Hydrogels for binding to proteins, formulations and uses thereof

Abstract

The present invention relates to 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.

Description

[0001] HYDROGELS FOR BINDING TO PROTEINS, FORMULATIONS AND USES THEREOF

[0002] FIELD OF THE INVENTION

[0003] 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.

[0004] BACKGROUND TO THE INVENTION

[0005] Measurement of low abundance proteins is key for enabling early detection of diseases1. A widely used method for the measurement of low abundance proteins is enzyme-linked immunosorbent assay (ELISA)2. 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.

[0006] However, ELISA requires multiple adding and washing steps, and hence is laborious. To address this challenge, ELISA has been implemented on microfluidic paper-based analytical devices by controlling the flow rate and hence arrival times of reagents to the detection zone3. The flow control requires patterning of paper and multiple layers which must be precisely aligned. Alternatively, label-free biosensors have been used to measure low abundance proteins. Unlike ELISA, label-free biosensors4require only capture antibodies and rely on changes in refractive index5'7or impedance8,9or mass10,11to determine the concentration of target proteins.

[0007] However, label-free biosensors are prone to errors because of changes in environmental conditions such as temperature12. To measure low abundance proteins using absorption or fluorescence, which are less sensitive but require simpler instrumentation and are more user-friendly than ELISA, proteins have been pre-concentrated using beads13and electrophoresis14'16. After pre-concentration, proteins are recovered, labelled with dyes or fluorophores, captured, and then detected. However, these techniques require a series of disparate steps, which is inefficient and impractical, especially for applications of testing at point of care or in the field.

[0008] There is a need for a simpler and efficient assay process which can detect proteins at low abundances. The present invention aims to address one or more of the above-mentioned problems in the art.

[0009] SUMMARY OF THE INVENTION

[0010] 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. As shown in Figure 1, 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. As FITC is fluorescent, proteins are pre-concentrated and labelled in a single step. Furthermore, as shown in Figure 1, the FITC is attached to the hydrogel’s backbone via a photolabile group, o-nitrobenzyl19'25, which exhibits controllable photoreactions with tunable absorption for wavelengths >300 nm23,25. Thus, the release of the fluorogenic labelled proteins can be triggered through the irradiation of the hydrogels with UV light (365 nm). As shown in Figure 1, the released proteins can then be captured and measured using standard fluorescence detection techniques.

[0011] To demonstrate the feasibility of the inventors’ hydrogel for pre-concentration, labelling, and controlled release, 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. Similar results have been shown with other exemplar proteins, (C-reactive protein) CRP, IL8, and IL6, showing that the 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. Further with the added advantages that 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.

[0012] In one aspect of the invention a hydrogel comprising a polymer formed of a plurality of inactive monomers and a plurality of active monomers is provided, 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.

[0013] Suitably the plurality of inactive monomers is selected from the group consisting of polyethylene glycol (PEG), acrylamide, N-isopropylacrylamide, methacrylamide, methacrylate, and PEG bis-azide.

[0014] Suitably 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.

[0015] Suitably 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.

[0016] Suitably the cleavable bond is o-nitrobenzyl.

[0017] In one embodiment of the hydrogel, the plurality of inactive monomers are acrylamide, the plurality of active monomers are polyethylene glycol methacrylamide, wherein 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.

[0018] In one aspect of the invention a formulation comprising a core and a shell is provided, wherein the core comprises the hydrogel of any one of the preceding aspects and embodiments, and the shell comprises a hydrogel having a pore size of less than 30 nm.

[0019] In one aspect of the invention 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.

[0020] Suitably the oral formulation is a pill, tablet, capsule, granule, troch, lozenge, a lollipop, or sampling material. Suitably the oral formulation is a disc. In one aspect there is provided a disc comprising the hydrogel of the invention.

[0021] In one aspect of the invention, a sampling device is provided, 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.

[0022] In another aspect there is provided an inert substrate comprising a coating thereon, wherein the coating comprises the hydrogel of the invention. Suitably the coating is a film.

[0023] In one aspect a container is provided, the 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.

[0024] In one embodiment the base layer is a polymer comprising free amine groups. Suitably 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. In one embodiment the base layer is chitosan. In one embodiment, 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. In one embodiment the blocking agent comprises PEG-methyl. Suitably the capture protein and the blocking agent bind to the base layer. Suitably the reactive layer comprises a majority of binding molecules, and a minority of blocking agent. Suitably the blocking agent is bound to the base layer only where the binding molecule is not bound to the base layer. Suitably the blocking agent binds to any free amine groups in the base layer. Suitably the blocking agent prevents free FITC binding to the base layer of the container, suitably when in use.

[0025] Suitably the container may be any container suitable for carrying out a method of the invention therein. In one embodiment, the container is a microwell or microtitre plate.

[0026] In one aspect of the invention a kit-of-parts is provided, said 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.

[0027] Optionally the kit may further comprise the container above.

[0028] In one aspect of the invention, 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.

[0029] In one aspect of the invention, there is provided a use of the biomaterial according to the previous aspect in cell culture.

[0030] In one aspect of the invention, there is provided a use of the 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.

[0031] In one aspect of the invention, 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. In one aspect of the invention, a method of concentrating and labelling proteins in a sample is provided, the method 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

[0032] In one aspect of the invention, a method of detecting proteins in a sample is provided, the method 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. 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.

[0033] In one aspect of the invention, a method of measuring a protein of interest in a sample is provided, the method 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. 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.

[0034] Suitably the protein of interest is a biomarker.

[0035] Suitably the biomarker is C-reactive protein (CRP), IL-6, IL-8, or cardiac troponin.

[0036] In one aspect of the invention, a method of determining whether a subject has a disease or disorder is provided, the method 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. 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.

[0037] Suitably the disease or disorder is cancer or a cardiovascular disease.

[0038] Suitably the cleavage inducer is UV light, or the cleavage inducer is selected from esterase or reducing agents.

[0039] Suitably in any of the above methods, 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.

[0040] In one embodiment of the method of measuring a protein of interest in a sample or the method of determining whether a subject has a disease, the container of the invention is used. Suitably in the step of contacting the fluorescently labelled proteins with a binding molecule capable of specifically binding to a protein of interest.

[0041] Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps.

[0042] Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0043] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

[0044] Various aspects of the invention are described in further detail below. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:

[0046] 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.

[0047] 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.

[0048] 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).

[0049] 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]tis the concentration of monomer as a function of irradiation time and [M]o is the starting monomer concentration.

[0050] 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 (mhydrogei) and precursor (mprecursor) solutions.

[0051] 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.

[0052] 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).

[0053] Figure 9: UV-Vis spectra of F-NVOC-allylamide (45, 30, 25, 10 and 5 pM) in DMSO where inset shows average absorbance between ± 5 nm of peak wavelength of 519 nm versus the monomer concentration (molar extinction coefficient at 519 nm = 49,600 ± 5,200 M"1cm"1).

[0054] Figure 10: UV-Vis spectra of F-NVOC-PEG4oo-methacrylamide (45, 30, 20, 10 and 5 pM) in PBS where inset shows average absorbance between ± 5 nm of peak wavelength of 492 nm versus the monomer concentration (molar extinction coefficient at 492 nm = 69,100 ± 4,200 M"1cm"1).

[0055] Figure 11: UV-Vis spectra of F-NVOC-PEG34oo-methacrylamide (45, 25, 10, 5 and 1 pM) in PBS where inset shows average absorbance between ± 5 nm of peak wavelength of 492 nm versus the monomer concentration (molar extinction coefficient at 492 nm = 63,900 ± 00 M"1cm"1)

[0056] Figure 12: UV-Vis spectra of 40 pM F-NVOC-PEG34oo-methacrylamide monomer in PBS with increasing irradiation time

[0057] 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

[0058] 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 15: (a) UV-Vis and (b) fluorescence emission spectra (excitation wavelength of 470 nm) of fluorescein in PBS where insets show peak absorbance (averaged over 485-495 nm, molar extinction coefficient at 490 nm = 52,600 ± 3,200 M"1cm"1) and emission (averaged over 509-519 nm, molar emission coefficient at 514 nm = (10.4 ± 0.1) x 109M"1cm"1) versus fluorescein concentration

[0059] Figure 16: (a) UV-Vis and (b) fluorescence emission spectra (excitation wavelength of 540 nm) of rhodamine-streptavidin in PBS where insets show peak absorbance (averaged over 572-582 nm, molar extinction coefficient at 577 nm = 650,602 ± 6,024 M"1cm"1) and emission (averaged over 585-595 nm, molar emission coefficient at 590 nm = (1.6 ± 0.04) x 109M"1cm"1) versus rhodamine-streptavidin concentration

[0060] 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

[0061] 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

[0062] 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

[0063] 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)

[0064] Figure 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)

[0065] 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)

[0066] 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

[0067] 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

[0068] 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)

[0069] 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)

[0070] 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)

[0071] 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

[0072] Figure 30: Reaction scheme for PEG hydrogels formed by alkyne-azide click chemistry between the crosslinker and active plus inactive monomers (n=11, m=75 and p=22)

[0073] Figure 31: Reaction scheme for the synthesis of 4-arm PEG alkyne (crosslinker, n=11)

[0074] Figure 32: Reaction scheme for the synthesis of PEG bis-azide (inactive monomer, m=75)

[0075] Figure 33: Reaction scheme for the synthesis of amine-PEG-azide (p=22)

[0076] Figure 34: Reaction scheme for the synthesis of NVOC-PEG-azide (p=22) Figure 35: Reaction scheme for the synthesis of FITC-NVOC-PEGwoo-azide (active monomer, p=22)

[0077] Figure 36: Reaction scheme showing the release of FITC when FITC-NVOC-PEGwoo-azide monomer is exposed to 365 nm light (p=22)

[0078] 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

[0079] 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)

[0080] 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)

[0081] 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)

[0082] 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

[0083] 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

[0084] The patent, scientific and technical literature referred to herein establish knowledge that was available to those skilled in the art at the time of filing. The entire disclosures of the issued patents published and pending patent applications, and other publications that are cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference. In the case of any inconsistencies, the present disclosure will prevail. Various aspects of the invention are described in further detail below.

[0085] DETAILED DESCRIPTION OF THE INVENTION

[0086] Hydrogels

[0087] In one aspect of the present invention, a hydrogel comprising a polymer formed of a plurality of inactive monomers and a plurality of active monomers is provided, 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.

[0088] As used herein, a “hydrogel” is a hydrogel comprising a polymer formed from a plurality of inactive monomers and a plurality of active monomers. Suitably, 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. Suitably the hydrogel is hydrophilic. Suitably the hydrogel is insoluble in water. Suitable crosslinkers are described elsewhere herein. Suitably, 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.

[0089] As used herein, 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.

[0090] In some preferred embodiments the “inactive monomer” is a monomer which does not comprise a fluorophore. Suitable inactive monomers are listed herein.

[0091] As used herein, an “active monomer” is a monomer comprising at least one fluorophore bonded thereto via a cleavable bond. Suitable active monomers are listed herein.

[0092] As used herein, a “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. In one embodiment, the crosslinker is bisacrylamide.

[0093] In one embodiment, the crosslinker is 4-arm PEG alkyne, suitably as shown below:

[0094] Suitably in embodiments where the cross linker is 4-arm PEG alkyne, then n is between 5-20, suitably between 8-15, suitably between 9-13, suitably n is 11.

[0095] As used herein, “fluorophore” refers to a compound, chemical group, or composition that is inherently fluorescent. Suitable fluorophores are listed herein.

[0096] As used herein “covalently binding” means that a covalent bond is formed by sharing of electrons between atoms. For example, the fluorophore covalently binds to the primary amines of the protein. In a non-limiting example, the isothiocyanate group of fluorescein isothiocyanate covalently binds to the primary amines in the protein. In a non-limiting example, the isothiocyanate group of fluorescein isothiocyanate covalently binds to the terminal amines in the protein. In a non-limiting example, one of the hydroxy groups of fluorescein isothiocyanate covalently binds to the hydrogel backbone via o-nitrobenzyl.

[0097] As used herein “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.

[0098] Suitably the protein is streptavidin. Suitably the protein is CRP. Suitably the protein is IL6. Suitably the protein is IL8.

[0099] In a non-limiting example, proteins described herein also encompass low abundance proteins. In a non-limiting example, proteins described herein are biomarkers. Suitable protein biomarkers are described elsewhere herein. Suitable biomarkers include inflammatory proteins, such as chemokines and cytokines. Suitably the protein may be a biomarker of inflammation. Suitably biomarkers of inflammation may be indicative of disease. Suitably the protein may be a chemokine or cytokine. Suitably the protein may be an interleukin or a C-reactive protein. In some embodiments, the protein is IL6, IL8, or CRP.

[0100] In a non-limiting example, proteins described herein can comprise additional functional groups such as fluorophores or chromophores.

[0101] Suitably the additional functional group is rhodamine. In some embodiments, the protein is streptavidin and the additional functional group is rhodamine.

[0102] “Cleavable bond” is defined elsewhere herein.

[0103] The pore size of a hydrogel is defined elsewhere herein.

[0104] Suitably the hydrogel comprises at least two or more inactive monomers and at least two or more active monomers. Suitably 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. Suitably the molar ratio of inactive monomers: active monomers is between 100:1 and 5:1. Suitably the molar ratio of inactive monomers to active monomers is 100: 1 , 40: 1 , 20: 1 , 10:1. Suitably the ratio of inactive monomer: active monomer is 40:1.

[0105] Suitably the hydrogel is a synthetic hydrogel. As used herein, “synthetic hydrogel” refers to a hydrogel which does not comprise natural polymers such as proteins and / or polysaccharides.

[0106] Suitably the hydrogel additionally comprises natural polymers such as proteins and / or polysaccharides. Non-limiting examples of proteins are collagen / or gelatine. Non-limiting examples of polysaccharides are starch, alginate, dextran, chitosan, hyaluronic acid and agarose.

[0107] Suitably the 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. Suitably 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. In one embodiment, hydrogels of the invention, and any formulations, or sampling devices thereof may be stored for at least 7 days. Suitably without degradation. Suitably therefore 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.

[0108] Inactive Monomer

[0109] Suitably 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. As discussed above, an inactive monomer is preferably a monomer which does not comprise a fluorophore. Suitably 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.

[0110] Suitably the plurality of inactive monomers substantially do not hydrolyse the cleavable bone (e.g. the o-nitrobenzyl-fluorescein carbonate link).

[0111] Suitably the plurality of inactive monomers are selected from the group consisting of polyethylene glycol, acrylamide, N-isopropylacrylamide, methacrylamide, methacrylate, and PEG bis-azide.

[0112] Suitably the plurality of inactive monomers are selected from the group consisting of acrylamide, N-isopropylacrylamide, methacrylamide, methacrylate and PEG bis-azide

[0113] Suitably the plurality of inactive monomers may comprise the same monomer, or different monomers.

[0114] Suitably 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.

[0115] Suitably 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.

[0116] Suitably the plurality of inactive monomers consist of acrylamide. Suitably therefore, in some embodiments) each inactive monomer in the plurality of inactive monomers is acrylamide.

[0117] Suitably the plurality of inactive monomers consist of PEG bis-azide. Suitably therefore, in some embodiments) each inactive monomer in the plurality of inactive monomers is PEG bis-azide.

[0118] Suitably the molar ratio of inactive monomer: crosslinker is between 2:1 and 75:1, in some embodiments between 50:1 and 75:1.

[0119] Suitably the molar ratio of inactive monomer: crosslinker is between 2:1 and 70:1 , in some embodiments, between 56:1 and 70:1.

[0120] Suitably 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.

[0121] Suitably the molar ratio of inactive monomer: crosslinker is 60: 1 , 61 : 1 , 62: 1 , 63: 1 , 64: 1 , 65: 1 , or 66:1. Suitably the molar ratio of the inactive monomer acrylamide: crosslinker bisacrylamide is between 50:1 and 75:1.

[0122] Suitably the molar ratio of the inactive monomer acrylamide: crosslinker bisacrylamide is between 56:1 and 70:1.

[0123] Suitably 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.

[0124] Suitably the molar ratio of the inactive monomer acrylamide: crosslinker bisacrylamide is 63:1.

[0125] Suitably the molar ratio of the inactive monomer PEG bis-azide: crosslinker 4-arm PEG alkyne is between 2:1 and 10:1.

[0126] Suitably the molar ratio of the inactive monomer PEG bis-azide: crosslinker 4-arm PEG alkyne is between 2:1 and 5:1.

[0127] Suitably 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.

[0128] Suitably the molar ratio of the inactive monomer PEG bis-azide: crosslinker 4-arm PEG alkyne is 2:1.

[0129] Suitably 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.

[0130] Suitably, for example, 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.

[0131] Suitably in a preferred embodiment, the molar ratio of inactive monomer: active monomer is 40:1

[0132] Accordingly, in a non-limiting example, for every 1000 molecules, 16 molecules are the crosslinker bisacrylamide, 98 are the active monomer and 886 molecules are the inactive monomer acrylamide.

[0133] Suitably 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.

[0134] Suitably 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.

[0135] Suitably the inactive monomer is water soluble.

[0136] Suitably the inactive monomer has a similar reactivity to the active monomer and cross-linker, e.g. to provide for suitable polymerisation.

[0137] Suitably the inactive monomer does not comprise any functional group that is capable of reacting with a protein.

[0138] Suitably the inactive monomer does not comprise any functional group that is capable of reacting with a fluorophore.

[0139] Suitably the inactive monomer substantially does not absorb UV light. Suitably the inactive monomer substantially does not absorb UV light at a wavelength which cleaves the cleavable bond. Suitable UV light is defined elsewhere herein.

[0140] Active Monomer

[0141] Suitably 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.

[0142] Suitably the plurality of active monomers are selected from for example but not limited to polyethylene glycol derivatives such as polyethylene glycol based monomers. The skilled person knows what polyethylene glycol based monomers are in this context and can readily identify suitable polyethylene glycol based monomers. One non-limiting example of such a polyethylene glycol based monomer is polyethylene glycol methacrylamide.

[0143] Suitably 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.

[0144] Suitably the active monomers acrylamide, N-isopropylacrylamide, methacrylamide, and methacrylate each comprise a polyethylene glycol spacer arm. Suitably, the polyethylene glycol spacer arm aids water solubility. Suitably the active monomers acrylamide, N-isopropylacrylamide, methacrylamide, and methacrylate each comprise a linear polyethylene glycol spacer arm.

[0145] Suitably the polyethylene glycol spacer arm is PEG400.

[0146] Suitably the polyethylene glycol spacer arm is PEG1000.

[0147] Suitably the polyethylene glycol spacer arm is PEG3400.

[0148] Suitably the plurality of active monomers may comprise the same monomer, or different monomers.

[0149] Suitably 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.

[0150] Suitably the plurality of active monomers consist of the same monomer.

[0151] Suitably the plurality of active monomers comprise methacrylamide. Alternatively the plurality of active monomers comprise allylamide.

[0152] Suitably the plurality of active monomers comprise polyethylene glycol methacrylamide.

[0153] Suitably the polyethylene glycol methacrylamide is prepared at a yield of at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%.

[0154] Suitably the plurality of active monomers each comprise a polyethylene glycol spacer arm.

[0155] Suitably the plurality of active monomers each contain a polyethylene glycol spacer arm.

[0156] Suitably the polyethylene glycol spacer arm is present between the o-nitrobenzyl derivative and methacrylamide.

[0157] Suitably the polyethylene ethylene glycol spacer arm is present between the 4-(4-(1- hydroxyethyl)-2-methoxy-5-nitrophenoxy) butanoic acid (NVOC) and methacrylamide.

[0158] Suitably the plurality of active monomers comprise PEG400-methacrylamide, PEG34oo- methacrylamide or PEGwoo-azide.

[0159] Suitable the plurality of active monomers comprise 4-(4-(1-hydroxyethyl)-2-methoxy-5- nitrophenoxy) butanoic acid (NVOC)-

[0160] In one embodiment, the plurality of active monomers comprise NVOC-PEG400- methacrylamide, NVOC-PEG34oo-methacrylamide, or NVOC-PEGwoo-azide. “NVOC” refers to 4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy) butanoic acid. Suitably the plurality of active monomers are selected from the group consisting of acrylamide, N-isopropylacrylamide, methacrylamide, and methacrylate.

[0161] In some cases, a polyethylene glycol spacer arm is not present.

[0162] In some cases, a polyethylene glycol spacer arm is not present between the NVOC and the methacrylamide. “NVOC” refers to 4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy) butanoic acid.

[0163] Suitably the plurality of active monomers each comprise at least one fluorophore.

[0164] Suitably 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.

[0165] Suitably 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.

[0166] Suitably 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.

[0167] Suitably 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.

[0168] Suitably 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.

[0169] Suitably the plurality of active monomers comprise or consist of FITC-NVOC-PEG3400- methacrylamide. Suitably therefore, in some embodiments, 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.

[0170] Suitably the plurality of active monomers comprise or consist of FITC-NVOC-allylamide. Suitably therefore, in some embodiments, 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.

[0171] Suitably the plurality of active monomers comprise or consist of FITC-NVOC-PEG400- methacrylamide. Suitably therefore, in some embodiments, 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.

[0172] Suitably the plurality of active monomers comprise or consist of FITC-NVOC- PEGwoo-azide. Suitably therefore, in some embodiments, 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.

[0173] Suitably 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.

[0174] Suitably 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.

[0175] Suitably the plurality of active monomers are hydrophilic.

[0176] In one example, 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.

[0177] In an alternative example, the plurality of active monomers according to the invention may comprise allylamide.

[0178] Suitably the active monomers are water soluble.

[0179] Suitably the active monomers have a similar reactivity to the inactive monomers and crosslinker, e.g. to provide for suitable polymerisation.

[0180] Suitably 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.

[0181] Cleavable Bond

[0182] Suitably the active monomers of the polymer comprised in the hydrogel each comprise one or more fluorophore groups attached thereto via a cleavable bond. As used herein, 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. Suitably the cleavable bond comprises a photolabile group. In the alternative, the cleavable bond does not comprise a photolabile group as defined herein.

[0183] Suitably the cleavable bond is a cleavable linkage. Suitably a cleavable linkage comprises a photolabile group as defined herein.

[0184] Suitably the cleavable bond is a cleavable moiety. Suitably a cleavable moiety comprises a photolabile group as defined herein.

[0185] Suitably the cleavable bond is a covalent bond.

[0186] Suitably the cleavable bond is cleaved by a cleavage inducer.

[0187] Suitably the cleavable linkage is cleaved by a cleavage inducer.

[0188] Suitably the cleavable moiety is cleaved by a cleavage inducer.

[0189] As used herein, a “cleavage inducer” is any agent or for example, light or radiation that can cleave the cleavable bond.

[0190] Suitably the cleavage inducer is light, radiation, enzymes, acids, alkalis, and / or heat.

[0191] Suitably the cleavage inducer is UV light, a reducing agent, or an esterase.

[0192] As used herein, “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. Such trialkylphosphine compounds include, but are not limited to, tri-n- butylphosphine (TBP) or tris[2-carboxyethyl] phosphine (TCEP).

[0193] Suitably the reducing agent is dithiothreitol (DTT) or 2-mercaptoethanol.

[0194] As used herein, “esterases” are enzymes that hydrolyse esters into alcohol and acids.

[0195] Suitably the esterase is pig liver esterase, or horse liver esterase.

[0196] Suitably the cleavable bond is cleaved by UV light.

[0197] As used herein, “UV light” comprises a wavelength range of between roughly 100 nm and 400 nm. Suitably between about 200nm to 400nm.

[0198] Suitably the UV light comprises a wavelength range of roughly at least 300 nm.

[0199] Suitably the UV light is UV-A and comprises a wavelength range of roughly at least 300 nm. Suitably the UV light is IIV-A and comprises a wavelength range between 315 and 400 nm.

[0200] Suitably the UV light comprises a wavelength range between 350 and 370 nm.

[0201] Suitably the UV light is 365 nm.

[0202] As used herein, a “photolabile group” or “photolabile protecting group” is a group that can be removed by light. For example, 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.

[0203] Suitably the cleavable bond (i.e. the photolabile group) may be for example nitrobenzyl- based or carbonyl-based.

[0204] 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.

[0205] Suitably the nitrobenzyl-based cleavable / photolabile group is o-nitrobenzyl.

[0206] Suitably the o-nitrobenzyl-based cleavable / photolabile group is NVOC. “NVOC” refers to 4- (4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy) butanoic acid.

[0207] Suitably the o-nitrobenzyl derivative is NVOC. “NVOC” refers to 4-(4-(1-hydroxyethyl)-2- methoxy-5-nitrophenoxy) butanoic acid..

[0208] Non-limiting examples of a carbonyl-based cleavable / photolabile group are phenacyl, 3’, 5’- dimethoxybenzoin, and p-hydroxyphenacyl.

[0209] Suitably the cleavable bond is nitrobenzyl-based.

[0210] Suitably the cleavable bond is m-nitrobenzyl. Suitably the cleavable bind is p-nitrobenzyl. Suitably the cleavable bond is o-nitrobenzyl.

[0211] Suitably the cleavable bond links the fluorophore to a polyethylene glycol chain of the polyethylene glycol spacer arm of each active monomer.

[0212] Suitably the cleavable bond o-nitrobenzyl links the fluorophore to a polyethylene glycol chain of the polyethylene glycol spacer arm of each active monomer.

[0213] Suitably the cleavable bond comprises a photolabile group attached to the hydrogel backbone at the one or more active monomers. Suitably 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

[0214] Suitably the cleavable bond comprises a nitrobenzyl-based photolabile group attached to the hydrogel backbone at the one or more active monomers.

[0215] Suitably the cleavable bond comprises a nitrobenzyl-based photolabile group by a polyethylene glycol spacer arm attached to the hydrogel backbone

[0216] Suitably the cleavable bond comprises an o-nitrobenzyl group attached to the hydrogel backbone at the one or more active monomers.

[0217] Suitably the cleavable bond comprises an o-nitrobenzyl group attached to the hydrogel backbone as illustrated in the following:

[0218] Suitably 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.

[0219] Suitably the isothiocyanate group of fluorescein isothiocyanate does not bind to the hydrogel backbone.

[0220] Suitably the polymer comprised in the hydrogel comprises at least one fluorophore bonded to the active monomers therein via cleavable bonds.

[0221] Suitably each active monomer comprises at least one fluorophore bonded thereto.

[0222] Suitably each active monomer comprises a plurality of fluorophores bonded thereto.

[0223] Suitably 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.

[0224] Suitably 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. Suitably each active monomer comprises at most 10 fluorophores bonded thereto.

[0225] Suitably 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.

[0226] Numerous fluorophores are known to those skilled in the art and 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. HAUGLAND, MOLECULAR PROBES HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS (9thedition, CD-ROM, September 2002). As used herein, “fluorescein” encompasses all kinds of conceivable derivatives such as fluorescein isothiocyanate.

[0227] Fluorophores may contain substituents that alter the solubility, spectral properties or physical properties of the fluorophore.

[0228] Suitably 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.

[0229] Suitably 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.

[0230] As used herein, “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. For example, 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, enamines, ynamines, ureas, pseudoureas, semicarbazides, carbodiimides, carbamates, imines, azides, azo compounds, azoxy compounds, and nitroso compounds.

[0231] Suitably the at least one fluorophore is rhodamine.

[0232] Suitably the at least one fluorophore is fluorescein isothiocyanate (FITC).

[0233] Suitably the at least one fluorophore is fluorescein (F).

[0234] Ratio of Monomers and Crosslinkers

[0235] Suitably the ratio of active: inactive monomers used in the hydrogel of the invention may be varied. Suitably 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.

[0236] Suitably the ratio of active monomers: inactive monomers is a molar ratio. Suitably there is an excess of inactive monomers. Suitably there is a molar excess of inactive monomers

[0237] Suitably the (molar) ratio of active monomers: inactive monomers is between 1 :100 to 1:5.

[0238] Suitably 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. Suitably the (molar) ratio of active: inactive monomer is 1: 100, 1 :40, 1 :20, or 1 : 10. Suitably the (molar) ratio of active: inactive monomer is 1 :40.

[0239] Suitably the ratio of inactive monomer: cross-linker is also a molar ratio.

[0240] Suitably the (molar) ratio of inactive monomer: cross-linker is 63:1. Alternatively the molar ratio of inactive monomer: cross-linker is 2:1.

[0241] Suitably the (molar) ratio of inactive monomer: crosslinker is between 2:1 and 75:1, or between 50:1 and 75:1.

[0242] Suitably the (molar) ratio of inactive monomer: crosslinker is between 2:1 and 70:1, or between 56:1 and 70:1.

[0243] Suitably 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. Suitably the (molar) ratio of inactive monomer: crosslinker is 60: 1 , 61 : 1 , 62: 1 , 63: 1 , 64: 1 , 65:1 , or 66:1.

[0244] Suitably the (molar) ratio of inactive monomer acrylamide: the crosslinker bisacrylamide is between 50:1 and 75:1.

[0245] Suitably the (molar) ratio of inactive monomer acrylamide: the crosslinker bisacrylamide is between 56:1 and 70:1.

[0246] Suitably 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.

[0247] Suitably the (molar) ratio of the inactive monomer acrylamide: the crosslinker bisacrylamide is 63:1.

[0248] Suitably the molar ratio of the inactive monomer PEG bis-azide: crosslinker 4-arm PEG alkyne is between 2:1 and 10:1.

[0249] Suitably the molar ratio of the inactive monomer PEG bis-azide: crosslinker 4-arm PEG alkyne is between 2:1 and 5:1.

[0250] Suitably 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.

[0251] Suitably the molar ratio of the inactive monomer PEG bis-azide: crosslinker 4-arm PEG alkyne is 2:1.

[0252] Preferred Hydrogels

[0253] Suitably 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.

[0254] Suitably the hydrogel comprises F-NVOC-PEG4oo-methacrylamide. “F” refers to fluorescein “NVOC” refers to 4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy) butanoic acid.

[0255] Suitably the hydrogel comprises F-NVOC-PEG34oo-methacrylamide. “F” refers to fluorescein “NVOC” refers to 4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy) butanoic acid. Suitably the hydrogel comprises F-NVOC-allylamide. “F” refers to fluorescein “NVOC” refers to 4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy) butanoic acid.

[0256] Suitably 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.

[0257] In a particularly preferred embodiment, the hydrogel comprises a polymer, wherein said polymer is a crosslinked FITC-NVOC-PEG34oo-methacrylamide / acrylamide copolymer wherein the crosslinker is bisacrylamide.

[0258] Suitably the hydrogel comprises acrylamide.

[0259] Suitably 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.

[0260] Suitably 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.

[0261] In a particularly preferred embodiment, 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.

[0262] Alternatively, 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.

[0263] In a particularly preferred embodiment, 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.

[0264] Suitably the polymer is a 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. Suitably 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.

[0265] In a particularly preferred embodiment, 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.

[0266] Formulations and Oral Formulations

[0267] In one aspect, a formulation comprising a core and a shell is provided, wherein the core comprises the hydrogel of any one of the preceding aspects and embodiments, and the shell comprises a hydrogel having a pore size of less than 30 nm.

[0268] As used herein, the “core” is substantially surrounded by the shell.

[0269] Suitably the core comprises the hydrogel of any one of the preceding aspects and embodiments. Accordingly, 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.

[0270] As used herein, the “shell” comprises a hydrogel, suitably which comprises a polymer. Suitably the hydrogel which forms the shell may be a different hydrogel to that of the core. Suitably the shell may be formed of any hydrogel having a pore size of less than 30nm.

[0271] Suitably the shell comprises a polymer formed of polyethylene glycol, acrylamide, N- isopropylacrylamide, methacrylamide, and / or methacrylate.

[0272] Suitably the formulation is a core-shell hydrogel.

[0273] Suitably the formulation is a core-shell structure.

[0274] As used herein, “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. The skilled person also knows that different 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.

[0275] Suitably 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.

[0276] Suitably 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.

[0277] Suitably the pore size of the hydrogel is 4 nm, 3.9 nm, 3.8 nm, 3.7 nm, 3.6 nm, 3.5 nm.

[0278] Suitably 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. Suitably 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).

[0279] Suitably 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.

[0280] Accordingly, 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.

[0281] Suitably 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.

[0282] Suitably 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.

[0283] Suitably 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. Suitably 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). Suitably the formulation comprises additional pharmaceutically acceptable excipients. Suitable additional excipients are for example, disintegrants, binders, lubricants, glidants and / or surfactants.

[0284] Suitably the shell comprises additional excipients.

[0285] Suitably the core comprises additional excipients

[0286] Suitably the shell or the core comprises additional excipients.

[0287] Suitably the shell and core comprise additional excipients.

[0288] As used herein “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.

[0289] As used herein “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.

[0290] As used herein “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.

[0291] As used herein “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.

[0292] As used herein “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.

[0293] In one aspect, 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.

[0294] As used herein “oral formulation” is a formulation suitable for administration / incubation by the oral route. In other words, the oral formulation of the invention is biocompatible. Oral route comprises buccal, sublingual, and sublabial.

[0295] Suitably the oral formulation may comprise a hydrogel formulated in a shell.

[0296] Suitably, the hydrogel is formulated in a hydrogel shell.

[0297] Suitably, the hydrogel is not formulated in a hydrogel shell.

[0298] Suitably 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. In a non-limiting example, the nanoparticles are formulated in a shell. Suitably, said shell is a hydrogel shell.

[0299] In one embodiment the oral formulation is a disc. In one embodiment the oral formulation is a sampling material, which is a disc.

[0300] Suitably the oral formulation is a lozenge, a lollipop, or sampling material.

[0301] Suitably the oral formulation is a lollipop.

[0302] As used herein 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. Furthermore 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.

[0303] Suitably the oral formulation comprising the hydrogel comprises additional excipients. Additional excipients are defined elsewhere herein.

[0304] 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.

[0305] Suitably a lozenge comprises a colourant and / or a flavouring agent.

[0306] Suitably a lollipop comprises a colourant and / or a flavouring agent.

[0307] Sampling device

[0308] In one aspect, a sampling device is provided 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.

[0309] As used herein a “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.

[0310] Suitably 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.

[0311] In one example, 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.

[0312] Accordingly, there might be one or more sampling devices, for example, the first sampling device is the oral formulation defined elsewhere herein and the second sampling device is a test tube.

[0313] The skilled person knows suitable sampling devices and test tubes and can readily identify such sampling devices and test tubes.

[0314] Suitably the sampling device is a test tube.

[0315] In other embodiments the hydrogel of the invention may be comprised upon a substrate, suitably an inert substrate. Suitably the hydrogel may be comprised in a film upon said substrate, suitably coated on said substrate. Suitably there is provided a substrate comprising the hydrogel of the invention. Suitably there is provided an inert substrate comprising a coating thereon, wherein the coating comprises the hydrogel of the invention. Suitably the coating is a film. Suitable such substrates maybe glass or metal. Suitably the substrate may be used as a sampling device. In other embodiments the hydrogel of the invention may be comprised in a disc. Suitably there is provided a disc comprising the hydrogel of the invention. Suitably the disc may be used as an oral formulation or a sampling device. Suitably the disc is between 1-10 mm in diameter, suitably between 2-8 mm in diameter, suitably around 6 mm in diameter. Suitably the disc may be around 1 mm in height. Suitably 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

[0316] Kit-of-parts

[0317] As used herein, “kits-of-parts” refers to a packaged set of related components, typically one or more hydrogel, formulation and / or oral formulation of the invention.

[0318] 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.

[0319] Accordingly, in one aspect, a kit-of-parts is provided, said 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.

[0320] In one aspect, the kit-of-parts comprises a. the hydrogel according to any of the preceding aspects and embodiments, and b. instructions for use.

[0321] In one aspect, the kit-of-parts comprises a. the formulation according to any of the preceding aspects and embodiments and b. instructions for use.

[0322] In one aspect, 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. In one aspect, the kit-of-parts comprises a. the sampling device according to any of the preceding aspects and embodiments, and b. instructions for use.

[0323] In one aspect, the kit-of-parts comprises a. the substrate according to any of the preceding aspects and embodiments, and b. instructions for use.

[0324] In one aspect, the kit-of-parts comprises a. the disc according to any of the preceding aspects and embodiments, and b. instructions for use.

[0325] Suitably the kit-of-parts can comprise further components such as a neutralising buffer, mouth wash, and / or tissues. Suitably the kit-of-parts can comprise a container according to an aspect of the present invention.

[0326] Suitably the sampling device is a testing tube.

[0327] Biomaterials

[0328] In one aspect, 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.

[0329] As used herein, “biomaterials” 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.

[0330] Suitably the biomaterial comprises additional excipients that confer desirable properties to a biomaterial or enhance desirable properties of the biomaterial. Suitably the additional excipients are disintegrants, binders, lubricants, glidants and / or surfactants as defined elsewhere herein.

[0331] Suitably non-limiting examples of additional excipients are colourants, diluents, buffering agents, antibiotics, growth factors and / or preservatives.

[0332] Suitably the additional excipient are antibiotics, growth factors and / or preservatives.

[0333] Suitably the additional excipient is a preservative.

[0334] Suitably the biomaterial is sterile.

[0335] Suitably the biomaterial is biocompatible.

[0336] Suitably the biomaterial is for use in cell culture.

[0337] Uses In one aspect, the invention provides use of a biomaterial of the invention in cell culture.

[0338] As used herein, “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.

[0339] Accordingly, the biomaterial is used to label cells in cell culture system and in for example, cell-based assays.

[0340] Suitably proteins expressed on the cell surface are labelled.

[0341] Suitably the proteins expressed on the cell surface are for example but not limited to annexin V.

[0342] Suitably the biomaterial is used in cell culture to label apoptotic cells.

[0343] Suitably the apoptotic cells express annexin V on the cell surface.

[0344] In an alternative aspect the invention provides use of a hydrogel of the invention as a cell support scaffold which simultaneously labels proteins expressed on the cell surface.

[0345] The term “scaffold”, as used herein, refers to any material that allows attachment of cells and subsequent proliferation and differentiation. “Attachment”, “attach” or “attaches” as used herein, refers to cells that adhere directly or indirectly to a substrate as well as to cells that adhere to other cells.

[0346] In one aspect, 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.

[0347] As used herein, “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.

[0348] In the alternative, “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.

[0349] As used herein, “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.

[0350] Suitably “concentrating and labelling proteins in a sample” occurs in a single step.

[0351] In one aspect, 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.

[0352] As used herein, “controlled release” refers to a release at predetermined intervals or a gradual release over a period of time.

[0353] In one aspect, 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.

[0354] As used herein, “diagnosing” 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.

[0355] As used herein, “disease” refers to a state resulting from a pathophysiological response to external or internal factors.

[0356] As used herein, “disorder” refers to the disruption to the normal or regular functions in the body or a part of the body.

[0357] Methods of concentrating and labelling proteins

[0358] In one aspect, a method of concentrating and labelling proteins in a sample is provided, the method 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

[0359] As used herein, “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.

[0360] As used herein, “suitable conditions” are temperatures between roughly about 15 and 40 °C, a pH between roughly 4 and 8 and atmospheric pressure.

[0361] Suitably the contacting step takes place for a suitable amount of time to allow the proteins in the sample to bind to the hydrogel. Sutiably the contacting step takes place for an incubation time. Suitably the incubation time is between 15 and 48 hours. Suitably the incubation time is between 24 and 48 hours. Suitably the incubation time is at least 24 hours, suitably about 24 hours. Suitably 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.

[0362] In general, 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).

[0363] However, in some examples, the methods may include the step of providing a biological fluid sample from a subject.

[0364] As used herein, “provide”, "obtain" or "obtaining" can be any means whereby one comes into possession of the sample by "direct" or "indirect" means. 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).

[0365] 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. The samples being tested in the methods described herein are also referred to as “test samples”.

[0366] As used herein, the terms "biological sample", “test sample”, "sample" and variations thereof refer to a sample obtained or derived from a subject. For the purposes described herein, the sample is, or comprises, a biological fluid (also referred to herein as a bodily fluid) sample. In alternative examples according to the present invention, 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.

[0367] Environmental samples concern both samples from nature, such as soil or water, and the surroundings, including the environment or surroundings which are not nature, such as the interior of buildings such as walls, floors, surfaces, ventilators, drains, ventilators, conveyor belts, and containers. 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.

[0368] As used herein, the term “biological fluid sample” encompasses a saliva sample. The term biological fluid sample also encompasses other bodily fluids such as a urine sample or a blood sample. All biological fluids and excretions are included.

[0369] As defined elsewhere herein, proteins of the invention also encompass low abundance proteins.

[0370] As used herein, “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.

[0371] Suitably the hydrogel pre-concentrates proteins.

[0372] Suitably 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. Suitably the hydrogel pre-concentrates the proteins by a preconcentration factor of between 190 and 300. In some embodiments, the hydrogel preconcentrates the proteins by a pre-concentration factor of up to 192. In some embodiments, the hydrogel pre-concentrates the proteins by a pre-concentration factor of up to 295. As used herein, “pre-concentration factor” refers to the increase in protein concentration before and after the sample is contacted with a hydrogel. By way of example, a preconcentration factor of 2 refers to a doubling of the concentration of the protein before and after the sample is contacted. In a non-limiting example, the protein to be concentrated and labelled is additionally tagged with a fluorophore. Suitably the protein to be concentrated is streptavidin and the fluorophore is rhodamine. Suitably, the fluorescence of the labelled protein is used to determine the protein concentration before and after the sample is contacted with the hydrogel. Thereby, the pre-concentration factor is provided in this nonlimiting example. Accordingly, as defined elsewhere herein “concentrating proteins” refers to the covalent capture of proteins in the hydrogel and said proteins are concentrated as demonstrated by the exemplary pre-concentration.

[0373] Suitably the protein is streptavidin. Suitably the protein is CRP. Suitably the protein is IL6. Suitably the protein is IL8.

[0374] Suitably the protein is rhodamine-streptavidin.

[0375] Suitably the fluorophore is fluorescein isothiocyanate.

[0376] Suitably upon release from the hydrogel, 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.

[0377] Suitably upon release from the hydrogel, the protein is labelled with at most 5, at most 6, at most 7, at most 8, at most 9, at most 10 fluorophores.

[0378] Suitably upon release from the hydrogel the protein is labelled with at most 10 fluorophores.

[0379] Suitably upon release from the hydrogel streptavidin, 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 fluorophores.

[0380] Suitably the fluorophore is fluorescein isothiocyanate.

[0381] Suitably upon release from the hydrogel streptavidin, 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. Suitably upon release from the hydrogel streptavidin, CRP, IL8, or IL6 is labelled with at least 50 fluorescein molecules.

[0382] Suitably upon release from the hydrogel streptavidin, CRP, IL8, or IL6 is labelled with 85 fluorescein molecules.

[0383] Suitably the hydrogel can provide a concentration factor of 192 for 0.1 ppm of protein in a sample. Suitably when using an inert substrate comprising the hydrogel.

[0384] Suitably the hydrogel can provide a concentration factor of 295 for 0.01 ppm of protein in a sample. Suitably when using an oral formulation comprising the hydrogel, suitably a disc.

[0385] Suitably, 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-5-nitrophenoxy) butanoic acid.

[0386] Suitably, 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) butanoic acid.

[0387] Suitably, 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) butanoic acid.

[0388] Methods of detecting proteins

[0389] In one aspect, a method of detecting proteins in a sample is provided, the method 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. 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.

[0390] “Contacting a sample” is defined elsewhere herein.

[0391] “Suitable conditions” is defined elsewhere herein.

[0392] “Fluorescently labelled proteins” is defined elsewhere herein.

[0393] “Cleavage inducer” is defined elsewhere herein. The suitable cleavage inducers UV light, reducing agents, or esterases are also defined elsewhere herein.

[0394] “Cleavable bond” is defined elsewhere herein.

[0395] “Fluorescently labelled proteins” is defined elsewhere herein.

[0396] Suitably the fluorescently labelled proteins are released from the hydrogel of the invention. Suitably 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.

[0397] Suitably 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. Suitably after a first exposure to a cleavage inducer.

[0398] In a non-limiting example, 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. For example, if roughly about 50 % of the fluorescently labelled proteins are released from the hydrogel of the invention in 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. Suitably after a second exposure to a cleavage inducer.

[0399] 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.

[0400] Suitably 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. Suitably the hydrogel is exposed to the cleavage inducer for this length of time.

[0401] Suitably the release of one or more fluorescently labelled proteins from the hydrogel of the invention takes less than 10 minutes. Suitably therefore the hydrogel is exposed to a cleavage inducer for less than 10 minutes. Suitably the fluorescently labelled proteins are released within 10 minutes. In some embodiments, 50 % of the fluorescently labelled proteins are released within at least about 100 seconds.

[0402] Suitably the fluorescently labelled streptavidin, CRP, IL8, or IL6 is released within 10 minutes. Suitably 50% of the fluorescently labelled streptavidin, CRP, IL8, or IL6 is released within at least about 100 seconds.

[0403] Suitably 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.

[0404] As used herein, “determining the presence of proteins in the sample” as used herein 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.

[0405] As defined elsewhere herein, proteins of the invention also encompass low abundance proteins.

[0406] Suitably, 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. Suitably 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. Suitably a lower limit of detection of 0.0020ppm.

[0407] Suitably, the method detects proteins having a concentration of 0.1 ppm.

[0408] Suitably, the method detects proteins having a concentration of 0.01 ppm.

[0409] Suitably the protein is streptavidin, and the limit of detection of the methods is 0.0033ppm.

[0410] Suitably the protein is CRP and the limit of detection of the methods is 0.0022ppm.

[0411] Suitably the protein is IL6 and the limit of detection of the methods is at least 0.005ppm.

[0412] Suitably the protein is IL8 and the limit of detection of the methods is at least 0.005ppm.

[0413] Suitably the method detects proteins of interest in the presence of interferents. Suitably 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. Suitably 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. Suitably the method detects proteins of interest in the presence of interferents at a concentration of up to 5ppm.

[0414] Suitably, 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, 0.0020ppm.

[0415] Suitably, 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.

[0416] Suitably 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.

[0417] Suitably 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.

[0418] “Sample” is defined elsewhere herein.

[0419] Methods of measuring a protein

[0420] In one aspect, a method of measuring the amount of a protein of interest in a sample is provided, the method 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. 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.

[0421] “Contacting a sample” is defined elsewhere herein.

[0422] 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. In various examples, the amount of the protein of interested may be determined, detected and / or quantified using spectrophotometry, such as for point-of-care use.

[0423] 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.

[0424] 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.

[0425] As defined elsewhere herein, proteins of the invention also encompass low abundance proteins.

[0426] “Fluorescently labelled proteins” is defined elsewhere herein.

[0427] Suitably the fluorescently labelled proteins comprise a large number of fluorophores relative to the proteins. This allows measuring low abundance proteins. Herein, the expression “low abundance protein” is synonymously used with “proteins present at a low level”.

[0428] Suitably 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.

[0429] Suitably the fluorescently labelled protein is streptavidin. Suitably the fluorescently labelled protein is CRP. Suitably the fluorescently labelled protein is IL6. Suitably the fluorescently labelled protein is IL8.

[0430] Suitably the fluorescently labelled protein is rhodamine-streptavidin.

[0431] Suitably the fluorescently labelled protein is labelled with the fluorophore fluorescein isothiocyanate.

[0432] Suitably 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.

[0433] Suitably 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. Suitably 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.

[0434] Suitably 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.

[0435] “Cleavage inducer” is defined elsewhere herein.

[0436] “Suitable conditions” is defined elsewhere herein.

[0437] As used herein, “isolating the fluorescently labelled proteins” refers to any suitable method in which the fluorescently labelled proteins are removed or separated from the hydrogel.

[0438] Suitably Isolating the fluorescently labelled proteins may comprise washing the hydrogel. Sutiably washing the hydrogel for a sufficient time to elute the fluorescently labelled proteins therefrom. Sutiably the washing may be performed with a buffer, such as PBS. Suitably the washing is performed for between 10 to 30 minutes. Suitably the washing is performed for at least 10 minutes.

[0439] As used herein, a “binding molecule” is a small molecule or antibody. In a non-limiting example, 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. The term "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. For example, polyclonal antibodies can be made by the conventional method of immunizing a mammal (e.g., rabbits, mice, rats, sheep, goats). Polyclonal 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).

[0440] Suitably the binding molecule may be located upon a surface. Suitably the binding molecule may be located in a container. Suitably therefore the contacting step takes place within a container comprising the binding molecule. Suitably 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. Suitably 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. Suitably 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.

[0441] Suitably the container may be any commercially available microwell plate coated with the binding molecule of interest. For example, a biotin-coated microwell plate (e.g. 15151, Thermo Fisher Scientific)

[0442] However in preferred embodiments, the container is a container of the invention as defined below.

[0443] Suitably 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.

[0444] In one embodiment the base layer a polymer comprising free amine groups. Suitably 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. In one embodiment the base layer is chitosan. . In one embodiment, 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. In one embodiment the blocking agent comprises PEG-methyl. Suitably the capture protein and the blocking agent bind to the base layer. Suitably the reactive layer comprises a majority of reactive proteins, and a minority of blocking agent. Suitably the blocking agent is bound to the base layer only where the reactive protein is not bound to the base layer. Suitably the blocking agent binds to any free amine groups in the base layer. Suitably the blocking agent prevents free FITC binding to the base layer of the container, suitably when in use.

[0445] Suitably the container may be any container suitable for carrying out a method of the invention therein. In one embodiment, the container is a microwell or microtitre plate.

[0446] Suitably the container of the invention reduces the background signal by a factor of 110 compared to commercially available microwell or microtitre plates. Suitably the container of the invention reduces the background signal caused by free fluorophore, specifically free FITC, binding directly to the container.

[0447] In one embodiment, 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. As used herein, “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.

[0448] Sutiably removing any unbound fluorescently labelled proteins comprises washing, suitably washing the container described above, suitably washing the microwell plate as described above.

[0449] By way of example, 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). In a non-limiting example, the level of fluorescence in sample is determined by spectrofluorometry.

[0450] Suitably the protein of interest is a biomarker.

[0451] 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.

[0452] Typically, the biomarker referred to herein is measured at the protein level.

[0453] Accordingly, in accordance with the invention the biomarker can be any biomarker which is detected or measured at the protein level.

[0454] Thus, in accordance with the invention the biomarker is a protein biomarker. Suitably the biomarker may be a marker of a diseases. Suitably the biomarker may be an infammatory marker. Suitably a biomarker of inflammation. Sutibaly such biomarkers of inflammation typically indicate disease. Suitably the biomarker may be a chemokine or cytokine.

[0455] Suitably 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-fetoprotein (AFP), human chorionic gonadotropin (HCG), CA 19-9, CA 15-3, CA 27- 29, lactate dehydrogenase (LDH), neuron-specific enolase (NSE), C-reactive protein (CRP).

[0456] Suitably the biomarker is IL-6, IL-8, cardiac troponin, or CRP.

[0457] Suitably the cardiac troponin is troponin C (TnC), troponin T (TnT), or troponin I (Tnl). Suitably the cardiac troponin is troponin T (TnT).

[0458] “Sample” is defined elsewhere herein.

[0459] Methods of determining whether a subject has a disease or disorder

[0460] In one aspect, a method of determining whether a subject has a disease or disorder is provided, the method 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. 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.

[0461] “Contacting a sample” is defined elsewhere herein.

[0462] “Fluorescently labelled proteins” is defined elsewhere herein.

[0463] “Cleavage inducer” is defined elsewhere herein.

[0464] “Suitable conditions” is defined elsewhere herein.

[0465] “Isolating the fluorescently labelled proteins” is defined elsewhere herein.

[0466] “Binding molecule” is defined elsewhere herein.

[0467] “Removing any unbound fluorescently labelled proteins” is defined elsewhere herein.

[0468] As defined elsewhere herein, proteins of the invention also encompass low abundance proteins.

[0469] As used herein, “detecting the presence of fluorescence” as used herein means conducting an assay to determine whether or not fluorescence is present, proteins are present in the sample. Suitably the presence of fluorescence is indicative of the presence of the protein biomarker in the sample. In a non-limiting example, 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. Suitably the level of fluorescence is indicative of the level of the protein biomarker in the sample. In a non-limiting example, 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 skilled person is aware of such thresholds and suitable reference ranges. The skilled person also knows that such thresholds and suitable reference ranges may depend on the particular assay that is used. In a non-limiting example, the level of fluorescence is indicative of the level of the protein biomarker IL-6, IL-8, or cardiac troponin. Suitably the level of fluorescence is indicative of the level of the protein biomarker troponin C (TnC), troponin T (TnT), or troponin I (Tnl). Suitably the level of fluorescence is indicative of the level of the protein biomarker troponin T (TnT).

[0470] “Diseases” and “disorders” are defined elsewhere herein.

[0471] Suitably the disease or disorder is cancer or cardiovascular disease.

[0472] Examples of 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. 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, glioblastomas, glioma (e.g., astrocytoma, oligodendroglioma), medulloblastoma); bronchus cancer; carcinoid tumour; cervical cancer (e.g. , 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. , head and neck squamous cell carcinoma, 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, 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. myelofibrosis (MF), chronic idiopathic myelofibrosis, chronic myelocytic leukaemia (CML), chronic neutrophilic leukaemia (CNL), hypereosinophilic syndrome (HES)); neuroblastoma; neurofibroma (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 (e.g. , 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 melanoma; vaginal cancer; and vulvar cancer (e.g., Paget' s disease of the vulva).

[0473] Examples of 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.

[0474] As used herein, “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.

[0475] “Sample” is defined elsewhere herein. EXAMPLES

[0476] 1. Experimental methods

[0477] 1.1 Materials and equipment

[0478] Allyltrichlorosilane (95%), acrylamide / bis-acrylamide (40% solution, 29:1), ammonium persulfate (>98%), ethanol (> 98%, Supelco), fluorescein, fluorescein isothiocyanate isomer I (FITC) (>90%), poly(ethylene glycol) (PEG) bis(amine) (average Mn400 and 3,400), 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 (HATU) (97%), / V, / V-diisopropylethylamine (DIPEA) (98%), triethylamine (TEA) (99%), methacryloyl chloride (97%), phosphate-buffered saline (PBS, 10 mM, pH 7.4), phosgene solution (15 wt. % in toluene), silica gel (high-purity grade, pore size 60 A), sodium sulphate, methanol, ethyl acetate, hexane, DMSO-de (99.9%, MagniSolv), CDCI3 (99.9%, MagniSolv), and acetone-de (99.9%, MagniSolv) were bought from Merck. Decon 90 was bought from Fisher Scientific. The remaining solvents were bought from Merck and prepared before use. Tetrahydrofuran (THF) and toluene were distilled under nitrogen from sodium using benzophenone as indicator. Dichloromethane (DCM), dimethylformamide (DMF) and acetonitrile were distilled under nitrogen from calcium hydride. Streptavidin tagged with rhodamine (S6366) was bought from Thermo Fisher.

[0479] Thin layer chromatography was performed on Macherey Nagel aluminum TLC-plates, precoated with 0.20 mm silica gel 60 and fluorescent indicator UV 254. Visualisation was performed with iodine on silica, followed by heating and UV light (A = 254 nm). Slide-A- Lyzer Dialysis Cassettes (MWCO 2K, Thermo Fisher) were used for purification of PEG 3,400 monomers. Pierce™ 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).

[0480] 1H and13C 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).1H and13C 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).

[0481] 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 mm2aluminum-cored PCB, which in turn was mounted to a 60x60 mm2heat 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.

[0482] 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 Vanquish™ multi wavelength absorbance detector and in a C18 column (1.8 pm particle size, 3 mm internal diameter and 50 mm long). A CLARIOstar Plus microplate reader was used to measure the fluorescence intensity of excited samples (fluorescein, Aexcitation = 472 nm, emission measured = 500 - 620 nm; rhodamine Aexcitation = 540 nm, emission measured = 570 - 670 nm).

[0483] 1.2 Monomer synthesis and purification

[0484] The reaction schemes used to synthesise F-NVOC-allylamide, F-NVOC-PEG400- methacrylamide, F-NVOC34oo-methacrylamide, and FITC-NVOC34oo-methacrylamide are shown in Figure 2. The reaction schemes were adapted from the procedure reported Landfester and Klinger25and our previous work26, 27. The details of reaction and purification conditions of monomers along with characteristic shifts in their NMR spectra are provided in the SI.

[0485] Further details on the synthesis and purification of F-NVOC-allylamide, NVOC-PEG400- methacrylamide monomer F-NVOC-PEG34oo-methacrylamide and FITC-NVOC-PEG3400- methacrylamide monomers are provided below:

[0486] F-NVOC-allylamide

[0487] NVOC (300 mg, 1.00 mmol, 1 equiv) and anhydrous DMF (5 mL) were added to a nitrogen purged oven dried 25 mL round-bottom flask (RBF). To this solution, allylamine (75 pL, 1.00 mmol, 1 equiv), HATLI (381 mg, 1.00 mmol, 1 equiv), and DIPEA (873 pL, 5.00 mmol, 5 equiv) were added. The reaction was left to stir for 18 h. The following day, the solution was diluted with ethyl acetate (50 mL) and transferred to an extraction vessel. The organic phase was washed with DI water (50 mL) three times. Finally, the organic phase was collected, dried over sodium sulphate, filtered, and concentrated in vacuo. The crude was purified via flash chromatography with an eluent of 5% methanol in DCM. This gave the expected product, N-allyl-4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy) butanamide (NVOC- allylamide), as a pale-yellow solid (322 mg, 95%).

[0488] 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) and TEA (83 pL, 0.60 mmol, 1.5 equiv) were dissolved in cold dry DCM (5 mL) in another dry, dark, two-necked RBF. To this fluorescein containing flask, 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. The volume was reduced by flushing the reaction vessel with nitrogen and then purified using flash chromatography (5% to 40% ethyl acetate: hexanes) in a dark fume hood, to give the product as a yellow solid (F-NVOC-allylamide, 225 mg).

[0489] 1H NMR (400 MHz, DMSO-d6): 5 8.03 (f, J = 5.7 Hz, 1 H), 7.52 (s, 1 H), 7.36 (s, 1 H), 5.79 (ddt, J = 17.2, 10.4, 5.3 Hz, 1 H), 5.47 (d, J = 4.4 Hz, 1 H), 5.26 (qd, J = 6.2, 4.3 Hz, 1 H), 5.11 (dq, J = 17.2, 1.8 Hz, 1 H), 5.03 (dq, J = 10.2, 1.6 Hz, 1 H), 4.04 (f, J = 6.5 Hz, 2H), 3.90 (s, 3H), 3.69 (ft, J = 5.5, 1.7 Hz, 2H), 2.69 (s, 2H), 2.28 (f, J = 7.4 Hz, 2H), 2.00 - 1.92 (m, 2H), 1.36 (d, J = 6.2 Hz, 3H).13C NMR (400 MHz, DMSO-d6) 5 171.23, 153.42, 146.27, 138.89, 137.98, 135.50, 114.95, 109.09, 108.36, 68.28, 63.90, 56.05, 40.84, 40.15, 39.94, 39.73, 39.52, 39.31 , 39.10, 38.89, 38.24, 31.48, 25.17, 24.67.

[0490] F-NVOC-PEGwo-methacrylamide monomer

[0491] First, PEG400 bis(amine) (500 mg, 1 equiv) was added to dry DCM (20 mL). The mixture was then cooled to 0 °C and TEA (15 uL, 0.1 mmol, 1.2 equiv) was added dropwise and the mixture stirred for 1 h under inert conditions. Following this, methacryloyl chloride (12 uL, 0.36 mmol, 0.6 equiv) was added dropwise. The mixture was stirred for 1 and purified by flash chromatography to yield amine-PEG4oo-methacrylamide (Mn= 504 g.mol-1) in 80% yield. To an ice-cooled suspension of NVOC (150 mg, 0.50 mmol, 1 equiv.), HATLI (229 mg, 0.70 mmol, 1.4 equiv) and 183 pL DIPEA (1.05 mmol, 2.1 equiv) in 10 mL of DMF were stirred. Amine-PEG4oo-methacrylamide (281 mg, 0.70 mmol, 1.4 equiv) was dissolved in minimal amount of DMF and added dropwise to the solution of NVOC, HATLI, and DI PEA. The reaction mixture was allowed to warm to room temperature and stirred for 18 h. After the reaction was completed, the mixture was treated with water (20 mL) and extracted with ethyl acetate (20 mL x 3). The combined organic layers were washed with brine, dried over anhydrous sodium sulphate, and concentrated to give a crude product, which was purified by column chromatography (100% ethyl acetate). NVOC-PEG4oo-methacrylamide was obtained at 20% yield as a crystallizing oil.

[0492] This oil was then redissolved in dry DCM (20 mL) in a dark RBF and stirred at 0 °C for 30 min under a constant flow of nitrogen. To this RBF, 4 mL of cold phosgene solution (15 wt.% in toluene, 1.5 equiv) was added dropwise over 20 min. The mixture was stirred under dark and inert conditions for 30 min. To another dry dark RBF, FITC (291 mg, 0.75 mmol) and TEA (60 uL, 0.41 mmol) were dissolved in cold dry DCM (5 mL) and stirred for 30 min. To this RBF, NVOC-PEG4oo-methacrylamide solution was added dropwise over 30 min. The mixture was stirred at room temperature for 18 h. The solvent was evaporated through purging with nitrogen in a fume hood and the residue purified with flash chromatography to yield the targeted monomer (F-NVOC-PEG4oo-methacrylamide, 169 mg, 35%).

[0493] 1H NMR (400 MHz, DMSO-d6) 5 8.01 - 7.88 (m), 7.61 (s), 7.27 (f, J = 5.7 Hz), 7.23 - 7.13 (m), 6.58 (d, J = 8.9 Hz), 6.37 (s), 5.66 (f, J = 1.3 Hz), 5.32 (p, J = 1.6 Hz), 4.08 - 4.02 (m), 3.93 - 3.87 (m), 3.68 (s), 3.36 - 3.30 (m), 3.33 (s), 3.16 (f, J = 6.1 Hz), 3.07 (g, J = 5.9 Hz), 2.43 (g, J = 7.1 Hz), 2.10 (d, J = 1.5 Hz), 1.99 (f, J = 6.5 Hz), 1.85 (f, J = 1.2 Hz), 1.64 (d, J = 0.9 Hz), 1.37 (dd, J = 6.2, 2.0 Hz), 1.35 (s), 1.24 (s), 0.93 (f, J = 7.1 Hz).13C NMR (400 MHz, DMSO-d6) 5 168.14, 156.83, 140.26, 119.67, 82.48, 70.24, 69.29, 63.70, 46.13, 40.47, 40.26, 40.05, 39.84, 39.63, 39.42, 39.22, 18.99.

[0494] F-NVOC-PEG34oo-methacrylamide and FITC-NVOC-PEG34oo-methacrylamide monomers Amine-PEG34oo-methacrylamide (Mn= 3604 g.mol-1) was prepared by reacting PEG3400 bis(amine) with methacryloyl chloride by following the same procedure as described above.

[0495] Amine-PEG34oo-methacrylamide (3 g, 1 equiv) was dissolved in 2 mL of DMF. To an ice- cooled suspension of NVOC (399 mg, 1.33 mmol, 1.5 equiv.), HATU (229 mg, 0.70 mmol, 1.4 equiv) and 183 pL DIPEA (1.05 mmol, 2.1 equiv) in 10 mL of DMF was stirred. The amine-PEG34oo-methacrylamide solution was added dropwise to the solution of NVOC, HATLI, and DIPEA. The reaction mixture was allowed to warm to room temperature and stirred for 18 h. The product, 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.

[0496] 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%).

[0497] 1H NMR (400 MHz, DMSO-d6) 5 7.91 (f, J = 5.6 Hz), 7.24 (f, J = 5.7 Hz), 5.65 (f, J = 1.2 Hz), 5.32 (p, J = 1.7 Hz), 4.13 - 4.00 (m), 3.50 (d, J = 141.5 Hz), 3.15 (f, J = 6.1 Hz), 3.07 (g, J = 6.1 Hz), 2.54 (s), 1.84 (f, J = 1.2 Hz), 1.63 (s), 1.36 (d, J = 10.2 Hz).13C NMR (400 MHz, DMSO-d6) 5 168.14, 156.83, 140.26, 119.67, 82.48, 70.24, 69.29, 63.70, 46.13, 40.47, 40.26, 40.05, 39.84, 39.63, 39.42, 39.22, 18.99.

[0498] 1.3 Preparation of hydrogel films

[0499] Glass microscope slides were cut into 25.4 ± 0.5 mm2and 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. 1 mL solutions containing either F-NVOC- allylamide dissolved in 250 pL DMSO or (F or FITC)- NVOC-PEG(4OO or 34oo)-methacrylamide dissolved in 250 pL of deionized water, 40% w:v acrylamide / bis-acrylamide (volume calculated to create a 10 wt.% gel), 1.25 pL of TEMED, and 12.5 pL of 10% w:v APS were prepared in N2-degassed water. The concentration of the NVOC containing monomer was 5, 10, or 15% of the total molar concentration of the monomers in the precursor solution. 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.

[0500] 1.4 Characterisation of monomers and hydrogel films The UV-Vis absorption spectra of monomers were measured at different concentrations to determine their molar extinction coefficient. Monomer stock solutions were irradiated to 365 nm light and samples were taken at fixed time intervals for subsequent analysis by UV- Vis spectroscopy and HPLC. For HPLC, 1 mg / ml monomer stock solutions were prepared in acetonitrile and collected samples solutions were chromatographed using a solvent gradient of acetonitrile / water from 90 / 10 to 10 / 90. The chromatograms were recorded at a wavelength of 491 nm. The HPLC data was used to determine the kinetics of light-triggered release of fluorescein from monomers.

[0501] The 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.

[0502] Table 1. Summary of percentage of the photolabile monomer incorporated in hydrogel films (where the total concentration of monomers in precursor solutions was 10% w: v). 1.5 Protein studies

[0503] 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-methacrylamidewas 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.

[0504] 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. To verify that the released RS was labelled with both rhodamine and fluorescein, 200 pl of PBS solutions collected after UV-light exposure was pipetted in a biotin coated well of microtiter plates and incubated for 30 min. Subsequently, the well was washed with 200 pL of PBS and the fluorescence emission spectra were measured at excitation wavelengths of 470 and 540 nm corresponding to fluorescein and rhodamine, respectively.

[0505] 2. Results

[0506] 2.1 Monomers

[0507] The chemical structures of the synthesised monomers are provided in Figure 3. 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. In contrast, as shown in Figures 10 and 11 , 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.

[0508] Photolysis occurs when the irradiation wavelength overlaps with the absorption band of the photolabile group28'30, in this case, NVOC. Thus, the monomers solutions were irradiated to 365 nm UV-light. A typical UV-Vis spectra of the monomers for different irradiation times (see Figure 12) 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. Thus, released fluorescein and the monomer were separated using HPLC with the resulting chromatograms provided in Figure 4(a) and Figures 13 and 14. HPLC measurements were carried out in acetonitrile at various irradiation times. The kinetics of light- triggered release of fluorescein was investigated by monitoring the rate of disappearance of the monomers ([M]t). Plots of ln[M]o / [M]t versus irradiation 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 kinetics of light-triggered release of fluorescein from PEG- methacrylamide monomers was faster than the allylamide monomer.

[0509] 2.2 Hydrogel films

[0510] 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. These spectra were obtained by changing the position of the slide relative to the beam of the spectrophotometer. 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 (mprecursor). 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). As shown in Figure 5(d), 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)31and the water solubility of the PEG-based monomers. There was a slight decrease in monomer incorporation with increasing PEG molecular weight.

[0511] The photoinduced release of fluorescein from hydrogels was investigated next. 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.

[0512] In summary, 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).

[0513] Considering these benefits offered by F-NVOC-PEG34oo-methacrylamide, the protein studies were carried out using a derivative of this monomer, FITC-NVOC-PEG34oo-methacrylamide, with isothiocyanate group that can react with the primary amines in proteins.

[0514] 2.3 Protein studies

[0515] Rhodamine-streptavidin (RS) 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.

[0516] 2.3.1 Protein capture and pre-concentration

[0517] The functionalized hydrogels, which were prepared by co-polymerizing acrylamide / bisacrylamide and FITC-NVOC-PEG34oo-methacrylamide monomers, were capable of protein capture because the isothiocyanate groups reacted with the terminal amine and primary amines in proteins32. 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.

[0518] Six functionalized hydrogels were prepared; three were incubated with 0.1 ppm RS solutions and the remaining three with 0.01 ppm RS solutions. As summarized in Table 2, the percentage of RS loaded in functionalized hydrogels was 77 ± 8% and 94 ± 6% for 0.1 and 0.01 ppm protein solutions, respectively. This is significant when compared to the light- responsive hydrogels prepared by Klinger and Landfester25, where proteins were trapped inside hydrogels as a result of decreased pore size arising from electrostatic interactions, where a protein loading of 53.2 ± 7.4% was reported. Subsequently, the concentration of RS in functionalized hydrogels was estimated and the results are provided in Table 2. The ratio of concentrations of RS in hydrogels and stock solutions provided the pre-concentration, which as shown in Figure 8(c), were estimated to be 192 and 236 for 0.1 and 0.01 ppm RS solutions, respectively.

[0519] Table 2. A summary of RS loading in hydrogels made by co-polymerizing FITC-NVOC- PEG3400- methacrylamide with acrylamide / bisacrylamide (molar ratio of FITC-NVOC-PEG3400- methacrylamide was 10%, volume of RS stock solution was 10 mL, volume of hydrogel was estimated to be 0.04 mL, and determined by change in fluorescence emission). RS concentration RS concentration before incubation after overnight % RS loaded in Amount of RS in RS concentration with incubation with functionalised functionalised in functionalised functionalized functionalized hydrogel hydrogel (pg) hydrogel (ppm) hydrogel (ppm) hydrogel (ppm)t

[0520] 0.0176 82% 0.824 20.6

[0521] 0.10 0.0205 80% 0.795 19.9

[0522] 0.0318 68% 0.682 17.1

[0523] 0.0003 97% 0.097 2.4

[0524] 0.01 0.0012 88% 0.088 2.2

[0525] 0.0002 98% 0.098 2.5

[0526] 2.3.2 Protein release and detection

[0527] Having demonstrated the loading of a model protein into the prepared hydrogels, the photolytic release of protein from the hydrogels was investigated. 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.

[0528] The fluorescence emission spectra of the PBS collected after each irradiation are shown in Figure 8(a). The resulting cumulative release of RS as a function of irradiation time is shown in Figure 8(b). The experimental data shown in Figure 8(b) when fitted to a single exponential rise did not give a good fit to the data (r = 0.966), but a double exponential rise to a maximum gave a good fit with a correlation coefficient of 0.9995, indicating that two processes take place during the photolytic release of captured RS. The first fast process accounts for about 54% of the RS released with a time constant of 1.124 min-1, while the second slower process accounts for the remaining 46% of the released RS with a time constant of 0.075 min-1. From this the inventors showed that 50% of the RS was released in -1.68 minutes, or -100 s. The origin of these two processes is currently unclear but may be a result of slow diffusion of released RS from the deeper layers of the hydrogel.

[0529] To validate that 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 resulting fluorescence spectra (see Figure 8(c)) showed emission peaks corresponding to fluorescein and rhodamine, confirming that the released RS was successfully labelled with fluorescein. Figures 15 and 16 show that the molar emission coefficient of fluorescein at 514 nm is ~10 times higher than rhodamine at 590 nm in RS. Based on this and considering the fluorescence emission intensities at 514 and 590 nm in Figure 5(c), the inventors estimate that the RS was labelled with -17 fluorescein molecules per one molecule of rhodamine. As each streptavidin was labelled with 5 rhodamine molecules, the inventors estimate that 85 molecules of fluorescein were attached to each streptavidin. One possible explanation of this observation is that the FITC was separated from the backbone of the functionalized hydrogels by PEG3400 spacer arm, making it easier for many FITC molecules to react with each RS molecule loaded in the hydrogels. The labelling of RS with large numbers of fluorescein is beneficial for measurement of low abundance proteins.

[0530] 2.3.3. Conclusion

[0531] This work exemplifies a hydrogel for pre-concentration, labelling, and controlled release of proteins for their subsequent detection by fluorescence. 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-1molecular 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.

[0532] By replacing fluorescein with FITC in the monomers and subsequently the hydrogels, proteins were captured by reaction of amines on the protein with the isothiocyanate group. 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.

[0533] 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.

[0534] 3. Further Polyacrylamide Hydrogel Experiments

[0535] Further exemplification was carried out to optimise variables such as the length of the PEG chain of the active monomer, ratio of active to inactive monomer, incubation time with protein solutions, and washing time to release proteins from hydrogels after exposure to 365 nm light. Equally, a protocol was developed to immobilise recognition elements (e.g., biotin and antibodies) to wells of microtitre plates. The in-house developed microtitre plates showed lower non-specific adsorption to free FITC released from hydrogels than commercial biotin coated microtitre plates. Furthermore, the polyacrylamide hydrogels of the invention were made as films on glass substrates as before, but also as discs, and it was shown that they can be stored in buffer for at least up to 7 days without any detrimental effects.

[0536] The further exemplification also showed that 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. These LCDs for proteins obtained using hydrogels of the invention is comparable to commercially available colorimetric ELISA kits [Analyst, 2014, 139, 439] with added advantages that the hydrogels do not require secondary antibodies, enzyme labels, and substrates as well as not requiring multiple adding / washing steps. Said further exemplification is described in full detail below. Materials and methods used are the same as described above in section 1 unless otherwise stated.

[0537] 3.1 Monomer synthesis and purification

[0538] 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.

[0539] 3.2 Composition of precursor solutions used to make hydrogels

[0540] Commercially available 40% (w:v) 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.

[0541] If the total crosslinker plus monomers concentration was reduced to 2.5% (w:v), after the addition of APS and TEMED, the solution became viscous but hydrogel was not formed. If the total crosslinker plus monomers concentration was increased to 10% (w:v), after the addition of APS and TEMED, hydrogels were formed. Thus, work was undertaken on hydrogels made of 5% and 10% (w:v) precursor solutions.

[0542] Table 3: Composition of 1 mL precursor solutions used to make polyacrylamide hydrogels (the total crosslinker plus monomers concentration was 5% (w:v)) 3.3 Hydrogel Films and Discs

[0543] 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.

[0544] 3.4 Biotin coated microtitre plates

[0545] Commercially available 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. 200 pL of 20 mg / mL NHS-PEG-biotin in PBS was then added to each well and allowed to react for 3 h. Subsequently, 200 pL of either 20 or 200 mg / ml NHS-PEG-methyl in PBS was added to each well and allowed to react for 3 h. NHS groups react with amines in chitosan and hence this procedure allowed immobilisation of biotin via the amines in chitosan deposited in wells followed by the blocking of the remaining amine groups in chitosan with PEG-methyl. Finally, the wells were washed with PBS to remove any unreacted NHS-PEG-biotin and NHS-PEG- methyl.

[0546] 3.5 Preliminary Studies

[0547] 3.5.1 Release kinetics of fluorescein using monomer solutions and chromatography It is important to study the release kinetics of FITC because it determines the rate of release of proteins captured in our hydrogels via the isothiocyanate groups of FITC. We studied the release kinetics of FITC by: (1) exposing monomer solutions to 365 nm light for different durations and analysing solutions using liquid chromatography, and (2) exposing hydrogel discs to 365 nm light for different durations and using fluorescence spectroscopy for analysis. The details are provided below.

[0548] Stock solutions of 1 mg / mL active monomers were prepared in acetonitrile (MeCN) and exposed to 365 nm light for selected duration. Afterwards, 20 pl solution was collected. The procedure was repeated to expose the solution for up to 30 mins. All collected solutions were analysed sequentially using reversed-phase high-performance liquid chromatography (RP-HPLC). RP-HPLC was used for analysis because after the active monomer solution is exposed to 365 nm light, it contains both released fluorescein and fluorescein bound to the monomer. As released fluorescein and that bound to the monomer absorbs at the same wavelength, it was essential to separate the two to determine their individual quantities.

[0549] For the RP-HPLC, 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]tversus 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.

[0550] 3.5.2 Release kinetics of fluorescein using hydrogel films and absorption and fluorescence spectroscopy

[0551] 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.

[0552] 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 (see inset in Figure 6(a)) 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. This suggests that fluorescein, once released, can rapidly diffuse out of the hydrogel, but the non-linearity after ~30 min exposure suggests that there might have been a small amount of irreversibly bound fluorescein remaining in the hydrogel. The time constant for release kinetics of fluorescein from F-NVOC-PEG34oo-methacrylamide containing hydrogel was 10.6 min.

[0553] 3.5.3 Effect of length of PEG in the active monomer

[0554] 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.

[0555] Table 4: Summary of percentage of the photolabile monomer incorporated in hydrogel films

[0556] (where the total concentration of monomers in precursor solutions was 10% w:v) 3.5.4 Reproducibility and storage stability of hydrogels

[0557] Hydrogel films made using 10% (w:v) precursor solutions were washed in 10 mL PBS overnight to remove any monomers that were not incorporated in hydrogels. A comparison of absorption spectra of 4 films after an overnight wash in PBS (Figure 17(a)) suggested that film-to-film reproducibility was excellent. These 4 films were stored in PBS for different durations (1, 3, 5 and 7 days) following which, their absorption spectra were measured. A comparison of Figures 17(a) and (b) shows that the absorption spectra of the films did not change significantly, suggesting that the storage stability of the films in PBS was at least 7 days.

[0558] Similar experiments were also performed for hydrogel films made using 5% (w:v) precursor solutions. The results are shown in Figure 18, which suggested that film-to-film reproducibility was excellent and that the films could be stored in PBS for at least 7 days. A comparison of Figures 17 and 18 highlights that the peak absorbance for hydrogel films made using 10% (w:v) precursor solution is significantly less than double that of hydrogels films made using 5% (w:v) precursor solution. This suggests that F-NVOC-PEG3400- methacrylamide was better incorporated in films made using 5% (w:v) precursor solutions. Furthermore, hydrogels made of 5% (w:v) precursor solution are expected to be less tightly knit than those prepared using 10% (w:v) precursor solution. For these reasons, the remaining work was carried out using hydrogels made of 5% (w:v) precursor solutions.

[0559] 3.5.5 Format of hydrogels - films versus discs

[0560] Hydrogels films deposited on glass slides are not an ideal format to be placed in people’s mouth. Hence, we made hydrogel discs with sizes comparable to mentos. For this purpose, we made fixtures where the bottom plastic had a -25x25 mm2square 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. Equally, the use of discs offered an additional benefit that proteins captured in hydrogels could be released in lower volumes of buffer than used for films (200 pL for discs versus 3 mL for films). The lower the volume of the buffer used to release proteins captured in our hydrogels, the higher is the resulting preconcentration factor. Thus, the remaining work was performed using hydrogel discs. 3.5.6 Pre-concentration factor

[0561] To determine the pre-concentration factor, we prepared 10 mL of a -0.005 ppm RS solution, and measured its fluorescence spectrum (black trace, Figure 19(a)). We then immersed a hydrogel disc in the unstirred RS solution for 24 h and measured the fluorescence spectrum of the solution (red trace, Figure 19(a)). Using the calibration curve of RS (shown in Figure 19(b)), we converted the peak fluorescence intensity of RS solutions before and after incubation with the hydrogel disc to determine RS captured by the hydrogel disc. We found that 85.6% RS was captured by our hydrogel after 24 h incubation. As the volume of the hydrogel disc was much smaller than the volume of RS solution, the concentration of RS in hydrogel discs was determined to be 1.2615 ppm (versus 0.00428 ppm in solution). Hence 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. Thus, the pre-concentration factor for the released proteins was -50.

[0562] 3.6 Optimisation Studies

[0563] 3.6.1 Effect of the ratio of active to inactive monomers in hydrogels

[0564] 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. The time constants for protein release kinetics did not follow a pattern, being 2.97, 2.03, 2.73 and 2.03 min for 1:10, 1:20, 1 :40 and 1 :100 active to inactive monomer ratios respectively, but the 1 :40 ratio gave a much higher signal than either the 1:10, 1:20 or 1:100 monomer ratios. This is because at lower monomer ratios the proteins that diffuse into the interior of the hydrogel are captured with multiple FITC groups, which all must be broken to release the labelled protein. For 1:100 monomer ratio, the lower concentration of FITC means that less protein is captured but it is released quickly because fewer bonds need to be cleaved. In contrast, proteins on the surface of the hydrogel will be captured by fewer FITCs, allowing rapid release but in much lower concentrations.

[0565] 3.6.2 Effect of incubation time of hydrogels in protein solutions

[0566] Based on the previous experiment, the 1 :40 molar ratio of active to inactive monomers was chosen for subsequent work. 10 mL of 0.1 ppm RS was incubated over hydrogel discs for 1, 15, 24 and 48 h. The hydrogel discs were exposed to 365 nm light for a total of 60 min and the results shown in Figure 21. For 0.1 ppm RS, the signal after 48 h was only slightly higher than for 24 h, so for subsequent work the incubation time was limited to 24 h maximum. Similar studies were conducted for 0.01 and 0.005 ppm RS solutions, but the maximum incubation time was limited to 24 h. After the selected incubation time, proteins were released from hydrogels by exposure to 365 nm light and fluorescence spectra of supernatants were measured. Plots of peak fluorescence versus exposure time for hydrogel discs incubated in after incubation in 0.01 and 0.005 ppm RS solutions for 24 h are shown in Figure 22 and Figure 23, respectively. Similar to Figure 21 , Figure 23 clearly shows that the fluorescence signal increased significantly as the incubation time of hydrogel discs in RS solutions increased from 15 to 24 h. Thus, all subsequent was carried out with 24 h incubation time.

[0567] Furthermore, a comparison of Figures 21 to 23 shows that for a selected incubation time, the fluorescence signal increases with the concentration of RS solution. This is promising because it implies that our hydrogels can be suited for quantitative measurements.

[0568] 3.6.3 Commercial versus in-house prepared biotin coated microtitre plates

[0569] Next, we studied the potential of PEG hydrogels for the measurement of proteins that are not pre-labelled with rhodamine. After incubation with protein solutions, when hydrogels are exposed to 365 nm light, free FITC and FITC-labelled proteins are released. Thus, to determine the protein concentration, it is essential to remove free FITC. One way to do this is to use microtitre plates with wells that are coated with recognition elements to capture proteins (e.g., biotin coated microtitre plates to capture streptavidin). We used commercially available biotin coated microtitre plates and did a control experiment where a 1:40 monomer molar ratio hydrogel disc was washed in PBS overnight and then exposed to 365 nm light for a maximum of 60 min, collecting released FITC in 200 pL of PBS over 30 minutes for each successive exposure. The 200 pL samples were pipetted into the wells of a commercially available biotinylated microtitre plate, allowed to incubate for 30 min and then washed out with PBS. The fluorescence spectrum from each well was recorded using a microtitre plate reader. Figure 24 shows the resulting peak fluorescence intensity as a function of exposure time. The released FITC reacts strongly with the biotinylated plate, giving a very high fluorescence background. For this reason, we developed our own biotinylated microtitre plate with low FITC binding. 200 pL of a 1.5% solution of chitosan in 0.1 M acetic acid was pipetted into the wells of a microtitre plate and dried at 50 °C for 2 hours. Each well was then treated with 200 pL of a 25 mg / mL solution of NHS-PEG-biotin in PBS, followed by treatment with 200 pL of either 25 or 250 mg / mL of NHS-PEG-methyl in PBS to block any unreacted amines. After this, the same procedure as for the commercial plate was followed to allow a direct comparison. The results for FITC release are shown in Figure 25. The higher concentration of blocking agent reduced the FITC signal by a factor of 110 compared to the commercial biotinylated microtitre plate.

[0570] 3.6.4 Effect of washing time after exposure to 365 nm light

[0571] 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.

[0572] 3.7 Measurement of Proteins

[0573] 3.7.1 Measurement of streptavidin

[0574] 10 mL of unlabelled streptavidin solutions of 0, 0.0025, 0.005, 0.0075 and 0.01 ppm in PBS were incubated over 1:40 active to inactive molar ratio hydrogel discs for 24 h, after which the discs were washed overnight in PBS and then exposed to 365 nm light for a total of 60 min in 200 pL of PBS. The released and now labelled streptavidin solutions were placed in the wells of a microtitre plate containing a biotinylated chitosan hydrogel and allowed to bind for 30 min, after which the streptavidin solutions were removed, and the chitosan hydrogels washed with PBS. The fluorescence emission of the bound fluorescein-labelled streptavidin was then measured in a microtitre plate reader at an excitation wavelength of 470 nm. Figure 27 summarises the results as a function of exposure time, showing that as the concentration of streptavidin increases, so does the fluorescence signal. At 60 min exposure time, a streptavidin calibration curve is shown in Figure 27(b). Based on the calibration curve, the limit of detection (LOD) for streptavidin was 0.0033 ppm, or 60 pM.

[0575] 3.7.2 Measurement of C-Reactive Protein (CRP)

[0576] 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. 200 pL of 20 mg / mL NHS-PEG-NHS in PBS was then added to each well and allowed to react for 2 h. Subsequently, 200 pL of 200 mg / ml NHS-PEG-methyl in PBS was added to each well and allowed to react for 2 h. The wells were washed with PBS to remove unreacted material, following which 200 uL of 0.005 ppm anti-CRP in PBS was added to each well and incubated for 2 h. Amines on the anti-CRP reacted with free NHS groups bound to chitosan to immobilise the antibody. Finally, the wells were washed with PBS to remove any unreacted NHS-PEG-biotin and NHS-PEG-methyl. 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. At 60 min exposure time, 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.

[0577] 3.7.3 Measurement of interleukin-8 (IL8)

[0578] To show that our hydrogels are suitable for pre-concentrating and labelling any protein, we performed an experiment where IL8 was captured, labelled and released using the 1:40 molar ratio active to inactive monomer hydrogel using the same method as described above for anti-CRP but using 0.005 ppm anti-IL8. Figure 29 shows the fluorescence signal from anti-IL8 coated microtitre plates for IL8 of original concentration 0.005 ppm in PBS as a function of exposure time, confirming that IL8 can be measured.

[0579] 4. Poly(ethylene glycol) (PEG) Hydrogel Experiments

[0580] Further experiments were performed to show the functionality to bind to proteins, and additionally concentrate and label proteins can be realised using poly(ethylene glycol) (PEG) in addition to polyacrylamide hydrogels. Optimum values for variables such as active to inactive monomer ratio and protein capture time was shown to be similar for both hydrogels. Furthermore it was found that PEG hydrogels were well suited for the measurement of low concentrations of proteins in the presence of high concentrations of a single and multiple interferents. This was shown by measuring 0.005 ppm concentration of an exemplar protein, streptavidin, in the presence of 0.5 and 5 ppm mucin. Furthermore, it was demonstrated that down to 0.005 ppm of IL6 can be measured in synthetic saliva containing multiple interferents.

[0581] The experiments described below show that the functionality of binding to proteins, and additionally concentrating and labelling proteins of the invention, can also be realised using hydrogels comprised of different types of monomers and crosslinkers, for example using a poly(ethylene glycol) (PEG) based hydrogel. In this case, the crosslinker was 4-arm PEG alkyne, inactive monomer was PEG bis-azide, and active monomer was FITC-NVOC- PEGwoo-azide. As shown in Figure 30, 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.

[0582] Said further exemplification is described in full detail below. Materials and methods used are the same as described above in section 1 unless otherwise stated.

[0583] 4.1 Synthesis of monomers

[0584] Synthesis of 4-arm PEG alkyne (crosslinker)

[0585] The synthesis of 4-arm PEG alkyne was adapted from literature [RSC Advances, 2016, 6, 36568], As shown in Figure 31, the 4-arm PEG2000 (1 g, 0.5 mmol, 1 equiv), propargyl bromide (1.67 mL of 80% in toluene, 15 mmol, 30 equiv) and sodium hydroxide (NaOH) pellets (0.6 g, 15 mmol, 30 equiv) were added to 4 mL of toluene and stirred for 24 h at 50 °C. The mixture was evaporated under vacuum and the residue was dissolved in 100 mL of water. The solution was extracted with dichloromethane (3x50 mL) and the collected organic layer was dried with anhydrous Na2SO4, concentrate. The final product, 4-arm PEG alkyne (molecular weight: -2050 g / mol), was obtained by precipitation from cold diethyl ether with a yield of 62% (652 mg, 0.311 mmol).

[0586] MALDI: found m / z for Cg7Hi8o044Na+: 2073, n=11

[0587] 1H NMR (300 MHz, CDCh) 64.20 (d, J = 2.4 Hz, 8H), 3.74 - 3.48 (m, 169H), 3.41 (d, J = 2.9 Hz, 8H), 2.44 (t, J = 2.4 Hz, 4H).

[0588] Synthesis of PEG bis-azide (inactive monomer)

[0589] The synthesis of PEG bis-azide was adapted from literature [Polymers, 2021, 13, 1403], As shown in Figure 32, 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. 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.

[0590] Mesylate-terminated PEG was dissolved in 25 mL DMF (5 mL per 1 g of polymer) in a round-bottomed flask and sodium azide (NaNs) (971 mg, 14.93 mmol, 10 equiv) was added. The mixture was heated to 65 °C for 16 h. After cooling to 20 °C, the mixture was filtered and the insoluble material was washed with 10 mL ethanol. The filtrate was concentrated using a rotary evaporator, and the resulting residue was dissolved in ultrapure water (5 mL per 1 g of polymer). The aqueous solution was extracted with DCM (3x10 mL), and the combined organic extracts were dried (MgSO4) and filtered using a sintered funnel under vacuum. The filtrate, PEGsssobis-azide (molecular weight: -3416 g / mol), was concentrated using rotary evaporator to obtain a yield of 52% (2.59 g, 0.774 mmol).

[0591] 1H NMR (400 MHz, CDCI3) 6 3.64 (s, 300H), 3.41 - 3.37 (m, 4H).

[0592] Synthesis of FITC-NVOC-PEGiooo-azide (active monomer)

[0593] To make the active monomer required the synthesis of starting materials as described below.

[0594] Synthesis of amine-PEG-azide

[0595] The synthesis of amine-PEG-azide was adapted from literature [Polymers, 2021, 13, 1403 and ACS Chemical Neuroscience, 2018, 9, 100], see Figure 33. The PEG1000 (5000 mg, 5 mmol, 1 equiv.) and TEA (5.5 mL, 40 mmol, 8 equiv.) were dissolved in 25 mL DCM (5 mL per 1 g of polymer) in a RBF, which was fitted with a septum and purged with nitrogen. MsCI (3.1 mL, 40 mmol, 8 equiv) was added dropwise at 0 °C, and the mixture was stirred at 20 °C for 16 h. 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. 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)

[0596] 1H NMR (400 MHz, CDCI3) 6 3.64 (s, 88H), 3.51 (t, J = 5.2 Hz, 2H), 3.42 - 3.36 (m, 2H), 2.86 (t, J = 5.2 Hz, 2H).

[0597] Synthesis of NVOC-PEG-azide

[0598] As shown in Figure 34, 4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy)butanoic acid (NVOC, 150 mg, 0.5 mmol, 1 equiv), 1-[Bis(dimethylamino)methylene]-1 H-1 ,2,3-triazolo[4,5- b]pyridinium 3-oxide hexafluorophosphate (HATU, 228 mg, 0.6 mmol, 1.2 equiv) and N,N- Diisopropylethylamine (DIPEA, 425 pL, 2.5 mmol, 5 equiv) were added in 5 mL DMF, stirred at 0 °C and purged with nitrogen. 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).

[0599] 'H NMR (400 MHz, CDCh) 6 7.57 (s, 1H), 7.32 (s, 1H), 6.37 (s, 1H), 5.55 (q, J= 6.1 Hz, 1H), 4.11 (t, J= 6.2 Hz, 2H), 3.98 (s, 3H), 3.64 (s, 94H), 3.48 - 3.43 (m, 4H), 3.39 (t, J= 5.1 Hz, 2H), 2.42 (t, J= 7.1 Hz, 2H), 2.24 - 2.15 (m, 3H), 1.54 (d, J= 6.3 Hz, 3H).

[0600] Synthesis of FITC-NVOC-PEGiooo-azide

[0601] As shown in Figure 35, NVOC-PEGwoo-azide (125 mg, 0.093 mmol, 1 equiv) was dissolved in 0.9 mL anhydrous THF and cooled to 0 °C. 6.6 mL of 15% phosgene (COCI2, 4.675 mmol, 100 equiv) was added and stirred (20 °C, 18 h) to this solution. Subsequently, 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. The aqueous solution was extracted with DCM, washed with brine, dried over Na2SC>4, filtered and concentrated using a rotary evaporator at 30 °C to obtain FITC-NVOC- PEGwoo-azide (molecular weight: -1752 g / mol) with a yield of 63% (103 mg, 0.0588 mmol)

[0602] 1H NMR (400 MHz, CDCh) 6 7.81 (s, 1H), 7.60 (s, 1 H), 7.50 (s, 1H), 7.07 (d, J = 18.3 Hz, 1 H), 6.84 (d, J = 54.2 Hz, 3H), 6.60 (s, 1H), 6.55 - 6.35 (m, 2H), 4.11 (s, 2H), 3.99 (s, 3H), 3.64 (s, 111 H), 3.41 - 3.35 (m, 2H), 2.19 (s, 3H), 1.88 (d, J = 6.6 Hz, 5H), 1.76 (d, J = 6.4 Hz, 2H).

[0603] 13C NMR (400 MHz, DMSO-d6) 5 168.14, 156.83, 140.26, 119.67, 82.48, 70.24, 69.29, 63.70, 46.13, 40.47, 40.26, 40.05, 39.84, 39.63, 39.42, 39.22, 18.99.

[0604] 4.2 Formation of PEG hydrogels

[0605] 4-arm PEG alkyne, PEG bis-azide and FITC-NVOC-PEG-azide monomers were added in required quantities (see Table X for details) into a vial. Ultrapure water was then added. The total concentration of crosslinker plus monomers in solutions used to make PEG hydrogels was 5% (w:v). Appropriate volumes of stock solutions of copper sulfate pentahydrate (CUSO4) and sodium ascorbate were added as stated in Table 5. The reaction mixture was vortexed and poured into cassettes to form 1.2 mm thick hydrogel sheets (20 °C, 3 h). 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 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.

[0606] If the total crosslinker plus monomers concentration was reduced to 2.5% (w:v), after the addition of CuSO4 and sodium ascorbate, the solution became viscous but a hydrogel was not formed. If the total crosslinker plus monomers concentration was increased to 10% (w:v), after the addition of CuSO4 and sodium ascorbate, hydrogels were formed. However, we used hydrogels made of 5% (w:v) crosslinker plus monomers concentration because these are expected to be less tightly knit and hence more porous than those prepared using 10% (w:v) crosslinker plus monomers concentration.

[0607] Table 5: Composition of 2 mL precursor solution used to make PEG hydrogels (the total crosslinker plus monomers concentration was 5% (w:v))

[0608] 4. 3 Preliminary Studies

[0609] 4.3.1 FITC Release Kinetics

[0610] FITC can be released from active monomer when it is exposed to 365 nm light (see Figure 36).

[0611] It is important to study the release kinetics of FITC because it determines the rate of release of proteins captured in our hydrogels via the isothiocyanate groups of FITC. We studied the release kinetics of FITC by exposing hydrogel discs to 365 nm light for different durations and using fluorescence spectroscopy for analysis. Hydrogel discs containing 1:10 molar ratio of active to inactive monomers were used. The total crosslinker plus monomers concentration was 5% (w:v). Hydrogel discs were immersed in 200 pL of fresh PBS and exposed to 365 nm light for the selected time. The gel was left in the PBS for 10 min, following which the buffer was collected and fluorescence was measured. The procedure was repeated to expose hydrogel discs for up to 60 min. A plot of the fluorescence spectra of PBS used to immerse the gel with different exposure times is provided in Figure 37 (a). Subsequently, fluorescence intensity at peak wavelength was plotted as a function of exposure time to obtain Figure 37 (b), where the solid line is a best fit to a three-parameter exponential rise to a maximum (r = 0.9952) to determine the time constant, which was ~2 min. 4.4 Optimisation Studies

[0612] To determine the optimum incubation time of hydrogels in protein solutions and active to inactive monomer ratio in PEG hydrogels, we used streptavidin that was already labelled with rhodamine (RS). This allowed us to carry out these optimisation studies by measuring the fluorescence of rhodamine independently of the labelling of the proteins with FITC while they are released from our hydrogels.

[0613] 4.4.1 Effect of incubation time of hydrogels in protein solutions

[0614] 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. 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.

[0615] Thus, remainder of the work was performed using 24 h incubation time in protein solutions. The data was fitted to a three-parameter exponential rise to a maximum (r = 0.9958) to determine the time constant, which was ~4 min for 1 h incubation and ~1.7 min for 15 and 24 h incubation. Overall, the release kinetics of RS from the hydrogel was comparable of FITC release kinetics from the hydrogel, which is to be expected.

[0616] 4.4.2 Effect of the ratio of active to inactive monomers in hydrogels

[0617] 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. The time constants for the release of RS from hydrogels comprising of different inactive to active monomer ratios were all similar (1.8, 1.7, 1.7 and 2.3 min for 1 :10, 1:20, 1 :40 and 1 :100 active to inactive monomer ratios, respectively) but the 1:40 ratio gave a much higher signal than either 1 :10, 1:20 or 1:100 monomer ratios. This is because at lower monomer ratios the proteins that diffuse into the interior of the hydrogel are captured with multiple FITC groups, which all must be broken to release the labelled protein. In contrast, proteins on the surface of the hydrogel will be captured by fewer FITCs, allowing rapid release but in much lower concentrations.

[0618] 4.5 Measurement of Proteins

[0619] Next, we studied the potential of PEG hydrogels for the measurement of proteins. For this purpose, we first used an exemplar protein, streptavidin and then interleukin 6 (IL6) that is considered as an important biomarker of oral cancer detection. Furthermore, we studied the effect of interferents on the performance of hydrogels for measuring proteins. In the first instance, we used mucin as interferent because it is present in abundance in saliva and hence is a key interferent in the measurement of salivary protein biomarkers for oral cancer detection. Subsequently, we studied the suitability of our hydrogels to measure IL6 in synthetic saliva. For selective detection of proteins i.e., streptavidin and IL6 released from PEG hydrogels, we used microtitre plates coated with biotin and anti-interleukin 6 (anti-l L6), respectively. As 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.

[0620] 4.5.1 Measurement of streptavidin

[0621] Hydrogels were incubated in one of the following solutions for 24 h:

[0622] • 0.005 ppm streptavidin without mucin (positive control)

[0623] • 0.005 ppm streptavidin with 0.5 ppm mucin (positive control with 100* interferent)

[0624] • 0.005 ppm streptavidin with 5 ppm mucin (positive control with 1000* interferent)

[0625] • Buffer i.e., no streptavidin and no mucin (negative control)

[0626] • 5 ppm mucin without streptavidin (negative control with 1000* interferent)

[0627] 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. A comparison of fluorescence intensity of wells treated with supernatants obtained after exposing hydrogels to 365 nm light where hydrogels were beforehand incubated in buffer and 0.005 ppm streptavidin (i.e., black and red traces, respectively in Figure 41) clearly shows that our hydrogels are well suited for the measurement of at least 0.005 ppm (or ~90 pM) streptavidin. The fluorescence intensity of wells treated with supernatants of 365 nm exposed hydrogels that were beforehand incubated in 5 ppm mucin was comparable to buffer (i.e., blue and red traces in Figure 41), suggesting that the interferent mucin was undetectable. Finally, comparison of supernatants of 365 nm exposed hydrogels that were beforehand incubated in streptavidin without and with mucin were comparable (black, magenta and dark yellow traces in Figure 41), suggesting that our PEG hydrogels were well suited for the measurement of streptavidin in the presence of the interferent.

[0628] 4.5.2 Measurement of lnterleukin-6 (IL6)

[0629] Hydrogels were incubated in one of the following solutions for 24 h:

[0630] • Buffer (negative control)

[0631] • Synthetic saliva (negative control with interferent)

[0632] • 0.005 ppm IL6 in buffer (positive control)

[0633] • 0.005 ppm IL6 in synthetic saliva (positive control with interferent)

[0634] 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. A comparison of fluorescence intensity of wells treated with supernatants obtained after exposing hydrogels to 365 nm light where hydrogels were beforehand incubated in buffer and 0.005 ppm IL6 (i.e., black and blue traces, respectively in Figure 42) clearly shows that our hydrogels are well suited for the measurement of at least 0.005 ppm IL6. The fluorescence intensity of wells treated with supernatants of 365 nm exposed hydrogels that were beforehand incubated in synthetic saliva was comparable to buffer (i.e., black and red traces in Figure 42), suggesting that the synthetic saliva had no effect our hydrogels. Finally, comparison of supernatants of 365 nm exposed hydrogels that were beforehand incubated in IL6 in buffer and synthetic saliva were comparable (blue and dark yellow traces in Figure 42), suggesting that our PEG hydrogels were well suited for the measurement of IL6 in synthetic saliva. REFERENCES

[0635] (1) Surinova, S.; Schiess, R.; Huttenhain, R.; Cerciello, F.; Wollscheid, B.; Aebersold, R. On the development of plasma protein biomarkers. Journal of Proteome Research 2011 , 10 (1), 5. DOI: 10.1021 / pr1008515.

[0636] (2) M, A.; A., F. Enzyme linked immunosorbent assay; StatPearls Publishing, 2023.

[0637] (3) Carrell, C.; Kava, A.; Ngyuen, M.; Menger, R.; Munshi, Z.; Call, Z.; Nussbaum, M.; Henry, C. Beyond the lateral flow assay: A review of paper-based microfluidics. Microelectronic Engineering 2019, 206, 45.

[0638] (4) Chen, P.; Huang, N.-T.; Chung, M.-T.; Cornell, T. T.; Kurabayashi, K. Label-free cytokine micro-and nano-biosensing towards personalized medicine of systemic inflammatory disorders. Advanced Drug Delivery Reviews 2015, 95, 90.

[0639] (5) Goddard, N. J.; Gupta, R. A novel manifestation at optical leaky waveguide modes for sensing applications. Sensors and Actuators B 2020, 309, 127776. DOI:

[0640] 10.1016 / j.snb.2020.127776.

[0641] (6) Gupta, R.; Goddard, N. J. Leaky waveguides (LWs) for chemical and biological sensing - a review and future perspective. Sensors and Actuators B-Chemical 2020, 322, 128628. DOI:

[0642] 10.1016 / j.snb.2020.128628.

[0643] (7) Chen, C.; Wand, J. Optical biosensors: an exhaustive and comprehensive review. Analyst 2020, 145, 1605.

[0644] (8) Bahadir, E. B.; Sezginturk, M. F. A review on impedimetric biosensors. Artificial Cells,

[0645] Nanomedicine, and Biotechnology 2016, 44, 248.

[0646] (9) Kim, S.; Song, H.; Ahn, H.; Kim, T.; Jung, J.; Cho, S. K.; Shin, D.-M.; Choi, J.-R.; Hwang, Y.-H.; Kim, K. A review of advanced impedance biosensors with microfluidic chips for singlecell analysis. Biosensors 2021 , 11 , 412.

[0647] (10) Alanazi, N.; Almutairi, M.; Alodhayb, A. N. A review of quartz crystal microbalance for chemical and biological sensing applications. Sensing and Imaging 2023, 24, 10.

[0648] (11) Lim, H. J.; Saha, T.; Tey, B. T.; Tan, W. S.; OOi, C. W. Quartz crystal microbalancebased biosensors as rapid diagnostic devices for infectious diseases. Biosensors and Bioelectronics 2020, 168, 112513.

[0649] (12) Abbate, G.; Bernini, II.; Ragozzino, E.; Somma, F. The temperature dependence of the refractive index of water. Journal of Physics D: Applied Physics 1978, 11, 1167.

[0650] (13) Otieno, B. A.; Krause, C. E.; Latus, A.; Chikkaveeraiah, B. V.; Faria, R. C.; Rusling, J. F. On-line protein capture on magnetic beads for ultrasensitive microfluidic immunoassays of cancer biomarkers. Biosensors and Bioelectronics 2014, 53, 268.

[0651] (14) Osbourn, D. M.; Weiss, D. J.; Lunte, C. E. On-line preconcentration methods for capillary electrophoresis. Electrophoresis 2000, 21, 2768.

[0652] (15) Goddard, N. J.; Gupta, R. Speed and sensitivity - integration of electrokinetic preconcentration with a leaky waveguide biosensor. Sensors and Actuators B-Chemical 2019, 301, 127063. DOI: 10.1016 / j.snb.2019.127063.

[0653] (16) Gupta, R.; Baldock, S. J.; Fielden, P. R.; Prest, J. E.; Grieve, B. D. Isotachophoresisbased sample preparation of cellulases in sugarcane juice using bovine serum albumin as a model protein. Journal of Chromatography A 2010, 1217, 8026.

[0654] (17) Twining, S. S. Fluorescein isothiocyanate-labeled casein assay for proteolytic enzymes. Analytical biochemistry 1984, 143 (1), 30-34.

[0655] (18) Hungerford, G.; Benesch, J.; Mano, J. F.; Reis, R. L. Effect of the labelling ratio on the photophysics of fluorescein isothiocyanate (FITC) conjugated to bovine serum albumin. Photochemical & Photobiological Sciences 2007, 6 (2), 152-158.

[0656] (19) Xing, Y.; Zeng, B.; Yang, W. Light responsive hydrogels for controlled drug delivery. Frontiers in Bioengineering and Biotechnology 2022, 10.

[0657] (20) Kim, M. S.; Diamond, S. L. Photocleavage of o-nitrobenzyl ether derivatives for rapid biomedical release applications. Bioorganic & medicinal chemistry letters 2006, 16 (15), 4007-4010.

[0658] (21) Zhao, H.; Sterner, E. S.; Coughlin, E. B.; Theato, P. o-Nitrobenzyl alcohol derivatives: Opportunities in polymer and materials science. Macromolecules 2012, 45 (4), 1723-1736.

[0659] (22) Kloxin, A. M.; Kasko, A. M.; Salinas, C. N.; Anseth, K. S. Photodegradable hydrogels for dynamic tuning of physical and chemical properties. Science 2009, 324 (5923), 59-63. (23) Thomas III, S. W. New applications of photolabile nitrobenzyl groups in polymers. Macromolecular Chemistry and Physics 2012, 213 (23), 2443-2449.

[0660] (24) Rosales, A. M.; Vega, S. L.; DelRio, F. W.; Burdick, J. A.; Anseth, K. S. Hydrogels with reversible mechanics to probe dynamic cell microenvironments. Angewandte Chemie International Edition 2017, 56 (40), 12132-12136.

[0661] (25) Klinger, D.; Landfester, K. Dual stimuli-responsive poly (2-hydroxyethyl methacrylate- co- methacrylic acid) microgels based on photo-cleavable cross-linkers: pH-dependent swelling and light- induced degradation. Macromolecules 2011, 44 (24), 9758-9772.

[0662] (26) Pal, A. K.; Labella, E.; Goddard, N. J.; Gupta, R. Photofunctionalizable hydrogel for fabricating volume optical diffractive sensors. Macromolecular Chemistry and Physics 2019, 220 (16), 1900228.

[0663] (27) Gupta, R.; El Sayed, S.; Goddard, N. J. Hydrogel gratings with patterned analyte responsive dyes for spectroscopic sensing. RSC Advances 2021, 11, 40197.

[0664] (28) Holmes, C. P. Model studies for new o-nitrobenzyl photolabile linkers: Substituent effects on the rates of photochemical cleavage. The Journal of organic chemistry 1997, 62 (8), 2370-2380.

[0665] (29) Mathew, T.; Ajayaghosh, A.; Das, S.; Kamat, P. V.; George, M. A new photodegradable polyamide containing o-nitrobenzyl chromophore. Steady state and laser flash photolysis studies. Journal of Photochemistry and Photobiology A: Chemistry 1993, 71 (2), 181-189.

[0666] (30) Sternson, S. M.; Schreiber, S. L. An acid-and base-stable o-nitrobenzyl photolabile linker for solid phase organic synthesis. Tetrahedron letters 1998, 39 (41), 7451-7454.

[0667] (31) Buchmeiser, M. R. Polymeric monolithic materials: Syntheses, properties, functionalization and applications. Polymer 2007, 48 (8), 2187-2198.

[0668] (32) Davidson, R. S.; Hilchenbach, M. M. The use of fluorescent probes in immunochemistry. Photochemistry and photobiology 1990, 52 (2), 431-438.

Claims

CLAIMS1. 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.

2. The hydrogel according to claim 1 , wherein the plurality of inactive monomers is selected from the group consisting of polyethylene glycol, acrylamide, N- isopropylacrylamide, methacrylamide, methacrylate, and polyethylene glycol bisazide.

3. The hydrogel according to claim 1 , wherein 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 polyethylene glycol azide.

4. The hydrogel according to any of the preceding claims, wherein 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, preferably wherein the at least one fluorophore is fluorescein isothiocyanate (FITC).

5. The hydrogel according to any of the preceding claims wherein the (molar) ratio of inactive monomers to active monomers is between 100:1 and 5:1 , preferably wherein the ratio of inactive monomers to active monomers is 40:1.

6. The hydrogel according to any preceding claim wherein the plurality of inactive monomers are acrylamide or polyethylene glycol bis-azide.

7. The hydrogel according to any preceding claim wherein the plurality of active monomers are polyethylene glycol methacrylamide, preferably FITC-NVOC-PEG3400- methacrylamide.

8. The hydrogel according to any of claims 1-6 wherein the plurality of active monomers are polyethylene glycol azide, preferably FITC-NVOC-PEGwoo-azide.

9. The hydrogel according to any preceding claim wherein the cleavable bond is nitrobenzyl-based or carbonyl-based, preferably wherein the cleavable bond is selected from: o-nitrobenzyl, m-nitrobenzyl, and p-nitrobenzyl.

10. The hydrogel according to claims 1-7 or 9, wherein the plurality of inactive monomers are acrylamide, wherein the plurality of active monomers are polyethylene glycol methacrylamide, wherein 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, wherein said cleavable bond is optionally o-nitrobenzyl.

11. The hydrogel according to any of claims 1-6, 8 or 9, wherein the plurality of inactive monomers are polyethylene glycol bis-azide, wherein the plurality of active monomers are polyethylene glycol azide, wherein each polyethylene glycol azide 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 azide monomer by a cleavable bond, wherein said cleavable bond is optionally o-nitrobenzyl.

12. A formulation comprising a core and a shell, wherein the core comprises the hydrogel of any one of claims 1 to 11, and the shell comprises a second hydrogel having a pore size of less than 30 nm.

13. An oral formulation comprising the hydrogel according to any one claims 1 to 11, or the formulation according to claim 12.

14. The oral formulation according to claim 13, wherein the oral formulation is a pill, tablet, capsule, granule, troch, lozenge, a lollipop, sampling material, or a disc.

15. A sampling device comprising the hydrogel according to any one of claims 1 to 11, the formulation of claim 12, or the oral formulation according to claim 13 or 14, wherein the sampling device comprises a test tube.

16. An inert substrate comprising a coating thereon, wherein the coating comprises the hydrogel of any one of claims 1 to 11.

17. 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.

18. A kit-of-parts comprising: a. the hydrogel according to any one of claims 1 to 11, the formulation according to claim 12, the oral formulation according to claim 13 or 14, the sampling device according to claim 15, or the inert substrate according to claim 16; b. optionally the container according to claim 17; and c. instructions for use.

19. A biomaterial comprising the hydrogel according to any one of claims 1 to 11 , or the formulation according to claim 12.

20. Use of the biomaterial according to claim 19 in cell culture.

21. Use of a hydrogel according to any one of claims 1 to 11, the formulation according to claim 12, the oral formulation according to claim 13 or 14, the sampling device according to claim 15, or the inert substrate according to claim 16, for concentrating and labelling proteins in a sample.

22. Use of a hydrogel according to any one of claims 1 to 11, the formulation according to claim 12, the oral formulation according to claim 13 or 14, the sampling device according to claim 15, or the inert substrate according to claim 16 in a method of diagnosing a disease or disorder.

23. A method of concentrating and labelling proteins in a sample, the method comprising:a. Contacting a sample with the hydrogel of any one of claims 1 to 11 , the formulation according to claim 12, the oral formulation according to claim 13 or 14, the sampling device according to claim 15, or the inert substrate according to claim 16 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.

24. A method of detecting proteins in a sample, the method comprising: a. Contacting a sample with the hydrogel of any one of claims 1 to 11 , the formulation according to claim 12, the oral formulation according to claim 13 or 14, the sampling device according to claim 15, or the inert substrate according to claim 16 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. 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.

25. A method of measuring a protein of interest in a sample, the method comprising: a. Contacting a sample with the hydrogel of any one of claims 1 to 11 , the formulation according to claim 12, the oral formulation according to claim 13 or 14, the sampling device according to claim 15 or the inert substrate according to claim 16 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. 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.

26. The method according to claim 25, wherein the protein of interest is a biomarker.

27. The method according to claim 25, wherein the biomarker is C-reactive protein (CRP), IL6, IL8, or cardiac troponin.

28. A method of determining whether a subject has a disease or disorder, the method comprising: a. Contacting a sample from the subject with the hydrogel of any one of claims 1 to 11, the formulation according to claim 12, the formulation according to claim 13 or 14, the sampling device according to claim 15, or the inert substrate according to claim 16, 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. 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.

29. The method according to claim 28, wherein the disease or disorder is cancer or a cardiovascular disease.

30. The method according to any one of claims 24 to 29, wherein the cleavage inducer is UV light, or wherein the cleavage inducer is selected from esterase or reducing agents.

31. The method according to claim 30 wherein the UV light comprises a wavelength range of between roughly 200 nm and 400 nm, preferably wherein the UV light comprises a wavelength of about 365nm.

32. The method according to any one of claims 25 to 31 wherein the step of contacting the fluorescently labelled proteins with a binding molecule comprises contacting the fluorescently labelled proteins with a container according to claim 17.

Images