aspects and embodiments, or the formulation of any of any one of the preceding aspects and embodiments, or the oral formulation (optionally which is a disc) according to any one of the preceding aspects and embodiments, or the sampling device according to any one of the preceding aspects and embodiments, or the substrate according to any one of the preceding aspects and embodiments, under suitable conditions to allow proteins in the sample to bind to the hydrogel via the fluorophore, thereby obtaining fluorescently labelled proteins bound to said hydrogel; b.
  • step (a) Exposing the hydrogel of step (a) to a cleavage inducer under suitable conditions to cleave the cleavable bonds, thereby releasing said fluorescently labelled proteins from the hydrogel; and c. Determining the presence of proteins in the sample, wherein the presence of fluorescence is indicative of the presence of proteins in the sample.
  • a method of measuring a protein of interest in a sample comprising: a. Contacting a sample with the hydrogel of any one of the preceding aspects and embodiments or the formulation of any one of the preceding aspects and embodiments or the oral formulation (optionally which is a disc) according to any one of the preceding aspects and embodiments, or the sampling device according to any one of the preceding aspects and embodiments, or the substrate according to any one of the preceding aspects and embodiments, under suitable conditions to allow proteins in the sample to bind to the hydrogel via the fluorophore, thereby obtaining fluorescently labelled proteins bound to said hydrogel; b.
  • step (a) Exposing the hydrogel of step (a) to a cleavage inducer under suitable conditions to cleave the cleavable bonds, thereby releasing said fluorescently labelled proteins from the hydrogel; c. Isolating the fluorescently labelled proteins; d. Contacting the fluorescently labelled proteins with a binding molecule capable of specifically binding to a protein of interest; e. Removing any unbound fluorescently labelled proteins; and f. Measuring the level of fluorescence, wherein the level of fluorescence is indicative of the amount of the protein of interest in the sample.
  • the protein of interest is a biomarker.
  • the biomarker is C-reactive protein (CRP), IL-6, IL-8, or cardiac troponin.
  • CRP C-reactive protein
  • IL-6 IL-6
  • IL-8 cardiac troponin
  • a method of determining whether a subject has a disease or disorder comprising: a. Contacting a sample from the subject with the hydrogel of any one of the preceding aspects and embodiments, or the formulation of any one of the preceding aspects and embodiments, or the oral formulation (optionally which is a disc) according to any one of the preceding aspects and embodiments, or the sampling device according to any one of the preceding aspects and embodiments , or the substrate according to any one of the preceding aspects and embodiments, under suitable conditions to allow proteins in the sample to bind to the hydrogel via the fluorophore, thereby obtaining fluorescently labelled proteins bound to said hydrogel; b.
  • step (a) Exposing the hydrogel of step (a) to a cleavage inducer under suitable conditions to cleave the cleavable bonds, thereby releasing fluorescently labelled proteins from the hydrogel; c. Isolating the fluorescently labelled proteins; d. Contacting the fluorescently labelled proteins with a binding molecule capable of specifically binding to a protein biomarker of a disease or disorder; e. Removing any unbound fluorescently labelled proteins; f. Detecting the presence of fluorescence or measuring the level of fluorescence, wherein the presence of fluorescence is indicative of the presence of the protein biomarker in the sample, or wherein the level of fluorescence is indicative of the level of the protein biomarker in the sample, g. Determining based on f. that the subject has a disease or disorder, wherein the presence of fluorescence or the level of fluorescence is indicative of a disease or disorder.
  • the disease or disorder is cancer or a cardiovascular disease.
  • the cleavage inducer is UV light, or the cleavage inducer is selected from esterase or reducing agents.
  • the substrate according to any one of the preceding aspects and embodiments, or the disc according to any one of the preceding aspects and embodiments may be contacted with a sample from the subject.
  • the container of the invention is used.
  • the step of contacting the fluorescently labelled proteins with a binding molecule capable of specifically binding to a protein of interest is used.
  • Figure 1 Schematic showing pre-concentration, labelling and release of an exemplar protein, rhodamine-streptavidin, followed by fluorescence detection in a biotin coated microtiter plate.
  • Figure 2 Reaction schemes for the synthesis of monomers F-NVOC-allylamide, F-NVOC- PEG4oo-methacrylamide, F-NVOC-PEG 3400-methacrylamide, and FITC-NVOC-PEG3400- methacrylamide.
  • Figure 3 Chemical structures of monomers (rhombus, double star, and single star are groups for protein capture, fluorescent labelling, and light triggered release, respectively, circle is PEG, resulting in water soluble monomers, and rectangle is allylamide or methacrylamide, allowing monomers to be co-polymerised with acrylamide/bisacrylamide to obtain hydrogels).
  • Figure 4 (a) HPLC waterfall plot of F-PEG-NVOC-PEG34oo-methacrylamide showing increasing release of fluorescein (peak F) and decrease of starting reagent (peak M) as a function of irradiation time, and (b) rates of the light induced release of fluorescein from monomers with increasing irradiation time, where [M] t is the concentration of monomer as a function of irradiation time and [M]o is the starting monomer concentration.
  • Figure 5 Images of a hydrogel film on glass under (a) white, and (b) 365 nm light with a £1 coin as a size reference (distance across flats 22.5 mm), (c) UV-Vis spectra of the four different corners of the hydrogel film and (d) graph showing monomer incorporation factors, which is the ratio of the molar concentrations of the NVOC containing monomers in hydrogels (m hy drogei) and precursor (m pr ecursor) solutions.
  • Figure 6 a) UV-Vis spectra of a hydrogel film (co-polymer of acrylamide/bisacrylamide and F- NVOC- PEG34oo-methacrylam ide) with increasing irradiation times, and b) first-order reaction rate plot of the release of fluorescein from this hydrogel.
  • Figure 7 Emission spectra of (a) 0.1 ppm and (b) 0.01 ppm rhodamine-streptavidin (RS) solutions before and after overnight incubation with unfunctionalized and functionalized hydrogels, and (c) RS concentrations in solution and hydrogel.
  • RS rhodamine-streptavidin
  • Figure 8 (a) Emission spectra of RS (excitation wavelength of 540 nm) released when hydrogels were irradiated with UV-light, (b) cumulative release of protein as a function of irradiation time, and (c) emission spectra of released after incubation with biotin coated microtiter plates and buffer wash (peaks shown at excitation wavelengths of 470 and 540 nm).
  • Figure 12 UV-Vis spectra of 40 pM F-NVOC-PEG34oo-methacrylamide monomer in PBS with increasing irradiation time
  • Figure 13 HPLC waterfall plots of 1 mg/ml (a) F-NVOC-allylamide and (b) F-NVOC-PEG400- methacrylamide dissolved in acetonitrile showing increase in fluorescein (F) and decrease in photolabile monomer (M) concentrations for 0 to 30 min of irradiation time
  • Figure 14 Plot showing monomer concentration, determined by the peak area corresponding to M in chromatograms and molar extinction coefficient of monomer solutions, as a function of irradiation time
  • Figure 17 Absorption spectra of hydrogel films made using 10% (w:v) precursor solution (a) after an overnight wash in PBS, and (b) subsequent storage in PBS for 1 , 3, 5 and 7 days
  • Figure 18 Absorption spectra of hydrogel films made using 5% (w:v) precursor solution (a) after an overnight wash in PBS, and (b) subsequent storage in PBS for 1 , 3, 5 and 7 days
  • Figure 19 (a) Fluorescence spectra of 0.005 ppm RS solution before and after incubation with a hydrogel disc for 24 h and (b) RS calibration curve
  • Figure 20 Effect of varying the active to inactive monomer molar ratio on the release of RS (hydrogel disc was incubated in 0.005 ppm RS for 24 h)
  • FIG 21 Fluorescence at peak wavelength of PBS solutions used to immerse hydrogel discs while they are exposed to 365 nm light for different durations (hydrogel discs had been incubated in 10 mL of 0.1 ppm RS for 1 or 15 or 24 or 48 h)
  • Figure 22 Fluorescence at peak wavelength of PBS solutions used to immerse hydrogel discs while they are exposed to 365 nm light for different durations (hydrogel discs had been incubated in 10 mL of 0.01 ppm RS for 1 or 15 or 24 h)
  • Figure 23 Fluorescence at peak wavelength of PBS solutions used to immerse hydrogel discs while they are exposed to 365 nm light for different durations (hydrogel discs had been incubated in 10 mL of 0.005 ppm RS for 1 or 15 or 24 h)
  • Figure 24 Peak fluorescence intensity of released FITC in a commercial biotinylated microtitre plate as a function of exposure time
  • Figure 25 Peak fluorescence intensity of bound FITC for two concentrations of the NHS- PEG-methyl blocking reagent in the in-house developed biotinylated microtitre plate as a function of exposure time
  • Figure 26 Fluorescence at peak wavelength of PBS solutions used to immerse hydrogel discs versus exposure time for two different wash times (hydrogel discs had been incubated in 10 mL of 0.005 ppm RS for 24 h)
  • Figure 27 (a) Fluorescence signal from streptavidin bound to biotin coated in-house microtitre plates where streptavidin was pre-concentrated and released from polyacrylamide hydrogel, and streptavidin was labelled with FITC during the release process, and (b) the corresponding calibration curve (using data at 60 min exposure time)
  • Figure 28 (a) Fluorescence signal from CRP bound to anti-CRP coated in-house microtitre plates where CRP was pre-concentrated and released from polyacrylamide hydrogel, and CRP was labelled with FITC during the release process and (b) the corresponding calibration curve (using data at 60 min exposure time)
  • Figure 29 Fluorescence signal from IL8 bound to anti-IL8 coated in-house microtitre plates where IL8 was pre-concentrated and released from polyacrylamide hydrogel, and IL8 was labelled with FITC during the release process
  • Figure 37 (a) Fluorescence of solutions used to immerse gels exposed to 365 nm light for different durations and (b) plot of peak fluorescence intensity versus exposure time
  • Figure 38 Fluorescence spectra of PBS solutions (excitation wavelength was 540 nm) used to immerse hydrogel discs while they are exposed to 365 nm light for different durations (hydrogel discs had been incubated in 10 mL of 0.005 ppm RS for 24 h)
  • Figure 39 Fluorescence at peak wavelength of PBS solutions used to immerse hydrogel discs while they are exposed to 365 nm light for different durations (hydrogel discs had been incubated in 10 mL of 0.005 ppm RS for 1 or 15 or 24 h)
  • Figure 40 RS release kinetics from hydrogels comprising of different active: inactive monomer ratios (hydrogel discs were incubated in 10 mL 0.005 ppm RS solution for 24 h)
  • Figure 41 Fluorescence (excitation wavelength was 490 nm) of wells of microtitre plates after treatment with supernatants obtained after exposing PEG hydrogels to 365 nm light where different hydrogels were beforehand incubated in different solutions (i.e., streptavidin without and with mucin, buffer, mucin without streptavidin) for 24 h
  • Figure 42 Fluorescence (excitation wavelength was 490 nm) of wells of microtitre plates after treatment with supernatants obtained after exposing PEG hydrogels to 365 nm light where different hydrogels were beforehand incubated in different solutions (i.e., buffer, synthetic saliva, IL6 in buffer, and IL6 in synthetic saliva) for 24 h
  • a hydrogel comprising a polymer formed of a plurality of inactive monomers and a plurality of active monomers, wherein each active monomer comprises at least one fluorophore capable of covalently binding to a protein, wherein each fluorophore is attached to the active monomer by a cleavable bond.
  • a “hydrogel” is a hydrogel comprising a polymer formed from a plurality of inactive monomers and a plurality of active monomers.
  • the hydrogel comprises a plurality of polymers comprising both the active and inactive monomers, suitably which are cross-linked with a crosslinker to form an insoluble hydrophilic network.
  • the hydrogel is hydrophilic.
  • the hydrogel is insoluble in water.
  • Suitable crosslinkers are described elsewhere herein.
  • the active and inactive monomers are present in the polymer in a defined molar ratio. Said defined molar ratio is described elsewhere herein. The average length of the polymer chains is governed by the defined molar ratio of both the active and inactive monomers to the cross-linker.
  • an “inactive monomer” is a monomer which does not comprise a fluorophore bonded thereto via a cleavable bond, and suitably which does not comprise a fluorophore.
  • the “inactive monomer” is a monomer which does not comprise a fluorophore. Suitable inactive monomers are listed herein.
  • an “active monomer” is a monomer comprising at least one fluorophore bonded thereto via a cleavable bond. Suitable active monomers are listed herein.
  • crosslinker cross-links the polymers formed of inactive monomers and active monomers.
  • Suitable crosslinkers are bisacrylamide, polyethylene glycol diacrylamide, polyethylene glycol dimethacrylamide, polyethylene glycol diacrylate, polyethylene glycol dimethacrylate, or 4-arm PEG alkyne.
  • the crosslinker is bisacrylamide.
  • the crosslinker is 4-arm PEG alkyne, suitably as shown below:
  • n is between 5-20, suitably between 8-15, suitably between 9-13, suitably n is 11.
  • fluorophore refers to a compound, chemical group, or composition that is inherently fluorescent. Suitable fluorophores are listed herein.
  • covalently binding means that a covalent bond is formed by sharing of electrons between atoms.
  • the fluorophore covalently binds to the primary amines of the protein.
  • the isothiocyanate group of fluorescein isothiocyanate covalently binds to the primary amines in the protein.
  • the isothiocyanate group of fluorescein isothiocyanate covalently binds to the terminal amines in the protein.
  • one of the hydroxy groups of fluorescein isothiocyanate covalently binds to the hydrogel backbone via o-nitrobenzyl.
  • protein includes full length proteins, protein fragments, proteins in their native state or denatured proteins. Mixture of proteins can be a mixture of full length proteins, a mixture of protein fragments, or a mixture of full length proteins and protein fragments. Proteins can be acidic, neutral or basic.
  • the protein is streptavidin.
  • the protein is CRP.
  • the protein is IL6.
  • the protein is IL8.
  • proteins described herein also encompass low abundance proteins.
  • proteins described herein are biomarkers. Suitable protein biomarkers are described elsewhere herein. Suitable biomarkers include inflammatory proteins, such as chemokines and cytokines.
  • the protein may be a biomarker of inflammation.
  • biomarkers of inflammation may be indicative of disease.
  • the protein may be a chemokine or cytokine.
  • the protein may be an interleukin or a C-reactive protein. In some embodiments, the protein is IL6, IL8, or CRP.
  • proteins described herein can comprise additional functional groups such as fluorophores or chromophores.
  • the additional functional group is rhodamine.
  • the protein is streptavidin and the additional functional group is rhodamine.
  • the pore size of a hydrogel is defined elsewhere herein.
  • the hydrogel comprises at least two or more inactive monomers and at least two or more active monomers.
  • the hydrogel comprises up to 100, up to 1000, up to 10000, up to 100000, up to 1000000, up to 10000000 inactive monomers and up to 100, up to 1000, up to 10000, up to 100000, up to 1000000, up to 10000000 active monomers.
  • the molar ratio of inactive monomers: active monomers is between 100:1 and 5:1.
  • the molar ratio of inactive monomers to active monomers is 100: 1 , 40: 1 , 20: 1 , 10:1.
  • the ratio of inactive monomer: active monomer is 40:1.
  • the hydrogel is a synthetic hydrogel.
  • synthetic hydrogel refers to a hydrogel which does not comprise natural polymers such as proteins and/or polysaccharides.
  • the hydrogel additionally comprises natural polymers such as proteins and/or polysaccharides.
  • proteins are collagen/or gelatine.
  • polysaccharides are starch, alginate, dextran, chitosan, hyaluronic acid and agarose.
  • hydrogels of the invention, and any formulations, the inert substrate, or sampling devices thereof may be stored without any detrimental effects such as degradation, suitably they are stable.
  • the hydrogels of the invention, and any formulations, or sampling devices thereof may be stored for a period of at least 5 days, 6 days, 7 days, or more, optionally for 2 weeks, 3 weeks, 4 weeks.
  • hydrogels of the invention, and any formulations, or sampling devices thereof may be stored for at least 7 days.
  • hydrogels of the invention, and any formulations, or sampling devices thereof have an improved shelf-life of up to 5, 6, 7 days, or more.
  • the polymer comprised in the hydrogel of the invention is formed of a plurality of inactive monomers and a plurality of active monomers, polymerised together.
  • an inactive monomer is preferably a monomer which does not comprise a fluorophore.
  • the plurality of inactive monomers are selected from for example but not limited to polyethylene glycol, acrylamide, N-isopropylacrylamide, methacrylamide, methacrylate, and PEG bis-azide.
  • the plurality of inactive monomers substantially do not hydrolyse the cleavable bone (e.g. the o-nitrobenzyl-fluorescein carbonate link).
  • the plurality of inactive monomers are selected from the group consisting of polyethylene glycol, acrylamide, N-isopropylacrylamide, methacrylamide, methacrylate, and PEG bis-azide.
  • the plurality of inactive monomers are selected from the group consisting of acrylamide, N-isopropylacrylamide, methacrylamide, methacrylate and PEG bis-azide
  • the plurality of inactive monomers may comprise the same monomer, or different monomers.
  • the plurality of inactive monomers may comprise two or more, three or more, four or more etc. different inactive monomers selected from the list above.
  • the plurality of inactive monomers consist of the same monomer. That is to say, all inactive monomers in the plurality of inactive monomers are the same.
  • each inactive monomer in the plurality of inactive monomers is acrylamide.
  • each inactive monomer in the plurality of inactive monomers is PEG bis-azide.
  • the molar ratio of inactive monomer: crosslinker is between 2:1 and 75:1, in some embodiments between 50:1 and 75:1.
  • the molar ratio of inactive monomer: crosslinker is between 2:1 and 70:1 , in some embodiments, between 56:1 and 70:1.
  • the molar ratio of inactive monomer: crosslinker is 2:1, 3:1, 4:1 , 5:1 , 6:1, 7:1, 8:1 , 9:1 , or 10:1.
  • the molar ratio of inactive monomer: crosslinker is 60: 1 , 61 : 1 , 62: 1 , 63: 1 , 64: 1 , 65: 1 , or 66:1.
  • the molar ratio of the inactive monomer acrylamide: crosslinker bisacrylamide is between 50:1 and 75:1.
  • the molar ratio of the inactive monomer acrylamide: crosslinker bisacrylamide is between 56:1 and 70:1.
  • the molar ratio of the inactive monomer acrylamide: crosslinker bisacrylamide is 60:1, 61 :1, 62:1 , 63:1, 64:1 , 65:1, or 66:1.
  • the molar ratio of the inactive monomer acrylamide: crosslinker bisacrylamide is 63:1.
  • the molar ratio of the inactive monomer PEG bis-azide: crosslinker 4-arm PEG alkyne is between 2:1 and 10:1.
  • the molar ratio of the inactive monomer PEG bis-azide: crosslinker 4-arm PEG alkyne is between 2:1 and 5:1.
  • the molar ratio of the inactive monomer PEG bis-azide: crosslinker 4-arm PEG alkyne is 2:1, 3:1, 4:1, 5:1, 6:1 , 7:1, 8:1, 9:1, or 10:1.
  • the molar ratio of the inactive monomer PEG bis-azide: crosslinker 4-arm PEG alkyne is 2:1.
  • the molar ratio of inactive monomer: active monomer may be between 15:1 to 5:1, e.g. it may be 15:1, 10:1, or 5:1.
  • the molar ratio of inactive monomer: active monomer is 10:1. Therefore, for every 1000 molecules, 16 molecules are a crosslinker, 98 molecules are the active monomer and 886 molecules are the inactive monomer.
  • the molar ratio of inactive monomer: active monomer is 40:1
  • 16 molecules are the crosslinker bisacrylamide, 98 are the active monomer and 886 molecules are the inactive monomer acrylamide.
  • the polymer comprised in the hydrogel comprises at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% inactive monomers.
  • the polymer comprised in the hydrogel comprises at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% inactive monomers.
  • the inactive monomer is water soluble.
  • the inactive monomer has a similar reactivity to the active monomer and cross-linker, e.g. to provide for suitable polymerisation.
  • the inactive monomer does not comprise any functional group that is capable of reacting with a protein.
  • the inactive monomer does not comprise any functional group that is capable of reacting with a fluorophore.
  • the inactive monomer substantially does not absorb UV light.
  • the inactive monomer substantially does not absorb UV light at a wavelength which cleaves the cleavable bond. Suitable UV light is defined elsewhere herein.
  • the polymer comprised in the hydrogel of the invention is formed of a plurality of inactive monomers and a plurality of active monomers, polymerised together.
  • the active monomers contain fluorophores bonded thereto via a cleavable bond.
  • the plurality of active monomers are selected from for example but not limited to polyethylene glycol derivatives such as polyethylene glycol based monomers.
  • polyethylene glycol based monomers such as polyethylene glycol based monomers.
  • polyethylene glycol based monomers are in this context and can readily identify suitable polyethylene glycol based monomers.
  • polyethylene glycol based monomer is polyethylene glycol methacrylamide.
  • the plurality of active monomers are selected from the group consisting of polyethylene glycol acrylamide, polyethylene glycol N-isopropylacrylamide, polyethylene glycol methacrylamide, polyethylene glycol methacrylate, allylamide, and PEGwoo-azide.
  • the active monomers acrylamide, N-isopropylacrylamide, methacrylamide, and methacrylate each comprise a polyethylene glycol spacer arm.
  • the polyethylene glycol spacer arm aids water solubility.
  • the active monomers acrylamide, N-isopropylacrylamide, methacrylamide, and methacrylate each comprise a linear polyethylene glycol spacer arm.
  • the polyethylene glycol spacer arm is PEG400.
  • the polyethylene glycol spacer arm is PEG1000.
  • the polyethylene glycol spacer arm is PEG3400.
  • the plurality of active monomers may comprise the same monomer, or different monomers.
  • the plurality of active monomers may comprise two or more, three or more, four or more etc different active monomers selected from the list above.
  • the plurality of active monomers consist of the same monomer.
  • the plurality of active monomers comprise methacrylamide.
  • the plurality of active monomers comprise allylamide.
  • the plurality of active monomers comprise polyethylene glycol methacrylamide.
  • the polyethylene glycol methacrylamide is prepared at a yield of at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%.
  • the plurality of active monomers each comprise a polyethylene glycol spacer arm.
  • the plurality of active monomers each contain a polyethylene glycol spacer arm.
  • polyethylene glycol spacer arm is present between the o-nitrobenzyl derivative and methacrylamide.
  • polyethylene ethylene glycol spacer arm is present between the 4-(4-(1- hydroxyethyl)-2-methoxy-5-nitrophenoxy) butanoic acid (NVOC) and methacrylamide.
  • the plurality of active monomers comprise PEG 400 -methacrylamide, PEG 34 oo- methacrylamide or PEGwoo-azide.
  • Suitable the plurality of active monomers comprise 4-(4-(1-hydroxyethyl)-2-methoxy-5- nitrophenoxy) butanoic acid (NVOC)-
  • the plurality of active monomers comprise NVOC-PEG 400 - methacrylamide, NVOC-PEG34oo-methacrylamide, or NVOC-PEGwoo-azide.
  • NVOC refers to 4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy) butanoic acid.
  • the plurality of active monomers are selected from the group consisting of acrylamide, N-isopropylacrylamide, methacrylamide, and methacrylate.
  • a polyethylene glycol spacer arm is not present.
  • NVOC refers to 4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy) butanoic acid.
  • the plurality of active monomers each comprise at least one fluorophore.
  • the plurality of active monomers consist of F-NVOC-allylamide.
  • F refers to fluorescein.
  • NVOC refers to 4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy) butanoic acid.
  • the plurality of active monomers consist of F-NVOC-PEG4oo-methacrylamide.
  • F refers to fluorescein.
  • NVOC refers to 4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy) butanoic acid.
  • the plurality of active monomers consist of F-NVOC-PEG34oo-methacrylamide.
  • F refers to fluorescein.
  • NVOC refers to 4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy) butanoic acid.
  • the plurality of active monomers consist of F-NVOC-PEGwoo-azide.
  • F refers to fluorescein.
  • NVOC refers to 4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy) butanoic acid.
  • the F-NVOC-PEG34oo-methacrylamide is prepared at a yield of at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%.
  • F refers to fluorescein.
  • NVOC refers to 4-(4-(1- hydroxyethyl)-2-methoxy-5-nitrophenoxy) butanoic acid.
  • the plurality of active monomers comprise or consist of FITC-NVOC-PEG3400- methacrylamide.
  • each active monomer is FITC- NVOC-PEG34oo-methacrylamide.
  • FITC refers to fluorescein isothiocyanate.
  • NVOC refers to 4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy) butanoic acid.
  • the plurality of active monomers comprise or consist of FITC-NVOC-allylamide.
  • each active monomer is FITC-NVOC-allylamide.
  • FITC refers to fluorescein isothiocyanate.
  • NVOC refers to 4-(4-(1-hydroxyethyl)-2- methoxy-5-nitrophenoxy) butanoic acid.
  • the plurality of active monomers comprise or consist of FITC-NVOC-PEG400- methacrylamide.
  • each active monomer is FITC- NVOC-PEG4oo-methacrylamide.
  • FITC refers to fluorescein isothiocyanate.
  • NVOC refers to 4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy) butanoic acid.
  • each active monomer comprises or consist of FITC-NVOC- PEGwoo-azide.
  • each active monomer is FITC-NVOC-PEG3400- methacrylamide.
  • FITC refers to fluorescein isothiocyanate.
  • NVOC refers to 4-(4-(1- hydroxyethyl)-2-methoxy-5-nitrophenoxy) butanoic acid.
  • the polymer comprised in the hydrogel comprises at least 0.01%, at least 0.1 ,% at least 0.5%, at least 1 %, at least 2%, at least 3%, at least 4%, at least 5 %, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, a least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% of active monomers.
  • the polymer comprised in the hydrogel comprises at least 1%, at least 2%, at least 3%, at least 4%, at least 5 %, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11 %, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20% of active monomers.
  • the plurality of active monomers are hydrophilic.
  • the plurality of active monomers according to the invention suitably do not comprise allylamide since the addition of a cleavable bond resulted in a water insoluble monomer which cannot be incorporated in a hydrogel.
  • the plurality of active monomers according to the invention may comprise allylamide.
  • the active monomers are water soluble.
  • the active monomers have a similar reactivity to the inactive monomers and crosslinker, e.g. to provide for suitable polymerisation.
  • the active monomers each comprise at least one fluorophore, the fluorophore comprising a functional group that is capable of forming a covalent bond with a protein.
  • the active monomers of the polymer comprised in the hydrogel each comprise one or more fluorophore groups attached thereto via a cleavable bond.
  • a “cleavable bond” is a chemical bond which is cleaved, split, or fissioned. In general, a molecule is cleaved into two or more fragments via a cleavable bond.
  • the cleavable bond comprises a photolabile group. In the alternative, the cleavable bond does not comprise a photolabile group as defined herein.
  • the cleavable bond is a cleavable linkage.
  • a cleavable linkage comprises a photolabile group as defined herein.
  • the cleavable bond is a cleavable moiety.
  • a cleavable moiety comprises a photolabile group as defined herein.
  • the cleavable bond is a covalent bond.
  • the cleavable bond is cleaved by a cleavage inducer.
  • the cleavable linkage is cleaved by a cleavage inducer.
  • the cleavable moiety is cleaved by a cleavage inducer.
  • cleavage inducer is any agent or for example, light or radiation that can cleave the cleavable bond.
  • the cleavage inducer is light, radiation, enzymes, acids, alkalis, and/or heat.
  • the cleavage inducer is UV light, a reducing agent, or an esterase.
  • reducing agents include, but are not limited to, dithiothreitol (DTT), 2- mercaptoethanol (also known as p-mercaptoethanol), sodium bisulfite, thioglycolic acid, mercaptoethanesulfonic acid, glutathione and trialkylphosphine compounds or combinations thereof.
  • DTT dithiothreitol
  • 2- mercaptoethanol also known as p-mercaptoethanol
  • sodium bisulfite sodium bisulfite
  • thioglycolic acid mercaptoethanesulfonic acid
  • glutathione glutathione
  • trialkylphosphine compounds include, but are not limited to, tri-n- butylphosphine (TBP) or tris[2-carboxyethyl] phosphine (TCEP).
  • the reducing agent is dithiothreitol (DTT) or 2-mercaptoethanol.
  • esterases are enzymes that hydrolyse esters into alcohol and acids.
  • the esterase is pig liver esterase, or horse liver esterase.
  • the cleavable bond is cleaved by UV light.
  • UV light comprises a wavelength range of between roughly 100 nm and 400 nm. Suitably between about 200nm to 400nm.
  • the UV light comprises a wavelength range of roughly at least 300 nm.
  • the UV light is UV-A and comprises a wavelength range of roughly at least 300 nm.
  • the UV light is IIV-A and comprises a wavelength range between 315 and 400 nm.
  • the UV light comprises a wavelength range between 350 and 370 nm.
  • the UV light is 365 nm.
  • a “photolabile group” or “photolabile protecting group” is a group that can be removed by light.
  • the light can be UV light.
  • a “photolabile group” or “photolabile protecting group” is also known as photocleavable (protecting) group, photosensitive group, photoreleasable group or photoremovable group.
  • the cleavable bond i.e. the photolabile group
  • the cleavable bond may be for example nitrobenzyl- based or carbonyl-based.
  • Non-limiting examples of a nitrobenzyl-based cleavable/photolabile group are o-nitrobenzyl, 2-nitrobenzyl, 2,6-dinitrobenzyl, 4,5-dimethoxy-2-nitrobenzyl, 2,5-dihydroxybenzyl, 2-cyano- 6-nitrobenzyl, 2-nitroveratryl, 6-nitroveratryl, nitroveratryl, 4-(4-(1-hydroxyethyl)-2-methoxy-5- nitrophenoxy) butanoic acid (NVOC) or 6-nitropiperonylmethyl.
  • NVOC butanoic acid
  • nitrobenzyl-based cleavable/photolabile group is o-nitrobenzyl.
  • NVOC refers to 4- (4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy) butanoic acid.
  • NVOC refers to 4-(4-(1-hydroxyethyl)-2- methoxy-5-nitrophenoxy) butanoic acid.
  • Non-limiting examples of a carbonyl-based cleavable/photolabile group are phenacyl, 3’, 5’- dimethoxybenzoin, and p-hydroxyphenacyl.
  • the cleavable bond is nitrobenzyl-based.
  • the cleavable bond is m-nitrobenzyl.
  • the cleavable bind is p-nitrobenzyl.
  • the cleavable bond is o-nitrobenzyl.
  • the cleavable bond links the fluorophore to a polyethylene glycol chain of the polyethylene glycol spacer arm of each active monomer.
  • the cleavable bond o-nitrobenzyl links the fluorophore to a polyethylene glycol chain of the polyethylene glycol spacer arm of each active monomer.
  • the cleavable bond comprises a photolabile group attached to the hydrogel backbone at the one or more active monomers.
  • the cleavable bond comprises a photolabile group attached to the hydrogel backbone by a polyethylene glycol spacer arm at the one or more active monomers
  • the cleavable bond comprises a nitrobenzyl-based photolabile group attached to the hydrogel backbone at the one or more active monomers.
  • the cleavable bond comprises a nitrobenzyl-based photolabile group by a polyethylene glycol spacer arm attached to the hydrogel backbone
  • the cleavable bond comprises an o-nitrobenzyl group attached to the hydrogel backbone at the one or more active monomers.
  • the cleavable bond comprises an o-nitrobenzyl group attached to the hydrogel backbone as illustrated in the following:
  • the cleavable bond comprises an o-nitrobenzyl group attached to the hydrogel backbone by a polyethylene glycol spacer arm at the one or more active monomers.
  • the isothiocyanate group of fluorescein isothiocyanate does not bind to the hydrogel backbone.
  • the polymer comprised in the hydrogel comprises at least one fluorophore bonded to the active monomers therein via cleavable bonds.
  • each active monomer comprises at least one fluorophore bonded thereto.
  • each active monomer comprises a plurality of fluorophores bonded thereto.
  • each active monomer comprises at least 1 , at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 fluorophores, at least 15 fluorophores, at least 20 fluorophores bonded thereto.
  • each active monomer comprises at least 1 , at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 fluorophores bonded thereto.
  • each active monomer comprises at most 10 fluorophores bonded thereto.
  • the polymer comprised in the hydrogel comprises in total at least at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1500, at least 2000, at least 2500, at least 3000, at least 4000, at least 5000, at least at least 6000, at least 7000, at least 8000, at least 9000, at least 10.000, at least 15.000, at least 20.000, at least 25.000, at least 30.000, at least 40.000, at least 50.000, at least 60.000, at least 70.000, at least 80.000, at least 90.000, at least 100.000 fluorophores bonded thereto.
  • fluorophores include, but are not limited to coumarin, cyanine, benzofuran, a quinoline, a quinazolinone, an indole, a furan, a benzazole, a borapolyazaindacene and xanthenes including fluorescein, fluorescein isothiocyanate, tetrachlorofluorescein, carbofluoresceins, naphthofluoresceins, (semi)naphthofluoresceins, eosin Y eosin B, rhodamine and rhodol as well as other fluorophores described in RICHARD P.
  • fluorescein encompasses all kinds of conceivable derivatives such as fluorescein isothiocyanate.
  • Fluorophores may contain substituents that alter the solubility, spectral properties or physical properties of the fluorophore.
  • the at least one fluorophore is selected from the group consisting of fluorescein, fluorescein isothiocyanate, eosin Y, eosin B, tetrachlorofluorescein, carbofluoresceins, naphthofluoresceins, and (semi)naphthofluoresceins with suitable protein reactive groups.
  • the at least one fluorophore is selected from the group consisting of fluorescein, fluorescein isothiocyanate, eosin Y, eosin B, tetrachlorofluorescein, carbofluoresceins, naphthofluoresceins, and (semi)naphthofluoresceins with at least one suitable protein reactive group.
  • suitable protein reactive group refers to a group that is capable of reacting with another chemical group in a protein to form a covalent bond, i.e. is covalently reactive under suitable reaction conditions, and generally represents a point of attachment for another substance.
  • the “suitable protein reactive group” reacts with an amine containing molecule in a protein.
  • Reactive groups generally include nucleophiles, electrophiles and photoactivatable groups.
  • Exemplary reactive groups include, but not limited to, olefins, acetylenes, alcohols, phenols, ethers, oxides, halides, aldehydes, ketones, carboxylic acids, esters, amides, cyanates, isocyanates, thiocyanates, isothiocyanates, amines, hydrazines, hydrazones, hydrazides, diazo, diazonium, nitro, nitriles, mercaptans, sulfides, disulfides, sulfoxides, sulfones, sulfonic acids, sulfinic acids, acetals, ketals, anhydrides, sulfates, sulfenic acids isonitriles, amidines, imides, imidates, nitrones, hydroxylamines, oximes, hydroxamic acids thiohydroxamic acids, allenes, ortho esters, sulfites,
  • the at least one fluorophore is rhodamine.
  • the at least one fluorophore is fluorescein isothiocyanate (FITC).
  • the at least one fluorophore is fluorescein (F).
  • the ratio of active: inactive monomers used in the hydrogel of the invention may be varied.
  • the ratio may be varied to control the properties of the hydrogel, and to finetune said properties to different applications and uses of the hydrogel.
  • the ratio of active monomers: inactive monomers is a molar ratio.
  • the (molar) ratio of active monomers: inactive monomers is between 1 :100 to 1:5.
  • the (molar) ratio of active: inactive monomer is 1:100, 1:90, 1:80, 1:70, 1:60, 1:50, 1:40, 1:30, 1:20, 1 :10, 1:5.
  • the (molar) ratio of active: inactive monomer is 1: 100, 1 :40, 1 :20, or 1 : 10.
  • the (molar) ratio of active: inactive monomer is 1 :40.
  • the ratio of inactive monomer: cross-linker is also a molar ratio.
  • the (molar) ratio of inactive monomer: cross-linker is 63:1.
  • the molar ratio of inactive monomer: cross-linker is 2:1.
  • the (molar) ratio of inactive monomer: crosslinker is between 2:1 and 75:1, or between 50:1 and 75:1.
  • the (molar) ratio of inactive monomer: crosslinker is between 2:1 and 70:1, or between 56:1 and 70:1.
  • the (molar) ratio of inactive monomer: crosslinker is 2: 1 , 3: 1 , 4: 1 , 5: 1 , 6: 1 , 7: 1 , 8: 1 , 9:1 , or 10:1.
  • the (molar) ratio of inactive monomer: crosslinker is 60: 1 , 61 : 1 , 62: 1 , 63: 1 , 64: 1 , 65:1 , or 66:1.
  • the (molar) ratio of inactive monomer acrylamide: the crosslinker bisacrylamide is between 50:1 and 75:1.
  • the (molar) ratio of inactive monomer acrylamide: the crosslinker bisacrylamide is between 56:1 and 70:1.
  • the (molar) ratio of inactive monomer acrylamide: the crosslinker bisacrylamide is 60:1 , 61 :1 , 62:1 , 63:1 , 64:1 , 65:1 , or 66:1.
  • the (molar) ratio of the inactive monomer acrylamide: the crosslinker bisacrylamide is 63:1.
  • the molar ratio of the inactive monomer PEG bis-azide: crosslinker 4-arm PEG alkyne is between 2:1 and 10:1.
  • the molar ratio of the inactive monomer PEG bis-azide: crosslinker 4-arm PEG alkyne is between 2:1 and 5:1.
  • the molar ratio of the inactive monomer PEG bis-azide: crosslinker 4-arm PEG alkyne is 2:1 , 3:1 , 4:1 , 5:1 , 6:1 , 7:1 , 8:1 , 9:1 , or 10:1.
  • the molar ratio of the inactive monomer PEG bis-azide: crosslinker 4-arm PEG alkyne is 2:1.
  • the hydrogel comprising a polymer may be formed of a plurality of inactive monomers which are acrylamide, and a plurality of active monomers which are polyethylene glycol methacrylamide, wherein each polyethylene glycol methacrylamide monomer comprises at least one fluorescein isothiocyanate group capable of covalently binding to a protein, and the (molar) ratio of inactive monomers to active monomers is between 100 to 1 and 5 to 1 , and each fluorescein isothiocyanate is attached to said polyethylene glycol methacrylamide by a cleavable bond, wherein said cleavable bond is o-nitrobenzyl.
  • the hydrogel comprises F-NVOC-PEG4oo-methacrylamide.
  • F refers to fluorescein
  • NVOC refers to 4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy) butanoic acid.
  • the hydrogel comprises F-NVOC-PEG34oo-methacrylamide.
  • F refers to fluorescein “NVOC” refers to 4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy) butanoic acid.
  • the hydrogel comprises F-NVOC-allylamide.
  • F refers to fluorescein “NVOC” refers to 4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy) butanoic acid.
  • the hydrogel comprises FITC-NVOC-PEG34oo-methacrylamide.
  • FITC refers to fluorescein isothiocyanate.
  • NVOC refers to 4-(4-(1-hydroxyethyl)-2-methoxy-5- nitrophenoxy) butanoic acid.
  • the hydrogel comprises a polymer, wherein said polymer is a crosslinked FITC-NVOC-PEG34oo-methacrylamide/acrylamide copolymer wherein the crosslinker is bisacrylamide.
  • the hydrogel comprises acrylamide.
  • the polymer is a FITC-NVOC-PEG34oo-methacrylamide/acrylamide copolymer.
  • FITC refers to fluorescein isothiocyanate.
  • NVOC refers to 4-(4-(1-hydroxyethyl)-2-methoxy-5- nitrophenoxy) butanoic acid.
  • the polymer is a crosslinked FITC-NVOC-PEG34oo-methacrylamide/acrylamide copolymer.
  • FITC refers to fluorescein isothiocyanate.
  • NVOC refers to 4-(4-(1- hydroxyethyl)-2-methoxy-5-nitrophenoxy) butanoic acid.
  • the polymer is a crosslinked FITC-NVOC-PEG3400- methacrylamide/acrylamide copolymer wherein the crosslinker is bisacrylamide.
  • FITC refers to fluorescein isothiocyanate.
  • NVOC refers to 4-(4-(1-hydroxyethyl)-2-methoxy-5- nitrophenoxy) butanoic acid.
  • the hydrogel comprising a polymer may be formed of a plurality of inactive monomers which are PEG bis-azide, and a plurality of active monomers which are FITC- NVOC-PEGwoo-azide, wherein each polyethylene glycol monomer comprises at least one fluorescein isothiocyanate group capable of covalently binding to a protein, and the (molar) ratio of inactive monomers to active monomers is between 100 to 1 and 5 to 1 , and each fluorescein isothiocyanate is attached to said polyethylene glycol by a cleavable bond, wherein said cleavable bond is o-nitrobenzyl.
  • the hydrogel comprises a polymer, wherein said polymer is a crosslinked FITC-NVOC-PEG1000 azide /PEG bis-azide copolymer wherein the crosslinker is 4-arm PEG alkyne.
  • the polymer is a FITC-NVOC-PEGwoo-azide /PEG bis-azide copolymer.
  • FITC fluorescein isothiocyanate.
  • NVOC 4-(4-(1-hydroxyethyl)-2-methoxy-5- nitrophenoxy) butanoic acid.
  • the polymer is a crosslinked FITC-NVOC-PEGwoo-azide /PEG bis-azide copolymer.
  • FITC refers to fluorescein isothiocyanate.
  • NVOC refers to 4-(4-(1-hydroxyethyl)-2- methoxy-5-nitrophenoxy) butanoic acid.
  • the polymer is a crosslinked FITC-NVOC-PEGwoo- azide /PEG bis-azide copolymer wherein the crosslinker is 4-arm PEG alkyne.
  • FITC refers to fluorescein isothiocyanate.
  • NVOC refers to 4-(4-(1-hydroxyethyl)-2-methoxy-5- nitrophenoxy) butanoic acid.
  • a formulation comprising a core and a shell
  • the core comprises the hydrogel of any one of the preceding aspects and embodiments
  • the shell comprises a hydrogel having a pore size of less than 30 nm.
  • the “core” is substantially surrounded by the shell.
  • the core comprises the hydrogel of any one of the preceding aspects and embodiments.
  • the hydrogel comprises a polymer comprising a plurality of inactive monomers and a plurality of active monomers, polymerised together.
  • the active monomers contain fluorophores bonded thereto via a cleavable bond.
  • the “shell” comprises a hydrogel, suitably which comprises a polymer.
  • the hydrogel which forms the shell may be a different hydrogel to that of the core.
  • the shell may be formed of any hydrogel having a pore size of less than 30nm.
  • the shell comprises a polymer formed of polyethylene glycol, acrylamide, N- isopropylacrylamide, methacrylamide, and/or methacrylate.
  • the formulation is a core-shell hydrogel.
  • the formulation is a core-shell structure.
  • pore size characterises the size of the openings of the hydrogel described herein.
  • the skilled person is aware pore size is dependent on the monomer used to produce polymers, i.e. methacrylamide yields a different pore size compared to N- isopropylacrylamide yielding a different pore size, respectively.
  • concentrations of a given monomer can yield different pore sizes in the resulting hydrogel. For example, a higher concentration of methacrylamide results in a hydrogel having smaller pore sizes compared to a lower concentration of methacrylamide which results in a hydrogel having larger pore sizes.
  • the pore size of the hydrogel is less than 30 nm, less than 29 nm, less than 28 nm, less than 27 nm, less than 26 nm, less than 25 nm, less than 24 nm, less than 23 nm, less than 22 nm, less than 21 nm, less than 20 nm, less than 19 nm, less than 18 nm, less than 17 nm, less than 16 nm, less than 15 nm, less than 14 nm, less than 13 nm, less than 12 nm, less than 11 nm, less than 10 nm, less than 9 nm, less than 8 nm, less than 7 nm, less than 6 nm, less than 5 nm, less than 4 nm, less than 3 nm, less than 2 nm.
  • the pore size of the hydrogel is less than 7 nm, less than 6 nm, less than 5 nm, less than 4 nm, less than 3 nm.
  • the pore size of the hydrogel is 4 nm, 3.9 nm, 3.8 nm, 3.7 nm, 3.6 nm, 3.5 nm.
  • the pore size of the hydrogel is adapted to the size of the detected protein.
  • a nonlimiting example of the detected protein is cardiac troponin.
  • the pore size of the hydrogel is 4 nm, 3.9 nm, 3.8 nm, 3.7 nm, 3.6 nm, or 3.5 nm to match the diameter of cardiac troponin T (3.37 nm).
  • the pore size of the hydrogel of the shell is less than 30 nm, less than 29 nm, less than 28 nm, less than 27 nm, less than 26 nm, less than 25 nm, less than 24 nm, less than 23 nm, less than 22 nm, less than 21 nm, less than 20 nm, less than 19 nm, less than 18 nm, less than 17 nm, less than 16 nm, less than 15 nm, less than 14 nm, less than 13 nm, less than 12 nm, less than 11 nm, less than 10 nm, less than 9 nm, less than 8 nm, less than 7 nm, less than 6 nm, less than 5 nm, less than 4 nm, less than 3 nm, less than 2 nm.
  • the pore size of the hydrogel of the shell is controlled to limit intake of abundant larger proteins from sample.
  • a non-limiting example of such a larger protein is mucin.
  • the pore size of the hydrogel of the shell is less than 7 nm, less than 6 nm, less than 5 nm, less than 4 nm, less than 3 nm.
  • the pore size of the hydrogel of the shell is 4 nm, 3.9 nm, 3.8 nm, 3.7 nm, 3.6 nm, 3.5 nm.
  • the pore size of the hydrogel is adapted to the size of the detected protein.
  • a nonlimiting example of the detected protein is cardiac troponin T.
  • the pore size of the hydrogel is 4 nm, 3.9 nm, 3.8 nm, 3.7 nm, 3.6 nm, or 3.5 nm to match the size of cardiac troponin T (3.37 nm).
  • the formulation comprises additional pharmaceutically acceptable excipients. Suitable additional excipients are for example, disintegrants, binders, lubricants, glidants and/or surfactants.
  • the shell comprises additional excipients.
  • the core comprises additional excipients
  • the shell or the core comprises additional excipients.
  • the shell and core comprise additional excipients.
  • disintegrants are excipients that facilitate dissolution and enhance availability. Suitable disintegrants are for example, starch, starch derivatives and crosslinked polymers such as polyvinyl pyrrolidone or crospovidone.
  • binders are excipients that agglomerate the hydrogel and the other excipients, where present. They also improve compressibility. Suitable binders are for example cellulose derivatives such as microcrystalline cellulose, methylcellulose, carboxymethylcellulose sodium, hydroxypropyl methylcellulose, hydroxyethyl cellulose, and hydroxypropyl cellulose. Other binders include polyvidone, polyvinyl pyrrolidone, gelatin, natural gums, starch paste, pregelatinized starch, sucrose, corn syrup, polyethylene glycols, and sodium alginate, ammonium calcium alginate and polyethylene glycols.
  • lubricants are excipients that prevent sticking of the hydrogel and reduce friction during a potential compression stage in hydrogel formulation.
  • Suitable lubricants are vegetable oils, mineral oils, polyethylene glycols, salts of stearic acid (such as calcium stearate, magnesium stearate, and sodium stearyl fumarate), mineral salts (such as talc), organic salts (such as sodium benzoate, sodium acetate, and sodium oleate) and polyvinyl alcohols.
  • glidants are excipients that reduce inter-particle friction in a formulation, thereby improving flow.
  • Suitable glidants are alkali stearates (such as magnesium stearate or calcium stearate), silicate salts (such as magnesium silicate, magnesium trisilicate, magnesium silicate anhydrous, calcium silicate), starches, mineral salts (such as talc), and colloidal silicon dioxide.
  • surfactants are amphiphilic excipients that decrease the surface tension or interfacial tension between two phases such as between wo liquids or a liquid and a solid.
  • Suitable surfactants can be ionic, non-ionic and amphoteric.
  • Anionic surfactants include, but are not limited to, sodium lauryl sulphate, sodium laurate, dialkyl sodium sulfosuccinates, sodium stearate, potassium stearate, sodium oleate, deoxycholic acid, sodium deoxycholate, cholic acid, and sodium taurocholate.
  • Non-ionic surfactants include, but are not limited to, one or more of polyoxyethylene, sorbitan, fatty acid esters, fatty alcohols, glyceryl esters, fatty acid esters of fatty alcohols and alcohols.
  • an oral formulation comprising the hydrogel according to any of the preceding aspects and embodiments is provided, or the formulation according to the preceding aspect.
  • oral formulation is a formulation suitable for administration/incubation by the oral route.
  • the oral formulation of the invention is biocompatible.
  • Oral route comprises buccal, sublingual, and sublabial.
  • the oral formulation may comprise a hydrogel formulated in a shell.
  • the hydrogel is formulated in a hydrogel shell.
  • the hydrogel is not formulated in a hydrogel shell.
  • the oral formulation is a pill, tablet, capsule, granule, troch, lozenge, a lollipop, or sampling material.
  • Suitable sampling materials can be without limitation sheets, spherical beads, discs, or nanoparticles.
  • the nanoparticles are formulated in a shell.
  • said shell is a hydrogel shell.
  • the oral formulation is a disc. In one embodiment the oral formulation is a sampling material, which is a disc.
  • the oral formulation is a lozenge, a lollipop, or sampling material.
  • the oral formulation is a lollipop.
  • a “lollipop” comprises a hydrogel on a stick.
  • the hydrogel has for example the shape of a disc or ball.
  • the shape is constructed such that the collection of a biological sample is facilitated.
  • the shape is constructed such that the hydrogel can take up the biological sample and therefore, the proteins in a time and resource efficient manner.
  • the oral formulation comprising the hydrogel comprises additional excipients. Additional excipients are defined elsewhere herein.
  • Suitable additional excipients are for example, disintegrants, binders, lubricants, glidants and/or surfactants as defined elsewhere herein.
  • Further suitable additional excipients of the oral formulation comprise colourants, diluents, buffering agents, preservatives, flavouring agents, and pharmacologically compatible carriers.
  • a lozenge comprises a colourant and/or a flavouring agent.
  • a lollipop comprises a colourant and/or a flavouring agent.
  • a sampling device comprising the hydrogel according to any of the preceding aspects and embodiments, comprising the formulation of any of the preceding aspects and embodiments, or comprising the oral formulation according to any of the preceding aspects and embodiments, wherein the sampling device is a test tube.
  • sampling device is a piece of equipment which is used to collect a biological sample.
  • Biological samples are defined elsewhere herein and also encompass fluid samples such as saliva. Accordingly, the sampling device can contain both the hydrogel of the invention and the biological sample at the same time.
  • the sampling device is constructed such that the biological sample can be deposited within such sampling device and is brought into contact with the hydrogel.
  • the sampling device is the oral formulation defined elsewhere herein and said oral formulation is deposited in another sampling device such as a test tube.
  • the first sampling device is the oral formulation defined elsewhere herein and the second sampling device is a test tube.
  • sampling devices and test tubes can readily identify such sampling devices and test tubes.
  • the sampling device is a test tube.
  • the hydrogel of the invention may be comprised upon a substrate, suitably an inert substrate.
  • the hydrogel may be comprised in a film upon said substrate, suitably coated on said substrate.
  • a substrate comprising the hydrogel of the invention.
  • an inert substrate comprising a coating thereon, wherein the coating comprises the hydrogel of the invention.
  • the coating is a film. Suitable such substrates maybe glass or metal.
  • the substrate may be used as a sampling device.
  • the hydrogel of the invention may be comprised in a disc.
  • a disc comprising the hydrogel of the invention.
  • the disc may be used as an oral formulation or a sampling device.
  • the disc is between 1-10 mm in diameter, suitably between 2-8 mm in diameter, suitably around 6 mm in diameter.
  • the disc may be around 1 mm in height.
  • said discs are easy to fit into the oral cavity of subjects, and further provide the benefit that a lower volume of buffer is required to release any captured proteins compared to hydrogel films. The lower the volume of the buffer used to release proteins captured in the hydrogels, the higher is the resulting preconcentration factor
  • kits-of-parts refers to a packaged set of related components, typically one or more hydrogel, formulation and/or oral formulation of the invention.
  • Kits-of-parts are provided which are used for protein labelling and analysis using the hydrogel according to any of the preceding aspects and embodiments, the formulation according to any of the preceding aspects and embodiments, or the oral formulation according to any of the preceding aspects and embodiments, or the sampling device according to any of the preceding aspects and embodiments.
  • kit-of-parts comprising a. the hydrogel according to any of the preceding aspects and embodiments, the formulation according to any of the preceding aspects and embodiments, the oral formulation (optionally which may be a disc) according to any of the preceding aspects and embodiments, the sampling device according to any of the preceding aspects and embodiments, the substrate according to any of the preceding aspects and embodiments, and b. instructions for use.
  • the kit-of-parts comprises a. the hydrogel according to any of the preceding aspects and embodiments, and b. instructions for use.
  • the kit-of-parts comprises a. the formulation according to any of the preceding aspects and embodiments and b. instructions for use.
  • the kit-of-parts comprises a. the oral formulation according to any of the preceding aspects and embodiments, and b. instructions for use, optionally wherein the oral formulation may be a disc.
  • the kit-of-parts comprises a. the sampling device according to any of the preceding aspects and embodiments, and b. instructions for use.
  • the kit-of-parts comprises a. the substrate according to any of the preceding aspects and embodiments, and b. instructions for use.
  • the kit-of-parts comprises a. the disc according to any of the preceding aspects and embodiments, and b. instructions for use.
  • kit-of-parts can comprise further components such as a neutralising buffer, mouth wash, and/or tissues.
  • kit-of-parts can comprise a container according to an aspect of the present invention.
  • the sampling device is a testing tube.
  • a biomaterial comprising the hydrogel according to any one of the preceding aspects and embodiments is provided, or comprising the formulation according to any previous aspect and embodiment.
  • biological materials are synthetic materials that are used and adapted for a biological purpose. Said biological purpose is but is not limited to for example, labelling proteins and therefore, labelling cells, or labelling proteins in biological samples such as biological fluid samples which are further defined elsewhere herein.
  • the biomaterial comprises additional excipients that confer desirable properties to a biomaterial or enhance desirable properties of the biomaterial.
  • additional excipients are disintegrants, binders, lubricants, glidants and/or surfactants as defined elsewhere herein.
  • additional excipients are colourants, diluents, buffering agents, antibiotics, growth factors and/or preservatives.
  • the additional excipient are antibiotics, growth factors and/or preservatives.
  • the additional excipient is a preservative.
  • the biomaterial is sterile.
  • the biomaterial is biocompatible.
  • the biomaterial is for use in cell culture.
  • the invention provides use of a biomaterial of the invention in cell culture.
  • cell culture refers to any application in which cells are cultivated such cell culture systems also known as tissue culture systems and for example, cell-based assays.
  • the biomaterial is used to label cells in cell culture system and in for example, cell-based assays.
  • proteins expressed on the cell surface are labelled.
  • proteins expressed on the cell surface are for example but not limited to annexin V.
  • the biomaterial is used in cell culture to label apoptotic cells.
  • the apoptotic cells express annexin V on the cell surface.
  • the invention provides use of a hydrogel of the invention as a cell support scaffold which simultaneously labels proteins expressed on the cell surface.
  • attach refers to cells that adhere directly or indirectly to a substrate as well as to cells that adhere to other cells.
  • the hydrogel according to any one of the preceding aspects and embodiments, a formulation or an oral formulation (optionally a disc) according to any one of the preceding aspects and embodiments, or a sampling device according to any one of the preceding aspects and embodiments, or a substrate according to any one of the preceding aspects and embodiments is used for concentrating and labelling proteins in a sample.
  • concentrating proteins refers to the covalent capture of proteins in the hydrogel. Said covalent capture of proteins is facilitated by the reaction between primary amines in proteins with at least one fluorophore capable of covalently binding to a protein present in the hydrogel. Thereby, proteins are concentrated and labelled in a single step.
  • concentrating proteins refers to concentrating protein by methods well known to the person skilled in the art such as dialysis, precipitation, chromatography and using cellulose membrane concentrators. Said alternative can be combined with the concentrating step according to the invention.
  • labelling proteins refers to labelling of active reactive sites on the protein or protein fragments.
  • the label is a fluorophore and therefore, directly detectable. Fluorescent labels are detected by the excitation of a suitable molecular adduct that can be visualised by excitation with light that is absorbed by the dye or can be measured with standard fluorometers or imaging systems, for example.
  • concentrating and labelling proteins in a sample occurs in a single step.
  • the hydrogel according to any one of the preceding aspects and embodiments, a formulation according to any one of the preceding aspects and embodiments, an oral formulation (optionally a disc) according to any one of the preceding aspects and embodiments, or a sampling device according to any one of the preceding aspects and embodiments, or a substrate according to any one of the preceding aspects and embodiments is used for concentrating and labelling proteins in a sample, followed by controlled release of the concentrated and labelled proteins.
  • controlled release refers to a release at predetermined intervals or a gradual release over a period of time.
  • the hydrogel according to any one of the preceding aspects and embodiments, a formulation according to any one of the preceding aspects and embodiments, an oral formulation (optionally a disc) according to any one of the preceding aspects and embodiments, a sampling device according to any one of the preceding aspects and embodiments, or a substrate according to any one of the preceding aspects and embodiments, or a container according to any one of the preceding aspects and embodiments is used in a method of diagnosing a disease or disorder.
  • diagnosis refers to any quantitative or semi-quantitative determination of the existence of disease or disorder or the extent of its progression (prognosis) and is to be construed liberally accordingly.
  • disease refers to a state resulting from a pathophysiological response to external or internal factors.
  • disorder refers to the disruption to the normal or regular functions in the body or a part of the body.
  • a method of concentrating and labelling proteins in a sample comprising: a. Contacting a sample with the hydrogel of any one of the preceding aspects and embodiments or the formulation, the formulation of any one of the preceding aspects and embodiments, the oral formulation (optionally which may be a disc) of any one of the preceding aspects and embodiments, the sampling device of any one of the preceding aspects and embodiments, the substrate of any one of the preceding aspects and embodiments under suitable conditions to allow proteins in the sample to bind to the hydrogel via the fluorophore, thereby obtaining fluorescently labelled proteins bound to said hydrogel
  • contacting a sample refers to bringing a sample into contact with the hydrogel of the invention, or a formulation thereof under suitable conditions to allow proteins in the sample to bind to the hydrogel.
  • suitable conditions are temperatures between roughly about 15 and 40 °C, a pH between roughly 4 and 8 and atmospheric pressure.
  • the contacting step takes place for a suitable amount of time to allow the proteins in the sample to bind to the hydrogel.
  • the contacting step takes place for an incubation time.
  • the incubation time is between 15 and 48 hours.
  • the incubation time is between 24 and 48 hours.
  • the incubation time is at least 24 hours, suitably about 24 hours.
  • the contacting step comprises contacting a sample with the hydrogel for at least 24 hours to allow proteins in the sample to bind to the hydrogel via the fluorophore, thereby obtaining fluorescently labelled proteins bound to said hydrogel.
  • the method described herein are in vitro methods that are performed using a sample that has already been obtained from the subject (i.e. the sample is provided for the method, and the steps taken to obtain the sample from the subject are not included as part of the method).
  • the methods may include the step of providing a biological fluid sample from a subject.
  • Directly obtaining a sample means performing a process (e.g., performing a physical method such as extraction) to obtain the sample.
  • Indirectly obtaining a sample refers to receiving the sample from another party or source (e.g., a third party laboratory that directly acquired the sample).
  • the methods provided herein comprise providing a biological fluid sample (for example saliva, a blood sample, such as a serum or plasma sample, urine sample) from a subject.
  • a biological fluid sample for example saliva, a blood sample, such as a serum or plasma sample, urine sample
  • test samples for example saliva, a blood sample, such as a serum or plasma sample, urine sample.
  • biological sample refers to a sample obtained or derived from a subject.
  • the sample is, or comprises, a biological fluid (also referred to herein as a bodily fluid) sample.
  • a “biological sample” or “sample” also encompasses environmental samples or samples from food product sources.
  • Biological, environmental and food product samples contain a protein of interest.
  • the samples can be fresh samples, frozen samples, or preserved samples, for example, preserved in formalin.
  • the samples according to the invention can contain further compounds such as metabolites of drugs, antibiotics, anticoagulants, chemicals such as preservatives, fixatives or buffers, nutrients, fertilisers or the like. In some instances, the sample is from a contaminated source.
  • Food product samples are samples from food or beverage sources at any stage.
  • the sample can be a raw material, a material that is being processed, an “in- process sample”, or a sample from the finalised food product.
  • biological fluid sample encompasses a saliva sample.
  • biological fluid sample also encompasses other bodily fluids such as a urine sample or a blood sample. All biological fluids and excretions are included.
  • proteins of the invention also encompass low abundance proteins.
  • fluorescently labelled proteins refers to a protein comprising the fluorophore. Said fluorophore is attached chemically to the protein, or in other words bound to the protein.
  • the hydrogel pre-concentrates proteins.
  • the hydrogel pre-concentrates the proteins by a pre-concentration factor of up to 10, up to 20, up to 30, up to 40, up to 50, up to 100, up to 150, up to 200, up to 250, up to 300, up to 350, up to 400, up to 450.
  • the hydrogel pre-concentrates the proteins by a preconcentration factor of between 190 and 300.
  • the hydrogel preconcentrates the proteins by a pre-concentration factor of up to 192.
  • the hydrogel pre-concentrates the proteins by a pre-concentration factor of up to 295.
  • pre-concentration factor refers to the increase in protein concentration before and after the sample is contacted with a hydrogel.
  • a preconcentration factor of 2 refers to a doubling of the concentration of the protein before and after the sample is contacted.
  • the protein to be concentrated and labelled is additionally tagged with a fluorophore.
  • the protein to be concentrated is streptavidin and the fluorophore is rhodamine.
  • the fluorescence of the labelled protein is used to determine the protein concentration before and after the sample is contacted with the hydrogel.
  • the pre-concentration factor is provided in this nonlimiting example.
  • concentrating proteins refers to the covalent capture of proteins in the hydrogel and said proteins are concentrated as demonstrated by the exemplary pre-concentration.
  • the protein is streptavidin.
  • the protein is CRP.
  • the protein is IL6.
  • the protein is IL8.
  • the protein is rhodamine-streptavidin.
  • the fluorophore is fluorescein isothiocyanate.
  • the protein is labelled with at least 1 , at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 fluorophores.
  • the protein is labelled with at most 5, at most 6, at most 7, at most 8, at most 9, at most 10 fluorophores.
  • the protein is labelled with at most 10 fluorophores.
  • CRP IL8
  • IL6 is labelled with at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 fluorophores.
  • the fluorophore is fluorescein isothiocyanate.
  • CRP, IL8, or IL6 is labelled with at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 fluorescein molecules.
  • CRP, IL8, or IL6 is labelled with at least 50 fluorescein molecules.
  • CRP IL8
  • IL6 IL6
  • the hydrogel can provide a concentration factor of 192 for 0.1 ppm of protein in a sample.
  • a concentration factor of 192 for 0.1 ppm of protein in a sample can be provided.
  • an inert substrate comprising the hydrogel.
  • the hydrogel can provide a concentration factor of 295 for 0.01 ppm of protein in a sample.
  • an oral formulation comprising the hydrogel suitably a disc.
  • the hydrogel comprising a polymer formed of a plurality of inactive monomers which are acrylamide, and a plurality of active monomers which are fluorescein isothiocyanate-NVOC-polyethylene glycol methacrylamide, and the (molar) ratio of inactive monomers to active monomers is between 100 to 1 and 5 to 1, wherein each fluorescein isothiocyanate-NVOC-polyethylene glycol methacrylamide comprises at least one fluorescein isothiocyanate group capable of covalently binding to a protein such as streptavidin, CRP, IL8, or IL6, and each fluorescein isothiocyanate is attached to said polyethylene glycol methacrylamide by a cleavable bond wherein said cleavable bond is o- nitrobenzyl, provides a concentration factor of 192 for 0.1 ppm protein in a sample.
  • NVOC refers to 4-(4-(1-hydroxyethyl)-2-methoxy
  • the hydrogel comprising a polymer formed of a plurality of inactive monomers which are acrylamide, and a plurality of active monomers which are fluorescein isothiocyanate-NVOC-polyethylene glycol methacrylamide, and the (molar) ratio of inactive monomers to active monomers is between 100 to 1 and 5 to 1, wherein each fluorescein isothiocyanate-NVOC-polyethylene glycol methacrylamide comprises at least one fluorescein isothiocyanate capable of covalently binding to streptavidin, CRP, IL8, or IL6, and each fluorescein isothiocyanate is attached to said polyethylene glycol methacrylamide by a cleavable bond wherein said cleavable bond is o-nitrobenzyl provides a concentration factor of 295 for 0.01 ppm protein in a sample.
  • NVOC refers to 4-(4-(1-hydroxyethyl)-2- methoxy-5-nitrophenoxy) buta
  • the hydrogel comprising a polymer formed of a plurality of inactive monomers which are PEG bis-azide, and a plurality of active monomers which are fluorescein isothiocyanate-NVOC-polyethylene glycol azide, and the (molar) ratio of inactive monomers to active monomers is between 100 to 1 and 5 to 1 , wherein each fluorescein isothiocyanate- NVOC-polyethylene glycol azide comprises at least one fluorescein isothiocyanate capable of covalently binding to streptavidin, CRP, IL8, or IL6, and each fluorescein isothiocyanate is attached to said polyethylene glycol azide by a cleavable bond wherein said cleavable bond is o-nitrobenzyl provides a concentration factor of 295 for 0.01 ppm protein in a sample.
  • NVOC refers to 4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy) but
  • a method of detecting proteins in a sample comprising: a. Contacting a sample with the hydrogel of any one of the preceding aspects and embodiments, the formulation of any of any one of the preceding aspects and embodiments, the oral formulation (optionally which may be a disc) of any of any one of the preceding aspects and embodiments, the sampling device of any of any one of the preceding aspects and embodiments under suitable conditions, the substrate of any one of the preceding aspects and embodiments, to allow proteins in the sample to bind to the hydrogel via the fluorophore, or a sampling device of any of any one of the preceding aspects and embodiments, thereby obtaining fluorescently labelled proteins bound to said hydrogel; b.
  • step (a) Exposing the hydrogel of step (a) to a cleavage inducer under suitable conditions to cleave the cleavable bonds, thereby releasing said fluorescently labelled proteins from the hydrogel; and c. Determining the presence of proteins in the sample, wherein the presence of fluorescence is indicative of the presence of proteins in the sample.
  • Contacting a sample is defined elsewhere herein.
  • Fluorescently labelled proteins is defined elsewhere herein.
  • cleavage inducer is defined elsewhere herein.
  • the suitable cleavage inducers UV light, reducing agents, or esterases are also defined elsewhere herein.
  • Fluorescently labelled proteins is defined elsewhere herein.
  • the fluorescently labelled proteins are released from the hydrogel of the invention.
  • at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 80%, at least 90%, at least 95%, at least 99% of the fluorescently labelled proteins are released from the hydrogel of the invention.
  • the at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 80%, at least 90%, at least 95%, at least 99% fluorescently labelled proteins are released from the hydrogel of the invention in a first release.
  • a first exposure to a cleavage inducer Suitably after a first exposure to a cleavage inducer.
  • the release of the fluorescently labelled proteins from the hydrogel of the invention is repeated with the same hydrogel such that a second release takes place.
  • a second release takes place.
  • the release of the fluorescently labelled proteins from the hydrogel of the invention is repeated with the same hydrogel such that a second release takes place.
  • the first release suitably roughly about at least 10 %, at least 20%, at least 30%, at least 40%, at least 50% of the remaining fluorescently labelled proteins are released from the hydrogel of the invention in the second release.
  • at least 50% of the remaining fluorescently labelled proteins are released from the hydrogel of the invention in the second release.
  • Suitably fluorescently labeled proteins are released from the hydrogel of the invention in a third release, or fourth release. Suitably after a third or fourth exposure to a cleavage inducer.
  • the release of one or more fluorescently labelled proteins from the hydrogel of the invention takes at least about 5 seconds, at least about 10 seconds, at least about 15 seconds, at least about 20 seconds, at least about 25 seconds, at least about 30 seconds, at least about 35 seconds, at least about 40 seconds, at least about 45 seconds, at least about 50 seconds, at least about 55 seconds, at least about 60 seconds, at least about 65 seconds, at least about 70 seconds, at least about 75 seconds, at least about 80 seconds, at least about 85 seconds, at least about 90 seconds, at least about 95 seconds, at least about 100 seconds, at least about 105 seconds, at least about 110 seconds, at least about 115 seconds, at least about 120 seconds, at least about 180 seconds, at least about 240 seconds, at least about 300 seconds, at least about 600 seconds.
  • the hydrogel is exposed to the cleavage inducer for this length of time.
  • the release of one or more fluorescently labelled proteins from the hydrogel of the invention takes less than 10 minutes.
  • the hydrogel is exposed to a cleavage inducer for less than 10 minutes.
  • the fluorescently labelled proteins are released within 10 minutes.
  • 50 % of the fluorescently labelled proteins are released within at least about 100 seconds.
  • the fluorescently labelled streptavidin, CRP, IL8, or IL6 is released within 10 minutes.
  • 50% of the fluorescently labelled streptavidin, CRP, IL8, or IL6 is released within at least about 100 seconds.
  • the rhodamine labelled streptavidin, CRP, IL8, or IL6 is released within 10 minutes. In some embodiments, 50 % of the rhodamine labelled streptavidin, CRP, IL8, or IL6 are released within at least about 100 seconds.
  • determining the presence of proteins in the sample means conducting an assay to determine whether or not proteins are present in the sample. In other words, it is detected whether or not proteins are present in the sample.
  • proteins of the invention also encompass low abundance proteins.
  • the method detects proteins having a concentration of 1 ppm, 0.75 ppm, 0.5 ppm, 0.25 ppm, 0.1 ppm, 0.075 ppm, 0.05 ppm, 0.025 ppm, 0.01 ppm, 0.0075 ppm, 0.005 ppm, 0.0025 ppm, 0.0020ppm.
  • the methods have a lower limit of detection (LCD) of 1 ppm, 0.75 ppm, 0.5 ppm, 0.25 ppm, 0.1 ppm, 0.075 ppm, 0.05 ppm, 0.025 ppm, 0.01 ppm, 0.0075 ppm, 0.005 ppm, 0.0025 ppm, 0.0020ppm.
  • a lower limit of detection of 0.0020ppm.
  • the method detects proteins having a concentration of 0.1 ppm.
  • the method detects proteins having a concentration of 0.01 ppm.
  • the protein is streptavidin, and the limit of detection of the methods is 0.0033ppm.
  • the protein is CRP and the limit of detection of the methods is 0.0022ppm.
  • the protein is IL6 and the limit of detection of the methods is at least 0.005ppm.
  • the protein is IL8 and the limit of detection of the methods is at least 0.005ppm.
  • the method detects proteins of interest in the presence of interferents.
  • the method detects proteins of interest down to a low limit of detection, suitably down to the limits given above, in the presence of one or more interferents.
  • an interferent may be another protein which is not the protein of interest and which may bind to the hydrogel.
  • Suitable common interferents may be proteins present in biological samples, for example mucins, which are abundantly present in saliva.
  • the method detects proteins of interest in the presence of interferents at a concentration of up to 5ppm.
  • the hydrogel comprising a polymer formed of a plurality of inactive monomers which are acrylamide, and a plurality of active monomers which are polyethylene glycol methacrylamide, and the (molar) ratio of inactive monomers to active monomers is between 100 to 1 and 5 to 1, wherein each polyethylene glycol methacrylamide comprises at least one fluorescein isothiocyanate capable of covalently binding to a protein, and each fluorescein isothiocyanate is attached to said polyethylene glycol methacrylamide by a cleavable bond wherein said cleavable bond is o-nitrobenzyl, wherein the hydrogel is capable of detecting proteins having a concentration of 1 ppm, 0.75 ppm, 0.5 ppm, 0.25 ppm, 0.1 ppm, 0.075 ppm, 0.05 ppm, 0.025 ppm, 0.01 ppm, 0.0075 ppm, 0.005 ppm, 0.0025 ppm,
  • the hydrogel comprising a polymer formed of a plurality of inactive monomers which are acrylamide, and a plurality of active monomers which are polyethylene glycol methacrylamide, and the (molar) ratio of inactive monomers to active monomers is between 100 to 1 and 5 to 1, wherein each polyethylene glycol methacrylamide comprises at least one fluorescein isothiocyanate capable of covalently binding to a protein, and each fluorescein isothiocyanate is attached to said polyethylene glycol methacrylamide by a cleavable bond wherein said cleavable bond is o-nitrobenzyl, wherein the hydrogel is capable of detecting proteins having a concentration of 0.1 ppm.
  • the hydrogel comprising a polymer formed of a plurality of inactive monomers which are acrylamide, and a plurality of active monomers which are polyethylene glycol methacrylamide, and the (molar) ratio of inactive monomers to active monomers is between 100 to 1 and 5 to 1, wherein each polyethylene glycol methacrylamide comprises at least one fluorescein isothiocyanate capable of covalently binding to a protein, and each fluorescein isothiocyanate is attached to said polyethylene glycol methacrylamide by a cleavable bond wherein said cleavable bond is o-nitrobenzyl detects, wherein the hydrogel is capable of detecting proteins having a concentration of 0.01 ppm.
  • the hydrogel comprising a polymer formed of a plurality of inactive monomers which are acrylamide, and a plurality of active monomers which are polyethylene glycol methacrylamide, and the (molar) ratio of inactive monomers to active monomers is between 100 to 1 and 5 to 1, wherein each polyethylene glycol methacrylamide comprises at least one fluorescein isothiocyanate capable of covalently binding to a protein, and each fluorescein isothiocyanate is attached to said polyethylene glycol methacrylamide by a cleavable bond wherein said cleavable bond is o-nitrobenzyl detects, wherein the hydrogel is capable of detecting proteins having a concentration of 0.0020 ppm.
  • a method of measuring the amount of a protein of interest in a sample comprising: a. Contacting a sample with the hydrogel of any one of the preceding aspects and embodiments, the formulation of any one of the preceding aspects and embodiments, the oral formulation (optionally which may be a disc) of any one of the preceding aspects and embodiments, or sampling devices of any one of the preceding aspects and embodiments, the sampling device of any one of the preceding aspects and embodiments, or the substrate of any one of the preceding aspects and embodiments, under suitable conditions to allow proteins in the sample to bind to the hydrogel via the fluorophore, thereby obtaining fluorescently labelled proteins bound to said hydrogel; b.
  • step (a) Exposing the hydrogel of step (a) to a cleavage inducer under suitable conditions to cleave the cleavable bonds, thereby releasing said fluorescently labelled proteins from the hydrogel; c. Isolating the fluorescently labelled proteins; d. Contacting the fluorescently labelled proteins with a binding molecule capable of specifically binding to a protein of interest; e. Removing any unbound fluorescently labelled proteins; and f. Measuring the level of fluorescence, wherein the level of fluorescence is indicative of the amount of the protein of interest in the sample.
  • Contacting a sample is defined elsewhere herein.
  • the method described above includes the step of measuring the level of fluorescence of the protein of interest in the sample.
  • Conventional "measuring" methods may include sending a clinical sample(s) to a commercial laboratory for measurement the level of fluorescence in the protein of interest in the sample, or the use of commercially available assay kits for measuring the level of fluorescence in the sample. Exemplary kits and suppliers will be apparent to a person of skill in the art.
  • the amount of the protein of interested may be determined, detected and/or quantified using spectrophotometry, such as for point-of-care use.
  • the amount of the protein of interest in the sample may be determined by e.g. measuring the level of fluorescence present in the sample.
  • Assays for measuring the amount of a specified protein are well known in the art and include direct or indirect measures.
  • the amount of the protein of interest in a sample may also be determined by determining the level of protein of interest activity in a sample. Accordingly, fluorescence “level” encompasses both the amount of protein per se, or its level of activity.
  • proteins of the invention also encompass low abundance proteins.
  • Fluorescently labelled proteins is defined elsewhere herein.
  • the fluorescently labelled proteins comprise a large number of fluorophores relative to the proteins. This allows measuring low abundance proteins.
  • the expression “low abundance protein” is synonymously used with “proteins present at a low level”.
  • each fluorescently labelled protein is labelled with at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 fluorophores.
  • the fluorescently labelled protein is streptavidin.
  • the fluorescently labelled protein is CRP.
  • the fluorescently labelled protein is IL6.
  • the fluorescently labelled protein is IL8.
  • the fluorescently labelled protein is rhodamine-streptavidin.
  • the fluorescently labelled protein is labelled with the fluorophore fluorescein isothiocyanate.
  • the fluorescently labelled streptavidin, CRP, IL8, or IL6 is labelled with at least at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 fluorophores.
  • the fluorescently labelled streptavidin, CRP, IL8, or IL6 is labelled with at least at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 fluorescein molecules.
  • the fluorescently labelled streptavidin, CRP, IL8, or IL6 is labelled with at least 65 fluorescein molecules. Accordingly, the fluorescently labelled streptavidin comprises at least 65 fluorophores.
  • the fluorescently labelled streptavidin, CRP, IL8, or IL6 is labelled with 85 fluorescein molecules. Accordingly, the fluorescently labelled streptavidin , CRP, IL8, or IL6 comprises 85 fluorophores.
  • fluorescently labelled proteins refers to any suitable method in which the fluorescently labelled proteins are removed or separated from the hydrogel.
  • Isolating the fluorescently labelled proteins may comprise washing the hydrogel.
  • washing the hydrogel for a sufficient time to elute the fluorescently labelled proteins therefrom.
  • the washing may be performed with a buffer, such as PBS.
  • the washing is performed for between 10 to 30 minutes.
  • the washing is performed for at least 10 minutes.
  • binding molecule is a small molecule or antibody.
  • the small molecule is biotin.
  • Binding molecules also include non-immunoglobulin binding agents, such as phage display-derived peptide binders, and antibody mimics, e.g., affibodies, tetranectins (CTLDs), adnectins (monobodies), anticalins, DARPins (ankyrins), avimers, iMabs, microbodies, peptide aptamers, Kunitz domains, aptamers and affilins.
  • CTLs tetranectins
  • adnectins monobodies
  • anticalins DARPins (ankyrins)
  • DARPins ankyrins
  • avimers iMabs, microbodies, peptide aptamers, Kunitz domains, aptamers and affilins.
  • antibody includes, for example, both naturally occurring and non-naturally occurring antibodies, polyclonal and monoclonal antibodies, chimeric antibodies and wholly synthetic antibodies and fragments thereof, such as, for example, the Fab', F(ab')2, Fv or Fab fragments, or other antigen recognizing immunoglobulin fragments.
  • Antibodies which bind a particular epitope can be generated by methods known in the art.
  • polyclonal antibodies can be made by the conventional method of immunizing a mammal (e.g., rabbits, mice, rats, sheep, goats).
  • Monoclonal antibodies are then contained in the sera of the immunized animals and can be isolated using standard procedures (e.g., affinity chromatography, immunoprecipitation, size exclusion chromatography, and ion exchange chromatography). Monoclonal antibodies can be made by the conventional method of immunization of a mammal, followed by isolation of plasma B cells producing the monoclonal antibodies of interest and fusion with a myeloma cell (see, e.g., Mishell, et al., 1980).
  • Screening for recognition of the epitope can be performed using standard immunoassay methods including ELISA techniques, radioimmunoassays, immunofluorescence, immunohistochemistry, and Western blotting (Ausubel, et al., 1992).
  • In vitro methods of antibody selection such as antibody phage display, may also be used to generate antibodies (see, e.g., Schirrmann et al. 2011).
  • the binding molecule may be located upon a surface.
  • the binding molecule may be located in a container.
  • the contacting step takes place within a container comprising the binding molecule.
  • the contacting step may comprise contacting the fluorescently labelled proteins with a surface comprising the binding molecule capable of specifically binding to a protein of interest.
  • the contacting step may comprise contacting the fluorescently labelled proteins with a container comprising the binding molecule capable of specifically binding to a protein of interest.
  • the contacting step may comprise contacting the fluorescently labelled proteins with a microwell plate comprising the binding molecule capable of specifically binding to a protein of interest.
  • the container may be any commercially available microwell plate coated with the binding molecule of interest.
  • a biotin-coated microwell plate e.g. 15151, Thermo Fisher Scientific
  • the container is a container of the invention as defined below.
  • the container comprises an inner surface operable to be contacted with the fluorescently labelled proteins, wherein the inner surface is coated with a base layer upon which is coated a reactive layer, the reactive layer comprising a mixture of a binding molecule and a blocking agent, wherein the blocking agent comprises a compound which does not contain amine groups.
  • the base layer a polymer comprising free amine groups.
  • such polymers are, for example acrylamide/bisacrylamide optionally copolymerised with aminopropyl methacrylamide, or 4 arm PEG succinimidyl ester (NHS) copolymerised with PEG-bis amine, or chitosan.
  • the base layer is chitosan.
  • the binding molecule is a protein which recognises and binds to a protein of interest, for example biotin or an antibody, suitably an antibody or a binding fragment thereof which specifically binds to the protein of interest.
  • the blocking agent comprises PEG-methyl.
  • the capture protein and the blocking agent bind to the base layer.
  • the reactive layer comprises a majority of reactive proteins, and a minority of blocking agent.
  • the blocking agent is bound to the base layer only where the reactive protein is not bound to the base layer.
  • the blocking agent binds to any free amine groups in the base layer.
  • the blocking agent prevents free FITC binding to the base layer of the container, suitably when in use.
  • the container may be any container suitable for carrying out a method of the invention therein.
  • the container is a microwell or microtitre plate.
  • the container of the invention reduces the background signal by a factor of 110 compared to commercially available microwell or microtitre plates.
  • the container of the invention reduces the background signal caused by free fluorophore, specifically free FITC, binding directly to the container.
  • the binding molecule is biotin. In one embodiment the binding molecule is an anti-CRP antibody or binding fragment thereof. In one embodiment, the binding molecule is an anti-l L8 antibody or binding fragment thereof. In one embodiment, the binding molecule is an anti-IL6 antibody or binding fragment thereof.
  • “removing any unbound fluorescently labelled proteins” refers to any suitable method in which said unbound fluorescently labelled proteins are removed such as by one or more wash steps comprising for example a buffer.
  • Sutiably removing any unbound fluorescently labelled proteins comprises washing, suitably washing the container described above, suitably washing the microwell plate as described above.
  • the level of fluorescence in a sample can be determined (e.g., measured) by any suitable methods and materials known in the art, including, for example, a process selected from the group consisting of spectrofluorometry, protein microarrays, immunoprecipitation, immunofluorescence, Western blot analysis, Lateral Flow (using e.g. Lateral Flow Devices (LFDs) utilising a membrane bound antibody specific to the protein biomarker).
  • LFDs Lateral Flow Devices
  • the protein of interest is a biomarker.
  • a biomarker is an organic biomolecule (e.g. a protein, polypeptide, peptide, isomeric form thereof, immunologically detectable fragment thereof, which is differentially present in a sample taken from a subject having a disease as compared with a subject not having the disease.
  • a biomarker is differentially present if the mean or median level of the biomarker in the different groups is calculated to be statistically significant. Common tests for statistical significance include, among others, t-test (e.g., Student t-test), ANOVA, Kruskal-Wallis, Wilcoxon, Mann- Whitney, Receiver Operating Characteristic (ROC curve), accuracy and odds ratio.
  • Biomarkers alone or in combination, provide measures of relative risk that a subject belongs to one phenotypic status or another. Therefore, they are useful as markers for disease (diagnostics), therapeutic effectiveness of a drug and drug toxicity.
  • the biomarker referred to herein is measured at the protein level.
  • the biomarker can be any biomarker which is detected or measured at the protein level.
  • the biomarker is a protein biomarker.
  • the biomarker may be a marker of a diseases.
  • the biomarker may be an infammatory marker.
  • a biomarker of inflammation Sutibaly such biomarkers of inflammation typically indicate disease.
  • the biomarker may be a chemokine or cytokine.
  • the biomarker is cardiac troponin, creatinine kinase, creatinine kinase-MB, myoglobin, IL-1, IL-2, IL-3, IL4-, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL- 15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21 , IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL- 29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, IL-38, IL-39, IL-40, prostate-specific antigen (PSA), prostatic acid phosphatase (PAP), CA 125, carcinoembryonic antigen (CEA), alpha-fe
  • the biomarker is IL-6, IL-8, cardiac troponin, or CRP.
  • the cardiac troponin is troponin C (TnC), troponin T (TnT), or troponin I (Tnl).
  • the cardiac troponin is troponin T (TnT).
  • a method of determining whether a subject has a disease or disorder comprising: a. Contacting a sample from the subject with the hydrogel of any one of the preceding aspects and embodiments, the formulation of any one of the preceding aspects and embodiments, the oral formulation (optionally which may be a disc) of any one of the preceding aspects and embodiments under suitable conditions, the sampling devices of any one of the preceding aspects and embodiments, or the substrate of any one of the preceding aspects and embodiments, under suitable conditions to allow proteins in the sample to bind to the hydrogel via the fluorophore, thereby obtaining fluorescently labelled proteins bound to said hydrogel; b.
  • step (a) Exposing the hydrogel of step (a) to a cleavage inducer under suitable conditions to cleave the cleavable bonds, thereby releasing fluorescently labelled proteins from the hydrogel; c. Isolating the fluorescently labelled proteins; d. Contacting the fluorescently labelled proteins with a binding molecule capable of specifically binding to a protein biomarker of a disease or disorder; e. Removing any unbound fluorescently labelled proteins; f. Detecting the presence of fluorescence or measuring the level of fluorescence, wherein the presence of fluorescence is indicative of the presence of the protein biomarker in the sample, or wherein the level of fluorescence is indicative of the level of the protein biomarker in the sample, g. Determining based on f. that the subject has a disease or disorder, wherein the presence of fluorescence or the level of fluorescence is indicative of a disease or disorder.
  • Contacting a sample is defined elsewhere herein.
  • Fluorescently labelled proteins is defined elsewhere herein.
  • Binding molecule is defined elsewhere herein.
  • proteins of the invention also encompass low abundance proteins.
  • detecting the presence of fluorescence means conducting an assay to determine whether or not fluorescence is present, proteins are present in the sample.
  • the presence of fluorescence is indicative of the presence of the protein biomarker in the sample.
  • solely the presence of the protein biomarker in the sample is indicative of a disease or disorder. This means that if such a protein biomarker is present in the sample, the subject has a disease or disorder.
  • “Measuring the level of fluorescence” is defined elsewhere herein.
  • the level of fluorescence is indicative of the level of the protein biomarker in the sample.
  • the level of the protein biomarker in the sample is indicative of a disease or disorder. Accordingly, if a defined level or threshold of said protein biomarker is present in the sample, the subject has a disease or disorder.
  • the level of fluorescence is indicative of the level of the protein biomarker IL-6, IL-8, or cardiac troponin.
  • the level of fluorescence is indicative of the level of the protein biomarker troponin C (TnC), troponin T (TnT), or troponin I (Tnl).
  • the level of fluorescence is indicative of the level of the protein biomarker troponin T (TnT).
  • the disease or disorder is cancer or cardiovascular disease.
  • cancers include, but are not limited to, lung cancer (e.g. , bronchogenic carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung); kidney cancer (e.g., nephroblastoma, a.k.a.
  • SCLC small cell lung cancer
  • NSCLC non-small cell lung cancer
  • kidney cancer e.g., nephroblastoma, a.k.a.
  • Wilms' tumour, renal cell carcinoma acoustic neuroma; acute myeloid leukaemia; adenocarcinoma; adrenal gland cancer; anal cancer; angiosarcoma (e.g., lymphangio sarcoma, lymphangioendotheliosarcoma, hemangio sarcoma); appendix cancer; benign monoclonal gammopathy; biliary cancer (e.g. , cholangiocarcinoma); bladder cancer; breast cancer (e.g., adenocarcinoma of the breast, papillary carcinoma of the breast, mammary cancer, medullary carcinoma of the breast); brain cancer (e.g.
  • meningioma meningioma, glioblastomas, glioma (e.g., astrocytoma, oligodendroglioma), medulloblastoma); bronchus cancer; carcinoid tumour; cervical cancer (e.g.
  • cervical adenocarcinoma cervical adenocarcinoma
  • choriocarcinoma chordoma
  • craniopharyngioma colorectal cancer (e.g., colon cancer, rectal cancer, colorectal adenocarcinoma); connective tissue cancer; epithelial carcinoma; ependymoma; endothelio sarcoma (e.g., Kaposi' s sarcoma, multiple idiopathic haemorrhagic sarcoma); endometrial cancer (e.g.
  • uterine cancer uterine sarcoma
  • oesophageal cancer e.g., adenocarcinoma of the oesophagus, Barrett's adenocarcinoma
  • Ewing's sarcoma ocular cancer (e.g., intraocular melanoma, retinoblastoma); familiar hypereosinophilia; gall bladder cancer; gastric cancer (e.g., stomach adenocarcinoma); gastrointestinal stromal tumour (GIST); germ cell cancer; head and neck cancer (e.g.
  • oral cancer e.g., oral squamous cell carcinoma
  • throat cancer e.g., laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer
  • heavy chain disease e.g.
  • alpha chain disease alpha chain disease, gamma chain disease, mu chain disease; hemangioblastoma; hypopharynx cancer; inflammatory myofibroblastic tumours; immunocytic amyloidosis; liver cancer (e.g., hepatocellular cancer (HCC), malignant hepatoma); leiomyosarcoma (LMS); mastocytosis (e.g., systemic mastocytosis); muscle cancer; myelodysplastic syndrome (MDS); mesothelioma; myeloproliferative disorder (MPD) (e.g., polycythemia vera (PV), essential thrombocytosis (ET), agnogenic myeloid metaplasia (AMM) a.k.a.
  • MPD myeloproliferative disorder
  • MPD e.g., polycythemia vera (PV), essential thrombocytosis (ET), agnogenic myeloid metaplasia (AM
  • MF myelofibrosis
  • CML chronic myelocytic leukaemia
  • CNL chronic neutrophilic leukaemia
  • HES hypereosinophilic syndrome
  • neuroblastoma e.g.
  • neurofibromatosis NF
  • type 1 or type 2 schwannomatosis
  • neuroendocrine cancer e.g., gastroenteropancreatic neuroendoctrine tumour (GEP-NET), carcinoid tumour
  • osteosarcoma e.g., bone cancer
  • ovarian cancer e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma
  • papillary adenocarcinoma pancreatic cancer (e.g., pancreatic adenocarcinoma, intraductal papillary mucinous neoplasm (IPMN), Islet cell tumours)
  • penile cancer e.g., Paget' s disease of the penis and scrotum
  • pinealoma primitive neuroectodermal tumour (PNT); plasma cell neoplasia; paraneoplastic syndromes; intraepithelial neoplasms; prostate cancer (
  • prostate adenocarcinoma rectal cancer; rhabdomyosarcoma; salivary gland cancer; skin cancer (e.g., squamous cell carcinoma (SCC), keratoacanthoma (KA), melanoma, basal cell carcinoma (BCC)); small bowel cancer (e.g., appendix cancer); soft tissue sarcoma (e.g., malignant fibrous histiocytoma (MFH), liposarcoma, malignant peripheral nerve sheath tumour (MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma); sebaceous gland carcinoma; small intestine cancer; sweat gland carcinoma; synovioma; testicular cancer (e.g., seminoma, testicular embryonal carcinoma); thyroid cancer (e.g., papillary carcinoma of the thyroid, papillary thyroid carcinoma (PTC), medullary thyroid cancer); urethral cancer; uveal melanom
  • cardiovascular diseases include, but are not limited to coronary artery diseases (e.g. angina, heart attack), stroke, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, congenital heart disease, valvular heart disease, carditis, aortic aneurysms, peripheral artery disease, thromboembolic disease, and venous thrombosis.
  • the presence of fluorescence is the existence of fluorescence and may optionally also encompass determining the level of fluorescence, for example a low level of fluorescence, a medium level of fluorescence, or a high level of fluorescence.
  • the presence of fluorescence or level of fluorescence in a sample can be determined (e.g., measured) by any suitable methods and materials known in the art as defined elsewhere herein.
  • Slide-A- Lyzer Dialysis Cassettes MWCO 2K, Thermo Fisher
  • PierceTM Biotin Coated Plates (Clear, 8-Well Strip, Thermo Scientific) were used for protein release studies.
  • Microscope glass slides (1 mm thick low iron standard) were purchased from VWR (Leicestershire, UK).
  • 1 H and 13 C NMR spectra were obtained at 25 °C and recorded on a 300 MHz Varian VNMRS, 400 MHz Varian Unity Inova or 600 MHz Varian Unity Inova. Chemical shifts (5) are reported in parts per million (ppm), and splitting patterns are designated as s (singlet), d (doublet), dd (doublet of doublets), t (triplet), q (quartet), m (multiplet) and brs (broad singlet) and coupling constants (J) are expressed in Hertz (Hz).
  • 1 H and 13 C NMR spectra are referenced to the residual solvent signal: DMSO-de (2.50 or 39.52 ppm) and CDCI3 (7.26 or 77.16 ppm).
  • Irradiation at 365 nm was carried out using an in-house constructed source based on a UV LED (Nichia NVSU233A-U365, RS Components) with a radiant flux of 1030 mW at 1A forward current expanded to 25 mm diameter, giving a flux density of -210 mW cm -2 .
  • the LED and DC-DC converter components driving the LED were mounted on a 60x60 mm 2 aluminum-cored PCB, which in turn was mounted to a 60x60 mm 2 heat sink and fan (Thermo Electric Devices TDEX6015/TH12G, RS Components) using thermally conductive paste.
  • the LED driver and fan were powered by a 12 V 1A DC mains adaptor.
  • UV-Vis spectroscopy measurements were carried out on a Jenway 6715 UV-Vis spectrometer at 25 °C.
  • High-performance liquid chromatography (HPLC) was performed using Agilent 1260 Infinity coupled with a VanquishTM multi wavelength absorbance detector and in a C18 column (1.8 pm particle size, 3 mm internal diameter and 50 mm long).
  • NVOC 300 mg, 1.00 mmol, 1 equiv
  • anhydrous DMF 5 mL
  • allylamine 75 pL, 1.00 mmol, 1 equiv
  • HATLI 381 mg, 1.00 mmol, 1 equiv
  • DIPEA 873 pL, 5.00 mmol, 5 equiv
  • NVOC-allylamide (135 mg, 0.40 mmol, 1 equiv) was dissolved in dry DCM (5 mL) in a nitrogen purged oven dried 25 mL RBF wrapped in aluminum foil. The mixture was cooled to 0 °C and phosgene solution (15 wt.% in toluene) (455 mL, 0.60 mmol, 1.5 equiv) was added dropwise over 20 min and subsequently stirred at 0 °C for an hour.
  • Fluorescein sodium salt 450 mg, 1.20 mmol, 2 equiv
  • TEA 83 pL, 0.60 mmol, 1.5 equiv
  • the first reaction mixture was added dropwise under inert conditions for over 30 min. The reaction proceeded for 2 h in an ice bath and then allowed to cool to room temperature overnight.
  • Amine-PEG34oo-methacrylamide (3 g, 1 equiv) was dissolved in 2 mL of DMF.
  • NVOC 399 mg, 1.33 mmol, 1.5 equiv.
  • HATU 229 mg, 0.70 mmol, 1.4 equiv
  • DIPEA 183 pL DIPEA
  • NVOC-PEG34oo-methacrylamide was purified by immediate precipitation in diethyl ether, redissolving in DCM and final precipitation in diethyl ether. The resulting pellet was dried under vacuum at 40 °C.
  • This pellet was divided into two and reacted with either fluorescein or FITC using the same procedure described above.
  • the monomers were purified by dialysis and lyophilized, resulting in a sticky, off-white powder (F-NVOC-PEG34oo-methacrylamide, 680 mg, 59%; FITC-NVOC-PEG34oo-methacrylamide, 682 mg, 60%).
  • Glass microscope slides were cut into 25.4 ⁇ 0.5 mm 2 and cleaned in Decon 90, water, and ethanol for 30 min each in an ultrasonic bath.
  • the glass squares were immersed in 0.5% v: v allyltrichlorosilane in toluene for 30 min, washed in toluene, and dried before use.
  • the precursor solution was cast between glass squares and plastic covers separated with a spacer of 175 pm thickness (939-837-76, Goodfellow) at room temperature under dark. After 15 min, plastic covers were removed, and hydrogel films deposited on glass substrates were soaked in PBS overnight to remove any residual unreacted monomers.
  • UV-Vis absorption spectra of a hydrogel film (sample 8 in Table 1) deposited on glass slides were measured at different regions to determine area-to-area variability. Equally, absorbance at a wavelength of -490 nm of precursor solutions and hydrogel films (see T able 1) were recorded. Using these absorbance values and given the path length of cuvettes containing precursor solutions and thickness of films, we the inventors estimated the molar percentage of fluorescein containing monomer incorporated in the hydrogel films. To determine the kinetics of light-triggered release of fluorescein from hydrogel films, the films were exposed to 365 nm light for selected durations, and their UV-Vis spectra were measured after each irradiation time interval.
  • Protein studies were carried out using functionalised hydrogels made by co-polymerizing FITC-NVOC- PEG34oo-methacrylamide with acrylamide/bisacrylamide.
  • the percentage molar fraction of FITC- NVOC-PEG34oo-methacrylamide was 10% and total concentration of monomers in the precursor solution was 10% w: v
  • Hydrogel films made by polymerising 10% w: v acrylamide/bisacrylamide served as negative controls.
  • UV-Vis and fluorescence emission spectra of different concentration of rhodamine-streptavidin (RS) in PBS were measured.
  • Hydrogel films of acrylamide/bisacrylamide without and with FITC-NVOC- PEG34oo-methacrylamide were submerged in a 10 mL stock solution of 0.01 or 0.1 ppm RS in PBS overnight. Fluorescence emission spectra of PBS before and after incubation with each type of hydrogel films were measured. The data was used to determine the factor by which proteins were pre- concentrated.
  • the hydrogel films were washed in fresh PBS at room temperature in dark conditions overnight to remove any unbound RS. Subsequently, the films were immersed in 3 mL PBS and exposed to UV-light irradiation in short time bursts between 1 and 10 min. After each irradiation, the inventors waited for 30 min to allow the released RS to diffuse out of hydrogel films, and then collected PBS. The hydrogel films were then immersed in 3 mL of fresh PBS and the above process was repeated. The light-triggered release kinetics of RS was studied by monitoring the fluorescence emission corresponding to rhodamine with irradiation times. All measurements were performed in triplicate.
  • Figure 9 shows that the peak absorbance wavelength for F-NVOC-allylamide dissolved in DMSO was observed at 519 nm with a shoulder at -490 nm, suggesting aggregation. The aggregation was further supported by the observation that F-NVOC-allylamide solution in DMSO was largely non-fluorescent.
  • F-NVOC-PEG400- methacrylamide and F-NVOC-PEG34oo-methacrylamide monomers were water soluble and absorbed at wavelengths of -490 nm and -365 nm, which was attributed to fluorescein and NVOC groups.
  • Photolysis occurs when the irradiation wavelength overlaps with the absorption band of the photolabile group 28 ' 30 , in this case, NVOC.
  • the monomers solutions were irradiated to 365 nm UV-light.
  • a typical UV-Vis spectra of the monomers for different irradiation times showed that the peak attributed to the NVOC group was red shifted, indicating that nitrosobenzaldehyde was produced and hence the photoreactions were successful.
  • the absorption at -490 nm remained unchanged with irradiation times because both released fluorescein and fluorescein bound to the monomer remained in the same solution.
  • Hydrogels were prepared by free radical co-polymerisation of acrylamide/bisacrylamide with the different monomers.
  • the compositions of hydrogels studied in this work are summarized in Table 1.
  • the hydrogels were cast on a glass slide for support and the ability to place the slide inside a UV-Vis spectrophotometer.
  • Figure 5(a) shows a hydrogel film on a glass slide under white light with a £1 coin as a size reference
  • Figure 5(b) is the same slide under 365 nm illumination showing the emission of the fluorescein in the hydrogel.
  • Figure 5(c) gives the UV-visible absorption spectra of the four different corners of that slide, showing that the film is not quite uniform.
  • Figure 5(d) gives the monomer incorporation factors for the different types and molar ratios of the NVOC containing monomers.
  • the incorporation factor was defined as the ratio of the molar concentrations of the NVOC containing monomers in hydrogels (mhydrogei) and precursor solutions (m pr ecursor).
  • the mhydrogei and mprecursor were estimated based on the absorbance of the hydrogel and precursor solutions at -490 nm and using the molar extinction coefficient of fluorescein ( Figure 15).
  • the PEG-based methacrylamide monomers exhibited increased monomer incorporation factors when compared to the allylamide monomer. This was attributed to an increase in monomer reactivity (methacrylamide > allylamide) 31 and the water solubility of the PEG-based monomers. There was a slight decrease in monomer incorporation with increasing PEG molecular weight.
  • Figure 6(a) shows the decrease of the absorption band of fluorescein in the hydrogel film of slide 8 (Table 1) as the irradiation time increases.
  • Figure 6(b) shows the first-order reaction rate plot of the decrease in fluorescein concentration in this hydrogel, indicating that the release is first-order up to 30 min of irradiation. Similar results were obtained for the other prepared hydrogels, showing first-order release kinetics up to 30 min of irradiation. This suggests that fluorescein, once released, can rapidly diffuse out of the hydrogel, but the non-linearity after -30 min irradiation suggests that there may be a small amount of irreversibly bound fluorescein remaining in the hydrogel.
  • F-NVOC-PEG34oo-methacrylamide could be prepared in high yield (59% compared to only 35% for F-NVOC-PEG4oo-methacrylamide) and did not require purification by flash chromatography. Furthermore, the kinetics of release of fluorescein in solutions of F- NVOC-PEG34oo-methacrylamide was the fastest. Finally, the percentage of monomer incorporated in the hydrogel with respect to the precursor solution was higher for F-NVOC- PEG34oo-methacrylamide than F-NVOC-allylamide (-32% and 65%, respectively).
  • Rhodamine-streptavidin was selected as an exemplar protein to study preconcentration, labelling and release of proteins followed by their quantification using fluorescence in biotin coated microtiter plates.
  • the streptavidin used in this work was tagged with rhodamine so that fluorescence of rhodamine can be used to determine the protein concentration before and after incubation with hydrogels. This in turn provided the pre- concentration factor.
  • Streptavidin was chosen because of its very strong and selective binding to biotin, which made it easy to capture in biotinylated microtiter plates after being released from hydrogels.
  • the functionalized hydrogels which were prepared by co-polymerizing acrylamide/bisacrylamide and FITC-NVOC-PEG 34 oo-methacrylamide monomers, were capable of protein capture because the isothiocyanate groups reacted with the terminal amine and primary amines in proteins 32 . This was confirmed by submerging the formed hydrogels in 10 mL of 0.10 and 0.01 ppm (1.66 and 0.166 nM, respectively) RS solutions in PBS. As shown in Figure 8(a) and (b), a majority of RS was lost from the solution after overnight incubation with a functionalized hydrogel. In contrast, control experiments with unfunctionalized polyacrylamide hydrogels resulted in no significant loss of RS from solutions. The negligible reduction in fluorescence from the stock solutions exposed to the unfunctionalized hydrogels shows that the RS conjugate does not bind significantly in the absence of the FITC moiety.
  • the loaded hydrogels were exposed to UV in short bursts of between 1 and 10 min while submerged in 3 mL PBS, kept in PBS for 30 min to allow for the released RS to diffuse out of the hydrogel, and then the PBS was collected. This process was repeated until no additional RS could be detected in the supernatant PBS.
  • the released RS was labelled with fluorescein
  • one of the PBS solutions containing labelled protein collected from a hydrogel incubated with 0.01 ppm RS and exposed to UV light was dispensed into a biotin coated well of a microtiter plate.
  • the released streptavidin was allowed to bind to biotin for 30 min and then a buffer wash was performed. This process allowed removal of any fluorescein dye, not bound to protein but released when the hydrogel was irradiated with UV-light.
  • the well of the microtiter plate was illuminated with 470 and then 540 nm light.
  • the hydrogel was formed by copolymerizing acrylamide/bisacrylamide with designed monomers.
  • the designed monomers in this example comprised of either fluorescein (F) or fluorescein isothiocyanate (FITC) attached to a polymerizable group via a light cleavable bond achieved using o-nitrobenzyl (NVOC).
  • the monomers polymerizable group was either allylamide or methacrylamide.
  • a polyethylene glycol (PEG) spacer arm may or may not be present between the NVOC and the polymerizable group.
  • the inventors showed that the incorporation of methacrylamide monomer with 3400 g mol -1 molecular weight PEG spacer arm (F-NVOC-PEG3400- methacrylamide) in hydrogels was -65%, which was double than the allylamide monomer without PEG.
  • the other benefits offered by the F-NVOC-PEG34oo-methacrylamide were water solubility, ease of preparation with high yield, and fast light-triggered release kinetics.
  • the designed hydrogels offered a pre-concentration factor of 192 and 236 for 0.1 and 0.01 ppm of an exemplar protein, streptavidin. Once pre-concentrated, the proteins were released by UV irradiation, leaving a free protein with a fluorescein label. The inventors showed that 50% of streptavidin was released from our hydrogels in -100 s and was labelled with 85 fluorescein molecules per one molecule of the protein. The labelling of proteins with large numbers of fluorophores can allow measurement of proteins present at low levels. Finally, the designed hydrogel when combined with capture of released streptavidin using biotin and fluorescence detection, allowed detection of at least 0.01 ppm (or -166 pM) of the protein.
  • the hydrogels are promising for protein preconcentration, labelling and on-demand release.
  • the hydrogels when used in combination with selective capture of labelled proteins and fluorescence detection, offer potential for measurement of low abundance proteins, enabling early detection of diseases.
  • the polyacrylamide hydrogels of the invention can pre-concentrate proteins by factor of -295. Proteins captured by the hydrogels can be released by illuminating with 365 nm light in ⁇ 10 min. The released proteins can be detected specifically and quantified using microtitre plates coated with recognition elements (e.g., biotin, anti-CRP and anti-IL8). Specific detection of proteins such as streptavidin, CRP and IL8 was shown, and calibration curves for streptavidin and CRP were developed to show that they can be quantified. The limit of detection (LOD) for streptavidin and CRP was proven to be 0.0033 ppm (or 60 pM) and 0.0022 ppm (or 19 pM), respectively.
  • LOD limit of detection
  • F-NVOC-allylamide, F-NVOC-PEG4oo-methacrylamide, F-NVOC-PEG34oo-methacrylamide and FITC-NVOC-PEG34oo-methacrylamide monomers were prepared as described above in section 1.2 and Figure 2.
  • acrylamide bisacrylamide solution was used as a stock solution.
  • the weight ratio of acrylamide to bisacrylamide was 29:1.
  • the required quantity of acrylamide: bisacrylamide stock solution was mixed with the active monomer (see Table 1.1 for details) and ultrapure water was added.
  • the total concentration of crosslinker and monomers in precursor solutions was 5% (w:v).
  • Required quantities of APS and TEMED shown in Table 1.1 were added, and the solution was thoroughly mixed. The solution was used to make either films on glass slides or discs using procedures described below.
  • Hydrogel films were prepared in a similar method to that used in section 1.3.
  • Hydrogel discs were prepared as follows: 2 mL of selected precursor solution (see Table 3) was poured into cassettes to form 1.2 mm thick hydrogel sheets. Discs were punched out from hydrogel sheets using a cork borer. The diameter and thickness of the discs were 6 mm and 1.2 mm, respectively. Discs were washed with 5 mL PBS (20 °C, overnight) and stored in PBS in the dark until use.
  • biotin coated microtitre plates (15151, Thermo Fisher Scientific) were used to perform a few studies.
  • the procedure for preparing in-house biotin coated microtitre plates was as follows: 1.5% chitosan was prepared by dissolving 0.015 g chitosan in 1 mL of 0.1 M acetic acid. The solution was stirred overnight. 300 pL of the solution was pipetted into a 48-well microtiter plate and oven dried at 75 °C for 2 h. 200 pL of 10 mM PBS, pH 7.4 was pipetted in each well, removed, and the process was repeated five times.
  • RP-HPLC reversed-phase high-performance liquid chromatography
  • the stationary phase was a C18 column.
  • the mobile phase gradient was varied from 10/90 to 90/10 MeCN/H2O from 0 to 10 min.
  • a UV detector was used, and absorbance was recorded at 491 nm.
  • the resulting chromatograms for the variants of the active monomer (F-NVOC-allylamide, F-NVOC-PEG4oo-methacrylamide and F-NVOC- PEG34oo-methacrylamide) are provided in Figures 13 and 4(a).
  • the peaks corresponding to released fluorescein and that attached to monomers are marked as F and M, respectively.
  • the kinetics of light-triggered release of fluorescein was investigated by monitoring the rate of disappearance of the fluorescein bound to monomers ([M] t ). Plots of ln[M]o/[M] t versus exposure time (see Figure 4(b)) show excellent linearity, indicating the expected first order kinetics. Furthermore, the rate plots provided in Figure 4(b) show that the kinetics of light- triggered release of fluorescein from PEG-methacrylamide monomers was faster than the allylamide monomer.
  • the time constant for release kinetics of fluorescein from F-NVOC- allylamide, F-NVOC-PEG4oo-methacrylamide and F-NVOC-PEG34oo-methacrylamide was 9.1 , 6.8 and 4.8 min, respectively.
  • Hydrogel films deposited on glass slides containing 1 :10 molar ratio of active to inactive monomers were used. The total crosslinker plus monomers concentration was 10% (w:v). Hydrogel films were immersed in fresh PBS and exposed to 365 nm light for selected time. The gel was left in the PBS for 10 mins following which the absorbance of hydrogel films was measured. The procedure was repeated to expose hydrogel discs for up to 60 min.
  • Figure 6(a) shows a decrease in the absorption peak of fluorescein (at -490 nm) in a hydrogel film as the exposure time to 365 nm light increases.
  • a plot of peak absorbance versus exposure time shows exponential decay to a minimum.
  • the corresponding semi-logarithmic plot of the decrease in fluorescein concentration in this hydrogel versus exposure time is provided in Figure 6(b), indicating that the release is first order up to 30 min of exposure. Similar results were obtained for the other prepared hydrogels, showing first-order release kinetics up to 30 min of exposure.
  • F-NVOC-PEG34oo-methacrylamide could be prepared in high yield (59% compared to only 35% for F-NVOC-PEG4oo-methacrylamide) and did not require purification by a tedious method, flash chromatography. Furthermore, as shown in Figure 4(b), the kinetics of the release of fluorescein in solutions of F-NVOC-PEG34oo-methacrylamide was the fastest. Finally, the percentage of monomer incorporated in the hydrogel with respect to the precursor solution was higher for F-NVOC-PEG34oo-methacrylamide than F-NVOC-allylamide (65% and -32%, respectively, see Table 4 and Figure 5d). Considering these benefits offered by F-NVOC-PEG34oo-methacrylamide, the remaining work was carried out using (F or FITC)-NVOC-PEG34oo-methacrylamide.
  • hydrogels films deposited on glass slides are not an ideal format to be placed in people’s mouth.
  • hydrogel discs with sizes comparable to mentos.
  • the bottom plastic had a -25x25 mm 2 square cavity with a depth of 1.2 mm.
  • the top plastic had two through holes that served fluidic inlet and outlet.
  • the top and bottom plastics were clamped together by screws following which, precursor solution was introduced in the cavity through the inlet in the top plastic.
  • the precursor solution was left in the cavity to polymerise, and 6 mm diameter discs were punched out using a cork borer.
  • the resulting discs had sufficient mechanical strength that they could be easily transferred from one solution to another using a spatula.
  • the concentration of RS in hydrogel discs was determined to be 1.2615 ppm (versus 0.00428 ppm in solution).
  • the pre-concentration factor was -295.
  • the proteins were released by exposing hydrogel discs to 365 nm light while being immersed in 200 pL PBS.
  • the pre-concentration factor for the released proteins was -50.
  • Hydrogels made using 5% (w:v) precursor solutions were used. The molar ratio of active to inactive monomers was varied between 1:10, 1:20, 1:40 and 1:100. Hydrogel discs were incubated in 10 mL of 0.005 ppm RS solution in PBS for 24 h, washed overnight in PBS and subsequently exposed to 365 nm light for a total of 60 min. After each exposure, the supernatant was collected and the fluorescence at peak wavelength versus exposure time was plotted and shown in Figure 20.
  • a 1:40 active to inactive molar ratio polyacrylamide hydrogel disc was incubated in 10 mL of 0.005 ppm streptavidin in PBS for 24 h, after which the hydrogel disc was washed overnight in PBS. The hydrogel disc was then exposed to 365 nm light for a total of 60 min. After each exposure, the disc was washed with 200 pL of PBS for either 10 or 30 min to determine if the wash time had any significant effect on the released streptavidin fluorescence signal.
  • Figure 26 shows that the wash time has no significant effect on the streptavidin fluorescence signal. This means that released proteins can rapidly diffuse out of hydrogels and hence a wash time of 10 min was sufficient.
  • CRP was captured, labelled and released using the 1:40 molar ratio active to inactive monomer hydrogel using the same method as described above.
  • Anti-CRP antibody was immobilised onto chitosan in the wells of a microtitre plate using a procedure similar to that used for the biotinylated wells. 1.5% chitosan was prepared by dissolving 0.015 g chitosan in 1 mL of 0.1 M acetic acid. The solution was stirred overnight. 300 pL of the solution was pipetted into a 48-well microtiter plate and oven dried at 75 °C for 2 h. 200 pL of 10 mM PBS, pH 7.4 was pipetted in each well, removed, and the process was repeated five times.
  • Figure 28 shows the fluorescence signal from anti-CRP coated microtitre plates for CRP of original concentrations 0, 0.0025, 0.005, 0.0075 and 0.01 ppm in PBS as a function of exposure time showing that as the concentration of CRP increases, so does the fluorescence signal.
  • a CRP calibration curve is shown in Figure 28(b). Based on the calibration curve, the LOD for CRP was 0.0022 ppm, or 19 pM.
  • hydrogels comprised of different types of monomers and crosslinkers, for example using a poly(ethylene glycol) (PEG) based hydrogel.
  • PEG poly(ethylene glycol)
  • the crosslinker was 4-arm PEG alkyne
  • inactive monomer was PEG bis-azide
  • active monomer was FITC-NVOC- PEGwoo-azide.
  • the crosslinker was mixed with active and inactive monomers in the presence of copper sulphate (CUSO4) and sodium ascorbate to trigger the formation of PEG hydrogels.
  • CUSO4 copper sulphate
  • PEG3350 (5 g, 1.493 mmol, 1 equiv) and triethylamine (TEA) (1.65 mL, 11.94 mmol, 8 equiv) were dissolved in 25 mL DCM (5 mL per 1 g of polymer) in a twonecked RBF under a flow of argon. The mixture was cooled in an ice bath, and methanesulfonyl chloride (MsCI, 0.92 mL, 11.94 mmol, 8 equiv) was added dropwise.
  • MsCI methanesulfonyl chloride
  • the flask was sealed, and the mixture was stirred at 20 °C for 16 h.
  • the reaction mixture was transferred to a centrifuge tube along with 250 mL ultrapure water (10 mL per 5 mL of DCM) and vortexed (1 min).
  • the mixture was centrifuged (5000 rpm, 3 min) and the aqueous phase was separated and discarded. This process was repeated three times, organic phase was dried with MgS04 and then filtered using a sintered funnel under vacuum. The filtrate was concentrated using rotary evaporator to obtain mesylate-terminated PEG.
  • the reaction mixture was transferred to a centrifuge tube along with ultrapure water (10 mL/g) and vortexed (1 min). The mixture was centrifuged (5000 rpm, 3 min) and the aqueous phase was separated and discarded. This process was repeated three times, after which the organic phase was dried with Na2SO4 and then filtered using a sintered funnel under vacuum. The filtrate was concentrated using rotary evaporator. NaNs (3.251 mg, 50 mmol, 10 equiv) and 25 mL DMF were added to the residue and stirred (65 °C, 3 d) and then cooled to 20 °C.
  • the mixture was centrifuged (5000 rpm, 3 min), and the precipitate was washed with 10 mL ethanol, concentrated using rotary evaporator, dissolved in water (5 mL/g) and extracted with DCM. The procedure was repeated three times, and the combined organic extracts were dried (Na2SO4) and concentrated using a rotary evaporator.
  • the polymer obtained from the procedure above was added to a round bottom flask. Subsequently, ethyl acetate (50 mL, 10 mL per 1 g of polymer) and 1 M HCI (15 mL, 3 mL per 1 g of polymer) was added, and the flask was purged with nitrogen and cooled to 0 °C.
  • Triphenylphosphine (PPhs, 1300 mg, 5 mmol, 1.1 equiv) was then added to the round bottom flask and the mixture was stirred under nitrogen (0 °C to 20 °C, 18 h). The aqueous layer was collected and washed with ethyl acetate (2x40 mL). Potassium hydroxide (KOH, 12.5 g, 2.5 g per 1 g of polymer) pellets were slowly added until dissolved to remove generated triphenylphosphine oxide (TPPO). The aqueous solution was extracted with DCM (5x40 mL), and the combined organic extracts were dried (Na2SO4) and filtered using a sintered funnel under vacuum.
  • KOH potassium hydroxide
  • the filtrate was concentrated using rotary evaporator and then lyophilized (18 h).
  • the filtrate was amine-PEGwoo-azide (molecular weight: -1055 g/mol) and was obtained with a yield of 66% (3.3 g, 3.3 mmol)
  • NVOC 4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy)butanoic acid
  • NVOC 1-[Bis(dimethylamino)methylene]-1 H-1 ,2,3-triazolo[4,5- b]pyridinium 3-oxide hexafluorophosphate
  • DIPEA N,N- Diisopropylethylamine
  • Amine-PEG-azide 500 mg, 0.5 mmol, 1 equiv was dissolved in minimal amount of DMF and added dropwise to the reaction mixture.
  • the reaction was warmed to 20 °C and stirred for 18 h, before adding water (20 mL) and extracted with DCM (3x20 mL).
  • the combined organic layers were washed with brine, dried over Na2SO4, filtered and concentrated using a rotary evaporator to obtain NVOC-PEG1000- azide (molecular weight: -1337 g/mol) with a yield of 58% (386 mg, 0.288 mmol).
  • NVOC-PEGwoo-azide 125 mg, 0.093 mmol, 1 equiv
  • aqueous THF 0.9 mL
  • 0.6 mL of 15% phosgene (COCI2, 4.675 mmol, 100 equiv) was added and stirred (20 °C, 18 h) to this solution.
  • nitrogen was bubbled in the solution (20 °C, 30 min) to evaporate THF and unreacted COCh.
  • the reside was dissolved in dry DCM (0.5 mL).
  • Fluorescein isothiocyanate (FITC, 36 mg, 0.093 mmol, 1 equiv) was dissolved in dry DCM (1 mL), then TEA (28 L, 0.2 mmol, 1.6 equiv) was added, and the reaction mixture was stirred (20 °C, 5 min). The first reaction mixture was added dropwise into second reaction mixture over 30 min and stirred (0 to 20 °C, 12 h). DCM was then added to the reaction mixture following which it was washed with NH4CI and then NaHCOs.
  • FITC-NVOC- PEGwoo-azide (molecular weight: -1752 g/mol) with a yield of 63% (103 mg, 0.0588 mmol)
  • the diameter and thickness of the discs were 6 mm and 1.2 mm, respectively. Discs were washed with 5 mL 10 mM ethylenediaminetetraacetic acid (EDTA) (20 °C, 6 h) and then 5 mL PBS (20 °C, overnight). Discs were stored in PBS in the dark until use.
  • EDTA ethylenediaminetetraacetic acid
  • Table 5 Composition of 2 mL precursor solution used to make PEG hydrogels (the total crosslinker plus monomers concentration was 5% (w:v))
  • FITC can be released from active monomer when it is exposed to 365 nm light (see Figure 36).
  • the incubation time is the time required for the protein to be captured by the FITC in the hydrogel.
  • Hydrogel discs containing 1:10 molar ratio of active to inactive monomers were used. The hydrogel discs were incubated in 10 mL of 0.005 ppm RS solution for different durations (1, 15 and 24 h). Subsequently, hydrogel discs were washed overnight and then immersed in 200 pL of fresh PBS and exposed to 365 nm light for a given exposure time. The gel was left in the PBS for 10 mins following which the buffer was collected, and fluorescence was measured. The procedure was repeated to expose hydrogel discs for up to a total of 60 min.
  • FIG 38 A plot of the fluorescence spectra of PBS used to immerse the gel with different exposure times is provided in Figure 38.
  • the fluorescence intensity at peak wavelength was plotted as a function of exposure time and plotted to obtain Figure 39, which shows that, as might be expected, the longest protein incubation time results in the largest fluorescence signal from the released labelled protein.
  • the molar ratio of active to inactive monomers was varied from 1:10, 1:20, 1:40 and 1:100.
  • Hydrogel discs were incubated in 10 mL of 0.005 ppm RS solution in PBS (0.005 ppm) for 24 h, washed overnight in PBS and subsequently exposed to 365 nm light for a total of 60 min. After each exposure, the supernatant was collected and the fluorescence at peak wavelength versus exposure time was plotted and shown in Figure 40.
  • proteins were labelled with FITC while being released from PEG hydrogels, after selected proteins were captured in wells of microtitre plates, they were measured using a microtitre plate reader with an excitation wavelength of 470 nm.
  • Hydrogels were washed overnight in PBS and then exposed to 365 nm light for 60 min in total. The supernatants were allowed to incubate in the biotinylated microtitre plate wells for 30 min, followed by a PBS wash to remove any unbound material, and fluorescence of each well was measured.
  • Figure 41 shows the fluorescence signal for these five solutions as a function of exposure time.
  • Hydrogels were washed overnight in PBS and then exposed to 365 nm light for 60 min in total. The supernatants were allowed to incubate in anti-l L6 coated microtitre plate wells for 30 min, followed by a PBS wash to remove any unbound material, and fluorescence of each well was measured.
  • Figure 42 shows the fluorescence signal for these four solutions as a function of exposure time.

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Abstract

La présente invention concerne des hydrogels qui sont capables de se lier à des protéines, de concentrer, marquer et libérer lesdites protéines marquées et concentrées. L'invention concerne également des formulations, des formulations orales, des biomatériaux, des utilisations, des procédés et des kits de pièces correspondants.
PCT/GB2024/051945 2023-07-24 2024-07-23 Hydrogels pour la liaison à des protéines, leurs formulations et utilisations Pending WO2025022116A1 (fr)

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