Hydrogels for binding to proteins, formulations and uses thereof

Abstract

The present invention relates to hydrogels capable of binding to proteins and providing additional concentration, labeling and release of the proteins. Corresponding formulations, oral formulations, biomaterials, uses, methods and kits of parts are also provided.

Description

Technical Field

[0001] Novel hydrogels capable of binding to proteins and further concentrating, labeling, and releasing said labeled and concentrated proteins are provided. Corresponding formulations, oral formulations, biomaterials, uses, methods, and multi-component kits are also provided. Background Technology

[0002] The determination of low-abundance proteins is key to achieving early disease detection. 1 The most widely used method for detecting low-abundance proteins is enzyme-linked immunosorbent assay (ELISA). 2 ELISA uses two types of antibodies: a capture antibody that allows the target protein to be captured from the sample, and a detection antibody labeled with an enzyme. A substrate is then added, which, through enzymatic action, produces a fluorescent or colored product. The fluorescence intensity, or absorbance, is proportional to the concentration of the target protein. Furthermore, because each enzyme can act on multiple substrate molecules, the fluorescence or absorbance signal is amplified to allow the determination of low-abundance proteins.

[0003] However, ELISA requires multiple addition and washing steps, making it laborious. To address this challenge, ELISA is implemented on microfluidic paper-based analytical devices by controlling the flow rate and the time it takes for the reagent to reach the detection zone. 3 Flow control requires patterning of paper and multiple layers that must be precisely aligned. Alternatively, label-free biosensors have been used to determine low-abundance proteins. Unlike ELISA, label-free biosensors... 4 Only antibody capture is required, and it depends on the refractive index. 5-7 or impedance 8,9 or quality 10,11 The changes are used to determine the concentration of the target protein.

[0004] However, label-free biosensors are prone to errors due to changes in environmental conditions (such as temperature). 12 For the determination of low-abundance proteins using absorbance or fluorescence, this absorbance or fluorescence assay has lower sensitivity but requires simpler instruments and is more user-friendly than ELISA, and has used beads. 13 and electrophoresis 14-16 Proteins are pre-concentrated. After pre-concentration, the proteins are recovered, labeled with dyes or fluorophores, captured, and then detected. However, these techniques require a series of different steps, which is inefficient and impractical, especially for point-of-care testing or field testing applications.

[0005] There is a need for a simpler and more efficient assay process for detecting low-abundance proteins. This invention aims to address one or more of the aforementioned problems in the art. Summary of the Invention

[0006] The inventors have unexpectedly discovered a novel hydrogel capable of pre-concentrating, fluorescently labeling, and light-triggered protein release. This novel hydrogel can be used in protein assays with far fewer steps than ELISA-based assays. Pre-concentration is achieved by covalently capturing the protein in the hydrogel, where the sample volume is much smaller than that required by currently available assays, allowing for direct use on patients, such as by aspirating saliva samples in the mouth, or in vitro use, such as in urine samples. Figure 1 As shown, covalent trapping of proteins involves the interaction between the primary amine in the protein and fluorescent groups present in the hydrogel (such as the isothiocyanate group in fluorescein isothiocyanate (FITC)). 17,18 The result of the reaction between FITC and other proteins. Because FITC is fluorescent, the protein is pre-concentrated and labeled in a single step. Furthermore, as... Figure 1 As shown, FITC is transmitted via a photoinstantaneous group (o-nitrobenzyl). 19-25 This photoinstability group, linked to the main chain of the hydrogel, exhibits a controllable photoreactivity and tunable absorbance for wavelengths >300 nm. 23,25 Therefore, the release of fluorescently labeled proteins can be triggered by irradiating the hydrogel with ultraviolet light (365 nm). Figure 1 As shown, the released proteins can then be captured and measured using standard fluorescence detection techniques.

[0007] To demonstrate the feasibility of the inventors' hydrogel for pre-concentration, labeling, and controlled release, streptavidin was initially used as an example protein. The streptavidin used in the example was labeled with rhodamine, allowing the fluorescence of rhodamine to be used to determine protein concentrations before and after incubation with the hydrogel. This, in turn, provided a pre-concentration factor. The designed hydrogel provided a pre-concentration factor of up to 295 times. Similarly, rhodamine labeling was used to study the phototriggered release kinetics of streptavidin from the hydrogel. The inventors showed that 50% of the streptavidin was released from the hydrogel within approximately 100 seconds, and generally, at least 50% of all proteins were released very rapidly from the hydrogel within 10 minutes. Finally, the inventors showed that the released streptavidin was labeled with 85 fluorophore molecules per protein molecule. When combined with biotin capture of the released streptavidin and fluorescence detection, the designed hydrogel allowed for the detection of very low protein concentrations in samples (at least 0.0033 ppm (or approximately 60 pM)). Similar results have been shown with other exemplary proteins (C-reactive proteins) CRP, IL8, and IL6, demonstrating that the hydrogel of the present invention can be used to achieve the pre-concentration, labeling, and controlled release of many different types of proteins, including those that may indicate disease. When combined with the capture and fluorescence detection of released CRP, the designed hydrogel allows for the detection of very low protein concentrations in the sample (at least 0.0022 ppm (or about 19 pM)). When combined with the capture and fluorescence detection of released IL6, the designed hydrogel allows for the detection of very low protein concentrations in the sample (at least 0.005 ppm (or about 240 pM)). Furthermore, an added advantage is that the hydrogel does not require secondary antibodies, enzyme labeling, and substrates, nor does it require the multiple addition / washing steps required by conventional ELISA assays.

[0008] In one aspect of the invention, a hydrogel is provided 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 linked to the active monomer via a cleavable bond.

[0009] Appropriately, the inactive monomers are selected from polyethylene glycol (PEG), acrylamide, N-isopropylacrylamide, methacrylamide, methacrylate and PEG bisazide.

[0010] Suitable, the active monomers are selected from polyethylene glycol acrylamide, polyethylene glycol N-isopropylacrylamide, polyethylene glycol methacrylamide, polyethylene glycol methacrylate, allylamide, and PEG. 1000 -Azides.

[0011] Suitablely, at least one fluorophore is selected from fluorescein, fluorescein isothiocyanate (FITC), eosin Y, eosin B, tetrachlorofluorescein, carbofluorescein, naphthalenefluorescein, and (semi)naphthalenefluorescein, which have suitable protein-reactive groups.

[0012] Appropriately, the cleavable bond is the ortho-nitrobenzyl group.

[0013] In one embodiment of the hydrogel, a plurality of inactive monomers are acrylamides and a plurality of active monomers are polyethylene glycol methacrylamide, wherein each polyethylene glycol methacrylamide monomer contains at least one fluorescein isothiocyanate capable of covalently binding to a protein, wherein the molar ratio of inactive monomers to active monomers is from 100:1 to 5:1, and wherein each fluorescein isothiocyanate is linked to the polyethylene glycol methacrylamide monomer via a cleavable bond, which is suitably o-nitrobenzyl.

[0014] In one aspect of the invention, a formulation comprising a core and a shell is provided, wherein the core comprises a hydrogel of any of the foregoing aspects and embodiments, and the shell comprises a hydrogel with a pore size of less than 30 nm.

[0015] In one aspect of the invention, an oral formulation comprising a hydrogel according to any one of the foregoing aspects and embodiments is provided, or a formulation according to the foregoing aspects is provided.

[0016] Suitable oral formulations are pills, tablets, capsules, granules, lozenges, lollipops, or sampling materials. Suitable oral formulations are round tablets. In one aspect, round tablets comprising the hydrogel of the present invention are provided.

[0017] In one aspect of the invention, a sampling device is provided comprising a hydrogel, a formulation of the foregoing aspects and embodiments, or an oral formulation of the foregoing aspects and embodiments, wherein the sampling device comprises a test tube.

[0018] On the other hand, an inert substrate is provided having a coating thereon, wherein the coating comprises the hydrogel of the present invention. Suitably, the coating is a thin film.

[0019] In one aspect, a container is provided comprising an inner surface operable to contact a reaction mixture, wherein the inner surface is coated with a base layer, and a reaction layer is coated on the base layer, the reaction layer comprising a mixture of binding molecules and a blocking agent, wherein the blocking agent comprises a compound without amine groups.

[0020] In one embodiment, the base layer is a polymer containing free amine groups. Suitably, such a polymer is, for example, acrylamide / bisacrylamide copolymerized optionally with aminopropylmethacrylamide, or 4-arm PEG succinimide ester (NHS) copolymerized with PEG-diamine, or chitosan. In one embodiment, the base layer is chitosan. In one embodiment, the binding molecule is defined below and is a protein (e.g., biotin or antibody) that recognizes and binds to the protein of interest, suitably an antibody or its binding fragment that specifically binds to the protein of interest. In one embodiment, the blocking agent contains PEG-methyl groups. Suitably, the capturing protein and the blocking agent bind to the base layer. Suitably, the reaction layer contains a majority of the binding molecules and a small amount of blocking agent. Suitably, the blocking agent binds to the base layer only if the binding molecules do not bind to the base layer. Suitably, the blocking agent binds to any free amine groups in the base layer. Suitably, the blocking agent suitably prevents free FITC from binding to the base layer of the container during use.

[0021] Suitablely, the container can be any container suitable for carrying out the methods of the invention therein. In one embodiment, the container is a microplate or a microtiter plate.

[0022] In one aspect of the invention, a multi-component kit is provided, the multi-component kit comprising:

[0023] a. A hydrogel according to any of the foregoing aspects and embodiments, a formulation according to any of the foregoing aspects and embodiments, an oral formulation (optionally a round tablet) according to any of the foregoing aspects and embodiments, a sampling device according to any of the foregoing aspects and embodiments, or a substrate according to any of the foregoing aspects and embodiments; and

[0024] b. Instruction manual.

[0025] Optionally, the kit may further include the aforementioned container.

[0026] In one aspect of the invention, a biomaterial is provided comprising a hydrogel according to any of the foregoing aspects and embodiments, or a formulation comprising any of the foregoing aspects and embodiments.

[0027] In one aspect of the invention, the use of biomaterials according to the foregoing aspect in cell culture is provided.

[0028] In one aspect of the invention, the use of a hydrogel according to any of the foregoing aspects and embodiments, or a formulation or oral formulation (optionally a round tablet) according to any of the foregoing aspects and embodiments, a sampling device according to any of the foregoing aspects and embodiments, or a substrate according to any of the foregoing aspects and embodiments, or a container according to any of the foregoing aspects and embodiments, for concentrating and labeling proteins in a sample is provided.

[0029] In one aspect of the invention, a hydrogel according to any of the foregoing aspects and embodiments, or a formulation or oral formulation (optionally a round tablet) according to any of the foregoing aspects and embodiments, or a sampling device according to any of the foregoing aspects and embodiments, or a substrate according to any of the foregoing aspects and embodiments, or a container according to any of the foregoing aspects and embodiments, is provided for use in a method of diagnosing a disease or condition.

[0030] In one aspect of the present invention, a method for concentrating and labeling proteins in a sample is provided, the method comprising:

[0031] a. Contacting a sample with a hydrogel of any of the foregoing aspects and embodiments, or a formulation of any of the foregoing aspects and embodiments, or an oral formulation (optionally a round tablet) of any of the foregoing aspects and embodiments, or a sampling device of any of the foregoing aspects and embodiments, or a substrate of any of the foregoing aspects and embodiments under suitable conditions to allow proteins in the sample to bind to the hydrogel via fluorophores, thereby obtaining fluorescently labeled proteins bound to the hydrogel.

[0032] In one aspect of the present invention, a method for detecting proteins in a sample is provided, the method comprising:

[0033] a. Contact the sample with a hydrogel of any of the foregoing aspects and embodiments, or a formulation of any of the foregoing aspects and embodiments, or an oral formulation (optionally a round tablet) of any of the foregoing aspects and embodiments, or a sampling device of any of the foregoing aspects and embodiments, or a substrate of any of the foregoing aspects and embodiments under suitable conditions to allow proteins in the sample to bind to the hydrogel via fluorophores, thereby obtaining fluorescently labeled proteins bound to the hydrogel.

[0034] b. Exposing the hydrogel from step (a) to a cleavage inducer under suitable conditions to cleave cleavable bonds, thereby releasing the fluorescently labeled protein from the hydrogel; and

[0035] c. Determine the presence of proteins in the sample, where the presence of fluorescence indicates the presence of proteins in the sample.

[0036] In one aspect of the present invention, a method for determining a protein of interest in a sample is provided, the method comprising:

[0037] a. Contacting a sample with a hydrogel of any of the foregoing aspects and embodiments, or a formulation of any of the foregoing aspects and embodiments, or an oral formulation (optionally a round tablet) of any of the foregoing aspects and embodiments, a sampling device of any of the foregoing aspects and embodiments, or a substrate of any of the foregoing aspects and embodiments under suitable conditions to allow proteins in the sample to bind to the hydrogel via fluorophores, thereby obtaining fluorescently labeled proteins bound to the hydrogel.

[0038] b. Exposing the hydrogel from step (a) to a cleavage inducer under suitable conditions to cleave cleavable bonds, thereby releasing the fluorescently labeled protein from the hydrogel;

[0039] c. Separate the fluorescently labeled protein;

[0040] d. Contact the fluorescently labeled protein with a binding molecule capable of specifically binding to the protein of interest;

[0041] e. Remove any unbound fluorescently labeled proteins; and

[0042] f. Measure the fluorescence level, where the fluorescence level indicates the amount of the protein of interest in the sample.

[0043] Appropriately, proteins of interest are biomarkers.

[0044] Appropriately, biomarkers are C-reactive protein (CRP), IL-6, IL-8, or cardiac troponin.

[0045] In one aspect of the invention, a method for determining whether a subject suffers from a disease or ailment is provided, the method comprising:

[0046] a. Contact a sample from the subject with a hydrogel of any of the foregoing aspects and embodiments, or a formulation of any of the foregoing aspects and embodiments, or an oral formulation (optionally a round tablet) of any of the foregoing aspects and embodiments, or a sampling device of any of the foregoing aspects and embodiments, or a substrate of any of the foregoing aspects and embodiments under suitable conditions to allow proteins in the sample to bind to the hydrogel via fluorophores, thereby obtaining fluorescently labeled proteins bound to the hydrogel.

[0047] b. Under suitable conditions, the hydrogel from step (a) is exposed to a cleavage inducer to cleave cleavable bonds, thereby releasing the fluorescently labeled protein from the hydrogel;

[0048] c. Separate fluorescently labeled proteins;

[0049] d. To bring fluorescently labeled proteins into contact with binding molecules that can specifically bind to protein biomarkers of diseases or symptoms;

[0050] e. Remove any unbound fluorescently labeled proteins;

[0051] f. Detecting the presence of fluorescence or measuring the level of fluorescence, wherein the presence of fluorescence indicates the presence of a protein biomarker in the sample, or wherein the level of fluorescence indicates the level of a protein biomarker in the sample.

[0052] g. Based on f., determine that the object suffers from a disease or condition, wherein the presence or level of fluorescence indicates the disease or condition.

[0053] Appropriately, the disease or ailment is cancer or cardiovascular disease.

[0054] Appropriately, the cleavage inducer is ultraviolet light, or the cleavage inducer is selected from esterases or reducing agents.

[0055] Suitablely, in any of the above methods, the substrate or the circular tablet according to any of the foregoing aspects and embodiments may be brought into contact with a sample from the object.

[0056] In one embodiment of a method for determining a protein of interest in a sample or for determining whether a subject has a disease, the container of the present invention is used. Suitably, in the step of contacting the fluorescently labeled protein with a binding molecule capable of specifically binding to the protein of interest...

[0057] Throughout the description and claims of this specification, the words “comprising” and “containing” and their variations mean “including but not limited to”, and they are not intended to exclude other parts, additives, components, integers or steps.

[0058] Throughout the description and claims of this specification, the singular encompasses the plural unless the context requires otherwise. In particular, where the indefinite article is used, the specification should be understood to consider both the plural and the singular unless the context requires otherwise.

[0059] Unless incompatible with the present invention, any features, integers, properties, compounds, chemical parts or groups described in connection with a particular aspect, embodiment or example of the invention shall be understood to be applicable to any other aspect, embodiment or example described herein.

[0060] The various aspects of the present invention will now be described in further detail. Attached Figure Description

[0061] Embodiments of the invention are further described below with reference to the accompanying drawings, wherein:

[0062] Figure 1 The schematic diagram illustrates the pre-concentration, labeling, and release of the exemplary protein rhodamine-streptavidin, followed by fluorescence detection in a biotin-coated microtiter plate.

[0063] Figure 2 Used for the synthesis of monomers F-NVOC-allylamide and F-NVOC-PEG. 400 - Methacrylamide, F-NVOC-PEG 3400 - Methacrylamide and FITC-NVOC-PEG 3400 1. Reaction scheme for methylacrylamide.

[0064] Figure 3 : The chemical structure of the monomer (rhombic, double-star, and single-star groups are used for protein capture, fluorescent labeling, and phototriggered release, respectively; round is PEG, which produces water-soluble monomers; rectangular is allylamide or methacrylamide, which allows the monomer to copolymerize with acrylamide / bisacrylamide to obtain hydrogels).

[0065] Figure 4 (a) F-PEG-NVOC-PEG 3400 HPLC waterfall plots of methacrylamide show that the release of fluorescein (peak F) increases with irradiation time, while the release of the initiating reagent (peak M) decreases with irradiation time, and (b) the rate of light-induced fluorescein release from the monomer increases with irradiation time, where [M] t [M]0 is the monomer concentration that varies with irradiation time, and [M]0 is the initial monomer concentration.

[0066] Figure 5 Images of a hydrogel film on glass under (a) white light and (b) 365 nm light, with a £1 coin as a size reference (side-to-side distance 22.5 mm), (c) UV-Vis spectra of the hydrogel film at four different angles, and (d) a graph showing the monomer doping factor, where the monomer doping factor is the hydrogel (m) 水凝胶 ) and precursor (m 前体 The molar concentration ratio of NVOC monomers in the solution.

[0067] Figure 6 a) Hydrogel films (acrylamide / bisacrylamide and F-NVOC-PEG) 3400 a) UV-Vis spectra of the copolymer of methacrylamide as a function of irradiation time, and b) first-order reaction rate plot of fluorescein release from the hydrogel.

[0068] Figure 7Emission 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 the solutions and hydrogels.

[0069] Figure 8 (a) Emission spectrum of RS released when the hydrogel is irradiated with ultraviolet light (excitation wavelength 540 nm), (b) Cumulative release of protein as a function of irradiation time, and (c) Emission spectrum of the release after incubation with a biotin-coated microtiter plate and washing with buffer (peaks at excitation wavelengths of 470 nm and 540 nm).

[0070] Figure 9 UV-Vis spectra of DMSO solutions of F-NVOC-allylamide (45µM, 30µM, 25µM, 10µM, and 5µM), with the inset showing the average absorbance between the peak wavelength of 519nm and ±5nm compared to the monomer concentration (molar extinction coefficient at 519nm = 49,600 ± 5,200M). -1 cm -1 ).

[0071] Figure 10 F-NVOC-PEG in PBS 400 UV-Vis spectra of methylacrylamide (45µM, 30µM, 20µM, 10µM, and 5µM), with the inset showing the average absorbance between the peak wavelength of 492nm and ±5nm relative to the monomer concentration (molar extinction coefficient at 492nm = 69,100±4,200M). -1 cm -1 ).

[0072] Figure 11 F-NVOC-PEG in PBS 3400 UV-Vis spectra of methylacrylamide (45µM, 25µM, 10µM, 5µM, and 1µM), with the inset showing the average absorbance between the peak wavelength of 492nm and ±5nm relative to the monomer concentration (molar extinction coefficient at 492nm = 63,900 ± 0.00M). -1 cm -1 ).

[0073] Figure 12 40µM F-NVOC-PEG in PBS 3400 - Ultraviolet-visible spectrum of methacrylamide monomer as irradiation time increases.

[0074] Figure 131 mg / ml of (a) F-NVOC-allylamide and (b) F-NVOC-PEG dissolved in acetonitrile 400 HPLC waterfall plot of methylacrylamide shows an increase in fluorescein (F) concentration and a decrease in photostable monomer (M) concentration during the irradiation period from 0 min to 30 min.

[0075] Figure 14 This plot shows the monomer concentration as a function of irradiation time, determined by the peak area corresponding to M in the chromatogram and the molar extinction coefficient of the monomer solution.

[0076] Figure 15 (a) UV-Vis spectrum and (b) fluorescence emission spectrum of fluorescein in PBS solution (excitation wavelength 470 nm), with the inset showing the peak absorbance (average value taken from 485 nm to 495 nm, molar extinction coefficient at 490 nm = 52,600 ± 3,200 M). -1 cm -1 ) and emission intensity (take the average value from 509nm to 519nm, the molar emission coefficient at 514nm = (10.4±0.1)×10 9 M -1 cm -1 (Compared to fluorescein concentration)

[0077] Figure 16 (a) UV-Vis spectrum and (b) fluorescence emission spectrum (excitation wavelength 540 nm) of rhodamine-streptavidin PBS solution, with the inset showing peak absorbance (average value from 572 nm to 582 nm, molar extinction coefficient at 577 nm = 650,602 ± 6,024 M). -1 cm -1 ) and emission intensity (taken as the average value in the range of 585nm to 595nm, molar emission coefficient at 590nm = (1.6±0.04)×10 9 M -1 cm -1 Compared to the concentration of rhodamine-streptavidin.

[0078] Figure 17 Absorption spectra of hydrogel films prepared using a 10% (w:v) precursor solution after (a) overnight washing in PBS and (b) subsequent storage in PBS for 1 day, 3 days, 5 days and 7 days.

[0079] Figure 18 Absorption spectra of hydrogel films prepared using a 5% (w:v) precursor solution after (a) overnight washing in PBS and (b) subsequent storage in PBS for 1 day, 3 days, 5 days and 7 days.

[0080] Figure 19 (a) Fluorescence spectra of 0.005 ppm RS solution before and after 24 hours of incubation with hydrogel round tablets and (b) RS calibration curve.

[0081] Figure 20 Effect of changing the molar ratio of active monomer to inactive monomer on RS release (hydrogel round tablets were incubated in 0.005 ppm RS for 24 hours).

[0082] Figure 21 The fluorescence intensity at the peak wavelength of the PBS solution used to immerse the hydrogel discs when exposed to 365 nm light for different durations (the hydrogel discs were incubated in 10 mL of 0.1 ppm RS for 1 h, 15 h, 24 h, or 48 h).

[0083] Figure 22 The fluorescence intensity at the peak wavelength of the PBS solution used to immerse the hydrogel discs when exposed to 365 nm light for different durations (the hydrogel discs were incubated in 10 mL of 0.01 ppm RS for 1 h, 15 h, or 24 h).

[0084] Figure 23 Fluorescence at peak wavelength of PBS solution used to immerse hydrogel discs when exposed to 365 nm light for different durations (hydrogel discs were incubated in 10 mL of 0.005 ppm RS for 1 h, 15 h, or 24 h).

[0085] Figure 24 : The peak fluorescence intensity of FITC released in a commercially available biotinylated microtiter plate as a function of exposure time.

[0086] Figure 25 The peak fluorescence intensity of FITC bound to two concentrations of NHS-PEG-methyl blocking agent as a function of exposure time in an internally developed biotinylated microtiter plate.

[0087] Figure 26 The change in fluorescence intensity at the peak wavelength of the PBS solution used to immerse the hydrogel discs relative to the exposure time under two different washing times (the hydrogel discs were incubated in 10 mL of 0.005 ppm RS for 24 hours).

[0088] Figure 27 (a) Fluorescent signal of streptavidin bound to a biotin-coated internal microtiter plate, wherein streptavidin was pre-concentrated and released from a polyacrylamide hydrogel, and streptavidin was labeled with FITC during release, and (b) the corresponding calibration curve (using data from a 60-min exposure time).

[0089] Figure 28 (a) Fluorescent signal of CRP bound to an internal microtiter plate coated with anti-CRP, wherein the CRP was pre-concentrated and released from a polyacrylamide hydrogel and the CRP was labeled with FITC during the release, and (b) the corresponding calibration curve (using data from a 60-min exposure time).

[0090] Figure 29 The fluorescence signal of IL8 is bound to an internal microtiter plate coated with an anti-IL8 coating, wherein IL8 is pre-concentrated and released from a polyacrylamide hydrogel, and IL8 is labeled with FITC during the release process.

[0091] Figure 30 : A reaction scheme for forming PEG hydrogels via alkyne-azide click chemistry between a crosslinking agent and active and inactive monomers (n=11, m=75 and p=22).

[0092] Figure 31 : A reaction scheme for the synthesis of 4-arm PEGyne (crosslinking agent, n=11).

[0093] Figure 32 : A reaction scheme for synthesizing PEG bisazides (inactive monomer, m=75).

[0094] Figure 33 : Reaction scheme for the synthesis of amine-PEG-azide (p=22).

[0095] Figure 34 : Reaction scheme for synthesizing NVOC-PEG-azides (p=22).

[0096] Figure 35 Used for the synthesis of FITC-NVOC-PEG 1000 - Reaction scheme for azide (active monomer, p=22).

[0097] Figure 36 : Displays when FITC-NVOC-PEG 1000 - The reaction scheme for FITC release when azide monomers are exposed to 365 nm light (p=22).

[0098] Figure 37 (a) A plot of fluorescence intensity of solutions used to immerse gels in 365 nm light for different durations and (b) A plot of peak fluorescence intensity relative to exposure time.

[0099] Figure 38Fluorescence spectra of the PBS solution used to immerse the hydrogel discs (excitation wavelength 540 nm) when the PBS solution used to immerse the hydrogel discs was exposed to 365 nm light for different durations (the hydrogel discs were incubated in 10 mL of 0.005 ppm RS for 24 hours).

[0100] Figure 39 The fluorescence intensity of the PBS solution used to immerse the hydrogel discs at the peak wavelength when the PBS solution used to immerse the hydrogel discs was exposed to 365 nm light for different durations (the hydrogel discs were incubated in 10 mL of 0.005 ppm RS for 1 h, 15 h, or 24 h).

[0101] Figure 40 RS release kinetics from hydrogels containing different ratios of active to inactive monomers (hydrogel discs were incubated in 10 mL of 0.005 ppm RS solution for 24 hours).

[0102] Figure 41 The fluorescence intensity of the wells of a microtiter plate after treatment with the supernatant obtained by exposing the PEG hydrogel to 365 nm light (excitation wavelength 490 nm), wherein different hydrogels were pre-incubated for 24 hours in different solutions (i.e., streptavidin but without mucin and streptavidin with mucin, buffer, mucin but without streptavidin).

[0103] Figure 42 The fluorescence intensity of the wells of a microtiter plate after treatment with the supernatant obtained by exposing the PEG hydrogel to 365 nm light (excitation wavelength 490 nm), wherein different hydrogels were pre-incubated for 24 hours in different solutions (i.e., buffer, synthetic saliva, buffer of IL6 and synthetic saliva solution of IL6).

[0104] The patents, scientific and technical documents mentioned herein establish the knowledge available to a person skilled in the art at the time of filing. All disclosures of published patents, pending patent applications, and other publications cited herein are incorporated herein by reference to the extent that each patent is specifically and individually cited and incorporated herein by reference. In any event of inconsistency, this disclosure shall prevail.

[0105] The various aspects of the present invention will now be described in further detail. Detailed Implementation

[0106] hydrogel

[0107] In one aspect of the invention, a hydrogel is provided 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 linked to the active monomer via a cleavable bond.

[0108] As used herein, a "hydrogel" is a hydrogel comprising a polymer formed of a plurality of inactive monomers and a plurality of active monomers. Suitably, the hydrogel comprises a variety of polymers, the polymers comprising active and inactive monomers, and suitably, the polymers are crosslinked with a crosslinking agent to form an insoluble hydrophilic network. Suitably, the hydrogel is hydrophilic. Suitably, the hydrogel is insoluble in water. Suitable crosslinking agents are described elsewhere herein. Suitably, the active and inactive monomers are present in the polymer in a defined molar ratio. This defined molar ratio is described elsewhere herein. The average length of the polymer chain depends on the defined molar ratio of the active and inactive monomers to the crosslinking agent.

[0109] As used herein, "inactive monomer" refers to a monomer that does not contain a fluorophore bound thereto via a cleavable bond, and suitably, a monomer that does not contain a fluorophore. In some preferred embodiments, "inactive monomer" refers to a monomer that does not contain a fluorophore. Suitable inactive monomers are listed herein.

[0110] As used herein, an "active monomer" is a monomer containing at least one fluorophore bound thereto via a cleavable bond. Suitable active monomers are listed herein.

[0111] As used herein, a "crosslinking agent" crosslinks a polymer formed from an inactive monomer and an active monomer. Suitable crosslinking agents are bisacrylamide, polyethylene glycol diacrylamide, polyethylene glycol dimethacrylamide, polyethylene glycol diacrylate, polyethylene glycol dimethacrylate, or 4-arm PEGyne. In one embodiment, the crosslinking agent is bisacrylamide. In another embodiment, the crosslinking agent is 4-arm PEGyne, suitably as shown below.

[0112]

[0113] Suitablely, in embodiments where the crosslinking agent is a 4-arm PEGyne, n is 5 to 20, suitablely 8 to 15, suitablely 9 to 13, and suitablely n is 11.

[0114] As used herein, a "fluorophore" refers to a compound, chemical group, or composition that has inherent fluorescence. Suitable fluorophores are listed herein.

[0115] As used herein, "covalent bonding" means the formation of a covalent bond through the sharing of electrons between atoms. For example, a fluorophore is covalently bonded to a primary amine of a protein. In one non-limiting example, the isothiocyanate group of fluorescein isothiocyanate is covalently bonded to a primary amine in a protein. In one non-limiting example, the isothiocyanate group of fluorescein isothiocyanate is covalently bonded to a terminal amine in a protein. In one non-limiting example, a hydroxyl group of fluorescein isothiocyanate is covalently bonded to the hydrogel backbone via an o-nitrobenzyl group.

[0116] As used herein, “protein” includes full-length protein, protein fragments, native protein, or denatured protein. A mixture of proteins may be a mixture of full-length proteins, a mixture of protein fragments, or a mixture of full-length proteins and protein fragments. Proteins may be acidic, neutral, or basic.

[0117] Appropriately, the protein is streptavidin. Appropriately, the protein is CRP. Appropriately, the protein is IL6. Appropriately, the protein is IL8.

[0118] In a non-limiting example, the proteins described herein also encompass low-abundance proteins. In a non-limiting example, the proteins described herein are biomarkers. Suitable protein biomarkers are described elsewhere herein. Suitable biomarkers include inflammatory proteins, such as chemokines and cytokines. Appropriately, the protein may be a biomarker of inflammation. Appropriately, a biomarker of inflammation may indicate disease. Appropriately, the protein may be a chemokine or a cytokine. Appropriately, the protein may be an interleukin or a C-reactive protein. In some embodiments, the protein is IL6, IL8, or CRP.

[0119] In a non-limiting example, the protein described herein may contain additional functional groups, such as fluorophores or chromophores.

[0120] Appropriately, the additional functional group is rhodamine.

[0121] In some implementations, the protein is streptavidin, and the additional functional group is rhodamine.

[0122] "Disintegrable bond" is defined elsewhere in this article.

[0123] The pore size of the hydrogel is defined elsewhere in this article.

[0124] 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 1000000, and up to 10000000 active monomers. Suitably, the molar ratio of inactive monomers to active monomers is from 100:1 to 5:1. Suitably, the molar ratio of inactive monomers to active monomers is 100:1, 40:1, 20:1, or 10:1. Suitably, the ratio of inactive monomers to active monomers is 40:1.

[0125] Appropriately, a hydrogel is a synthetic hydrogel. As used herein, a “synthetic hydrogel” means a hydrogel that does not contain natural polymers such as proteins and / or polysaccharides.

[0126] Where appropriate, the hydrogel may additionally contain natural polymers, such as proteins and / or polysaccharides. Non-limiting examples of proteins are collagen and / or gelatin. Non-limiting examples of polysaccharides are starch, alginate, dextran, chitosan, hyaluronic acid, and agarose.

[0127] Suitably, the hydrogels of the present invention, and any formulations thereof, inert substrates, or sampling devices thereof, can be stored without any harmful effects (such as degradation), and suitably, they are stable. Suitably, the hydrogels of the present invention, and any formulations thereof, or sampling devices thereof, can be stored for a period of at least 5 days, 6 days, 7 days, or longer (optionally 2 weeks, 3 weeks, or 4 weeks). In one embodiment, the hydrogels of the present invention, and any formulations thereof, or sampling devices thereof, can be stored for at least 7 days. Suitably, no degradation occurs. Therefore, the hydrogels of the present invention, and any formulations thereof, or sampling devices thereof, suitably have an improved shelf life of up to 5 days, 6 days, 7 days, or longer.

[0128] Inactive monomers

[0129] Suitablely, the polymer contained in the hydrogel of the present invention is formed from a plurality of inactive monomers and a plurality of active monomers polymerized together. As described above, the inactive monomers are preferably monomers that do not contain fluorophores.

[0130] Suitable, the plurality of inactive monomers are selected, for example but not limited to, polyethylene glycol, acrylamide, N-isopropylacrylamide, methacrylamide, methacrylate and PEG bisazide.

[0131] Appropriately, multiple inactive monomers are essentially not hydrolyzed into cleavable bonds (e.g., the o-nitrobenzyl-fluorescein carbonate bond).

[0132] Appropriately, the inactive monomers are selected from polyethylene glycol, acrylamide, N-isopropylacrylamide, methacrylamide, methacrylate and PEG bisazide.

[0133] Appropriately, the inactive monomers are selected from acrylamide, N-isopropylacrylamide, methacrylamide, methacrylate and PEG bisazide.

[0134] Appropriately, multiple inactive monomers may contain the same monomer or different monomers.

[0135] Appropriately, the plurality of inactive monomers may comprise two or more, three or more, four or more, or other different inactive monomers selected from the list above.

[0136] Appropriately, multiple inactive monomers are composed of the same monomers. That is, all inactive monomers in multiple inactive monomers are identical.

[0137] Suitablely, the plurality of inactive monomers are composed of acrylamide. Therefore, suitablely, in some embodiments, each of the plurality of inactive monomers is acrylamide.

[0138] Suitablely, the plurality of inactive monomers are composed of PEG bisazides. Thus, suitablely, in some embodiments, each of the plurality of inactive monomers is a PEG bisazide.

[0139] Appropriately, the molar ratio of inactive monomer to crosslinking agent is 2:1 to 75:1, and in some embodiments, it is 50:1 to 75:1.

[0140] Appropriately, the molar ratio of inactive monomer to crosslinking agent is 2:1 to 70:1, and in some embodiments, it is 56:1 to 70:1.

[0141] Appropriately, the molar ratio of inactive monomer to crosslinking agent is 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1.

[0142] Appropriately, the molar ratio of inactive monomer to crosslinking agent is 60:1, 61:1, 62:1, 63:1, 64:1, 65:1 or 66:1.

[0143] Appropriately, the molar ratio of the inactive monomer acrylamide to the crosslinking agent bisacrylamide is 50:1 to 75:1.

[0144] Appropriately, the molar ratio of the inactive monomer acrylamide to the crosslinking agent bisacrylamide is 56:1 to 70:1.

[0145] Appropriately, the molar ratio of the inactive monomer acrylamide to the crosslinking agent bisacrylamide is 60:1, 61:1, 62:1, 63:1, 64:1, 65:1 or 66:1.

[0146] Appropriately, the molar ratio of the inactive monomer acrylamide to the crosslinking agent bisacrylamide is 63:1.

[0147] Appropriately, the molar ratio of the inactive monomer PEG bisazide to the crosslinking agent 4-arm PEGyne is 2:1 to 10:1.

[0148] Appropriately, the molar ratio of the inactive monomer PEG bisazide to the crosslinking agent 4-arm PEGyne is 2:1 to 5:1.

[0149] Appropriately, the molar ratio of the inactive monomer PEG bisazide to the crosslinking agent 4-arm PEGyne is 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1.

[0150] Appropriately, the molar ratio of the inactive monomer PEG bisazide to the crosslinking agent 4-arm PEGyne is 2:1.

[0151] Appropriately, the molar ratio of inactive monomer to active monomer can be from 15:1 to 5:1, for example, 15:1, 10:1 or 5:1.

[0152] Appropriately, for example, the molar ratio of inactive monomer to active monomer is 10:1. Therefore, for every 1000 molecules, 16 molecules are crosslinking agents, 98 molecules are active monomers, and 886 molecules are inactive monomers.

[0153] Appropriately, in a preferred embodiment, the molar ratio of inactive monomer to active monomer is 40:1.

[0154] Therefore, in a non-limiting example, for every 1000 molecules, 16 molecules are the crosslinking agent bisacrylamide, 98 molecules are the active monomer, and 886 molecules are the inactive monomer acrylamide.

[0155] Suitable, the polymer contained in the hydrogel contains 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% of inactive monomers.

[0156] Suitablely, the polymer contained in the hydrogel contains 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% of inactive monomers.

[0157] Appropriately, the inactive monomer is water-soluble.

[0158] Appropriately, inactive monomers have similar reactivity to active monomers and crosslinking agents, for example, to provide suitable polymerization.

[0159] Appropriately, inactive monomers do not contain any functional groups that can react with proteins.

[0160] Appropriately, inactive monomers do not contain any functional groups capable of reacting with fluorophores.

[0161] Appropriately, inactive monomers substantially do not absorb ultraviolet light. Appropriately, inactive monomers substantially do not absorb ultraviolet light at wavelengths that break cleavable bonds. Suitable ultraviolet light is defined elsewhere in this document.

[0162] active monomers

[0163] Suitable of the invention, the polymer contained in the hydrogel is formed from a plurality of inactive monomers and a plurality of active monomers polymerized together. The active monomers contain fluorophores bound thereto via cleavable bonds.

[0164] Suitablely, the active monomers are selected from, for example, but not limited to, polyethylene glycol derivatives, such as polyethylene glycol-based monomers. Those skilled in the art will recognize what polyethylene glycol-based monomers are herein and can readily identify suitable polyethylene glycol-based monomers. A non-limiting example of such a polyethylene glycol-based monomer is polyethylene glycol methacrylamide.

[0165] Suitable, the active monomers are selected from polyethylene glycol acrylamide, polyethylene glycol N-isopropylacrylamide, polyethylene glycol methacrylamide, polyethylene glycol methacrylate, allylamide, and PEG. 1000 -Azides.

[0166] Suitable, the active monomers acrylamide, N-isopropylacrylamide, methacrylamide, and methacrylate each contain a polyethylene glycol spacer arm. Suitable, the polyethylene glycol spacer arm facilitates water solubility.

[0167] Appropriately, the active monomers acrylamide, N-isopropylacrylamide, methacrylamide, and methacrylate each contain a linear polyethylene glycol spacer arm.

[0168] Suitable, the polyethylene glycol spacer arm is PEG. 400 .

[0169] Appropriately, the polyethylene glycol spacer arm is PEG. 1000 .

[0170] Suitable, the polyethylene glycol spacer arm is PEG. 3400 .

[0171] Appropriately, multiple active monomers may contain the same monomer or different monomers.

[0172] Appropriately, the plurality of active monomers may include two or more, three or more, four or more, or other different active monomers selected from the list above.

[0173] Appropriately, multiple active monomers may be composed of the same monomer.

[0174] Suitable, the multiple active monomers include methacrylamide. Alternatively, the multiple active monomers include allylamide.

[0175] Appropriately, multiple active monomers include polyethylene glycol methacrylamide.

[0176] Suitable, polyethylene glycol methacrylamide is prepared in yields of at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, and 75%.

[0177] Appropriately, each of the multiple active monomers contains a polyethylene glycol spacer arm.

[0178] Appropriately, each of the multiple active monomers contains a polyethylene glycol spacer arm.

[0179] Appropriately, a polyethylene glycol spacer arm exists between the o-nitrobenzyl derivative and the methacrylamide.

[0180] Appropriately, the polyethylene glycol spacer arm is present between 4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy)butyric acid (NVOC) and methacrylamide.

[0181] Appropriately, multiple active monomers include PEG. 400 - Methacrylamide, PEG 3400 - Methacrylamide or PEG 1000 -Azides.

[0182] Suitablely, the active monomers include 4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy)butyric acid (NVOC).

[0183] In one embodiment, multiple active monomers comprise NVOC-PEG.400 - Methacrylamide, NVOC-PEG 3400 - Methacrylamide or NVOC-PEG 1000 -Azide. "NVOC" refers to 4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy)butyric acid.

[0184] Appropriately, the active monomers are selected from acrylamide, N-isopropylacrylamide, methacrylamide, and methacrylates.

[0185] In some cases, polyethylene glycol spacers are not present.

[0186] In some cases, the polyethylene glycol spacer arm is not present between NVOC and methacrylamide. "NVOC" refers to 4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy)butyric acid.

[0187] Appropriately, each of the multiple active monomers contains at least one fluorophore.

[0188] Appropriately, multiple active monomers are composed of F-NVOC-allylamide. "F" refers to fluorescein. "NVOC" refers to 4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy)butyric acid.

[0189] Appropriately, multiple active monomers are derived from F-NVOC-PEG. 400 - Composition: Methacrylamide. "F" refers to fluorescein. "NVOC" refers to 4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy)butyric acid.

[0190] Appropriately, multiple active monomers are derived from F-NVOC-PEG. 3400 - Composition: Methacrylamide. "F" refers to fluorescein. "NVOC" refers to 4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy)butyric acid.

[0191] Appropriately, multiple active monomers are derived from F-NVOC-PEG. 1000 - Composition of azide compounds. "F" refers to fluorescein. "NVOC" refers to 4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy)butyric acid.

[0192] Appropriately, F-NVOC-PEG 3400 - Methacrylamide was prepared in yields of at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, and 75%. "F" refers to fluorescein. "NVOC" refers to 4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy)butyric acid.

[0193] Appropriately, multiple active monomers contain FITC-NVOC-PEG. 3400 - Methacrylamide or FITC-NVOC-PEG 3400 - Methacrylamide composition. Therefore, suitably, in some embodiments, each active monomer is FITC-NVOC-PEG. 3400 - Methacrylamide. "FITC" refers to fluorescein isothiocyanate. "NVOC" refers to 4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy)butyric acid.

[0194] Suitablely, the plurality of active monomers comprise or consist of FITC-NVOC-allylamide. Therefore, suitablely, 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)butyric acid.

[0195] Appropriately, multiple active monomers contain FITC-NVOC-PEG. 400 - Methacrylamide or FITC-NVOC-PEG 400 - Methacrylamide composition. Therefore, suitably, in some embodiments, each active monomer is FITC-NVOC-PEG. 400 - Methacrylamide. "FITC" refers to fluorescein isothiocyanate. "NVOC" refers to 4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy)butyric acid.

[0196] Appropriately, multiple active monomers contain FITC-NVOC-PEG. 1000 -Azide or FITC-NVOC-PEG 1000 -Azide composition. Therefore, suitably, in some embodiments, each active monomer is FITC-NVOC-PEG. 3400 - Methacrylamide. "FITC" refers to fluorescein isothiocyanate. "NVOC" refers to 4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy)butyric acid.

[0197] Suitablely, the polymer contained in the hydrogel contains 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%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of an active monomer.

[0198] Suitable, the polymer contained in the hydrogel contains 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%, or at least 20% of an active monomer.

[0199] Appropriately, multiple active monomers are hydrophilic.

[0200] In one example, the plurality of active monomers according to the invention appropriately do not contain allyl amides because the addition of cleavable bonds produces water-insoluble monomers that cannot be incorporated into the hydrogel.

[0201] In an alternative example, the plurality of active monomers according to the invention may comprise allyl amides.

[0202] Appropriately, the active monomer is water-soluble.

[0203] Appropriately, the reactive monomer has similar reactivity to the inactive monomer and crosslinking agent, for example, to provide suitable polymerization.

[0204] Appropriately, each of the active monomers contains at least one fluorophore containing a functional group capable of forming a covalent bond with the protein.

[0205] Decomposable bonds

[0206] Suitable, the active monomers of the polymer contained in the hydrogel each contain one or more fluorophore groups attached thereto via cleavable bonds.

[0207] As used herein, a “cleavable bond” is a chemical bond that is cleaved, split, or fractured. Typically, a molecule is cleaved into two or more fragments via a cleavable bond. Suitablely, a cleavable bond contains a photoinstable group. Alternatively, a cleavable bond may not contain a photoinstable group as defined herein.

[0208] Appropriately, a cleavable bond is a cleavable linker group. Appropriately, a cleavable linker group includes photoinstantaneously unstable groups as defined herein.

[0209] Appropriately, a cleavable bond is a cleavable moiety. Appropriately, a cleavable moiety contains a photoinstantaneously unstable group as defined herein.

[0210] Appropriately, a cleavable bond is a covalent bond.

[0211] Appropriately, cleavable bonds can be cleaved by cleavage inducers.

[0212] Appropriately, the cleavable linker group is cleaved by a cleavage inducer.

[0213] Appropriately, the pyrolytic portion is pyrolyzed by a pyrolysis inducer.

[0214] As used in this article, "cleavage inducer" is any reagent or, for example, light or radiation, that can cleave cleavable bonds.

[0215] Appropriately, cleavage inducers are light, radiation, enzymes, acids, bases, and / or heat.

[0216] Appropriately, cleavage inducers are ultraviolet light, reducing agents, or esterases.

[0217] As used herein, “reducing agents” include, but are not limited to, dithiothreitol (DTT), 2-mercaptoethanol (also known as β-mercaptoethanol), sodium bisulfite, mercaptoacetic acid, mercaptoethanesulfonic acid, glutathione, and trialkylphosphine compounds or combinations thereof. These trialkylphosphine compounds include, but are not limited to, tri-n-butylphosphine (TBP) or tri[2-carboxyethyl]phosphine (TCEP).

[0218] Appropriately, the reducing agent is dithiothreitol (DTT) or 2-mercaptoethanol.

[0219] As used in this article, "esterase" is an enzyme that hydrolyzes esters into alcohols and acids.

[0220] Appropriately, the esterase is either a porcine liver esterase or a horse liver esterase.

[0221] Appropriately, cleavable bonds can be broken by ultraviolet light.

[0222] As used in this article, “ultraviolet light” includes wavelengths ranging from approximately 100 nm to 400 nm. More appropriately, between approximately 200 nm and 400 nm.

[0223] Appropriately, ultraviolet light includes a wavelength range of approximately 300 nm.

[0224] Appropriately, ultraviolet light is UV-A and includes a wavelength range of approximately 300 nm.

[0225] Appropriately, ultraviolet light is UV-A and includes a wavelength range between 315 nm and 400 nm.

[0226] Appropriately, ultraviolet light includes a wavelength range between 350 nm and 370 nm.

[0227] Appropriately, the ultraviolet light is 365nm.

[0228] As used herein, a "photoinstantaneous group" or "photoinstantaneous protecting group" is a group that can be removed by light. For example, the light can be ultraviolet light. A "photoinstantaneous group" or "photoinstantaneous protecting group" is also called a photolytic (protective) group, a photosensitizing group, a photorelease group, or a photoremovable group.

[0229] Appropriately, the cleavable bond (i.e., the photoinstantaneous group) can be, for example, based on nitrobenzyl or carbonyl.

[0230] Non-limiting examples of cleavable / photostable groups based on nitrobenzyl are o-nitrobenzyl, 2-nitrobenzyl, 2,6-dinitrobenzyl, 4,5-dimethoxy-2-nitrobenzyl, 2,5-dihydroxybenzyl, 2-cyano-6-nitrobenzyl, 2-nitroveratrol, 6-nitroveratrol, nitroveratrol, 4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy)butyric acid (NVOC), or 6-nitropiperylmethyl.

[0231] Appropriately, the cleavable / photostable group based on nitrobenzyl is o-nitrobenzyl.

[0232] Appropriately, the cleavable / photostable group based on the o-nitrobenzyl group is NVOC. "NVOC" refers to 4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy)butyric acid.

[0233] Appropriately, the o-nitrobenzyl derivative is NVOC. "NVOC" refers to 4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy)butyric acid.

[0234] Non-limiting examples of carbonyl-based cleavable / photostable groups are benzoylmethyl, 3',5'-dimethoxybenzoyl, and p-hydroxybenzoylmethyl.

[0235] Appropriately, the cleavable bond is based on the nitrobenzyl group.

[0236] Suitablely, the cleavable bond is m-nitrobenzyl. Suitablely, the cleavable bond is p-nitrobenzyl. Suitablely, the cleavable bond is ortho-nitrobenzyl.

[0237] Appropriately, cleavable bonds link the fluorophore to the polyethylene glycol chain of the polyethylene glycol spacer arm of each active monomer.

[0238] Appropriately, a cleavable bond (o-nitrobenzyl) links the fluorophore to the polyethylene glycol chain of the polyethylene glycol spacer arm of each active monomer.

[0239] Appropriately, the cleavable bond is contained in a photoinstantaneous group attached to the hydrogel backbone at one or more active monomers.

[0240] Appropriately, the cleavable bond is contained in one or more active monomers and linked to a photoinstantaneous group in the hydrogel backbone via a polyethylene glycol spacer arm.

[0241] Appropriately, the cleavable bond comprises a nitrobenzyl-based photoinstability group attached to one or more active monomers to the hydrogel backbone.

[0242] Appropriately, the cleavable bond comprises a nitrobenzyl-based photoinstability group linked to the hydrogel backbone via a polyethylene glycol spacer arm.

[0243] Appropriately, the cleavable bond comprises an o-nitrobenzyl group attached to one or more active monomers in the hydrogel backbone.

[0244] Appropriately, the cleavable bond includes an o-nitrobenzyl group attached to the hydrogel backbone, as shown below.

[0245]

[0246] Appropriately, the cleavable bond is contained in one or more active monomers linked to an o-nitrobenzyl group in the hydrogel backbone via a polyethylene glycol spacer arm.

[0247] Appropriately, the isothiocyanate groups of fluorescein isothiocyanate are not bound to the hydrogel backbone.

[0248] Fluorescent clusters

[0249] Suitable, the polymer contained in the hydrogel contains at least one fluorophore of an active monomer bound thereto via a cleavable bond.

[0250] Appropriately, each active monomer contains at least one fluorophore bound thereto.

[0251] Appropriately, each active monomer contains multiple fluorophores bound thereto.

[0252] Suitable, each active monomer contains 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 15, or at least 20 fluorophores bound thereto.

[0253] Suitable, each active monomer contains at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or at least 9 fluorophores bound thereto.

[0254] Appropriately, each active monomer contains up to 10 fluorophores bound thereto.

[0255] Suitablely, the polymer contained in the hydrogel comprises a total of 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 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.

[0256] Many fluorophores are well known to those skilled in the art, and include, but are not limited to, coumarin, anthocyanin, benzofuran, quinoline, quinazolinone, indole, furan, benzoazole, boron polyazainene, and xanthene (including fluorescein, fluorescein isothiocyanate, tetrachlorofluorescein, carbofluorescein, naphthalenefluorescein, (semi)naphthalenefluorescein, 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 (9th edition, CD-ROM, September 2002). As used herein, “fluorescein” includes all conceivable derivatives, such as fluorescein isothiocyanate.

[0257] Fluoresceins can contain substituents that alter their solubility, spectral properties, or physical properties.

[0258] Suitablely, at least one fluorophore is selected from fluorescein, fluorescein isothiocyanate, eosin Y, eosin B, tetrachlorofluorescein, carbofluorescein, naphthalenefluorescein, and (semi)naphthalenefluorescein, which have suitable protein-reactive groups.

[0259] Suitablely, at least one fluorophore is selected from fluorescein, fluorescein isothiocyanate, eosin Y, eosin B, tetrachlorofluorescein, carbofluorescein, naphthalenefluorescein, and (semi)naphthalenefluorescein, which have at least one suitable protein-reactive group.

[0260] As used herein, a “suitable protein reactive group” refers to a group that can react with another chemical group in a protein to form a covalent bond; that is, it is covalently reactive under suitable reaction conditions and usually represents a linker site for another substance. For example, a “suitable protein reactive group” reacts with amine-containing molecules in a protein. Reactive groups typically include nucleophiles, electrophiles, and photoactivated groups. Exemplary reactive groups include, but are not limited to, alkenes, alkynes, alcohols, phenols, ethers, oxides, halides, aldehydes, ketones, carboxylic acids, esters, amides, cyanates, isocyanates, thiocyanates, isothiocyanates, amines, hydrazines, hydrazones, acylhydrazines, diazo compounds, nitro groups, nitriles, thiols, sulfides, disulfides, sulfoxides, sulfones, sulfonic acids, sulfinic acids, acetals, ketals, acid anhydrides, sulfates, hyposulfonic acids, isonitriles, amidines, imides, imine esters, nitrones, hydroxylamines, oximes, isohydroxamic acids, thioisohydroxamic acids, propadiene, orthoesters, sulfites, enamines, alkynes, ureas, pseudoureas, aminoureas, carbodiimides, carbamates, imides, azides, azo compounds, azo oxides, and nitroso compounds.

[0261] Appropriately, at least one fluorophore is rhodamine.

[0262] Suitablely, at least one fluorophore is fluorescein isothiocyanate (FITC).

[0263] Suitable, at least one fluorophore is fluorescein (F).

[0264] The ratio of monomer to crosslinking agent

[0265] Suitablely, the ratio of active monomers to inactive monomers used in the hydrogel of the present invention can be varied. Suitablely, this ratio can be changed to control the properties of the hydrogel, and the properties can be fine-tuned for different applications and uses of the hydrogel.

[0266] Appropriately, the ratio of active monomer to inactive monomer is a molar ratio. Appropriately, an excess of inactive monomer is present. Appropriately, a molar excess of inactive monomer is present.

[0267] Appropriately, the molar ratio of active monomer to inactive monomer is 1:100 to 1:5.

[0268] Suitablely, the molar ratio of active monomer to inactive monomer is 1:100, 1:90, 1:80, 1:70, 1:60, 1:50, 1:40, 1:30, 1:20, 1:10, or 1:5. Suitablely, the molar ratio of active monomer to inactive monomer is 1:100, 1:40, 1:20, or 1:10. Suitablely, the molar ratio of active monomer to inactive monomer is 1:40.

[0269] Appropriately, the ratio of inactive monomer to crosslinking agent is also a molar ratio.

[0270] Appropriately, the molar ratio of inactive monomer to crosslinking agent is 63:1. Alternatively, the molar ratio of inactive monomer to crosslinking agent is 2:1.

[0271] Appropriately, the (molar) ratio of the inactive monomer to the crosslinking agent is 2:1 to 75:1, or 50:1 to 75:1.

[0272] Appropriately, the (molar) ratio of the inactive monomer to the crosslinking agent is 2:1 to 70:1, or 56:1 to 70:1.

[0273] Appropriately, the (molar) ratio of the inactive monomer to the crosslinking agent is 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1.

[0274] Appropriately, the (molar) ratio of the inactive monomer to the crosslinking agent is 60:1, 61:1, 62:1, 63:1, 64:1, 65:1 or 66:1.

[0275] Appropriately, the molar ratio of the inactive monomer acrylamide to the crosslinking agent bisacrylamide is 50:1 to 75:1.

[0276] Appropriately, the molar ratio of the inactive monomer acrylamide to the crosslinking agent bisacrylamide is 56:1 to 70:1.

[0277] Appropriately, the molar ratio of the inactive monomer acrylamide to the crosslinking agent bisacrylamide is 60:1, 61:1, 62:1, 63:1, 64:1, 65:1 or 66:1.

[0278] Appropriately, the molar ratio of the inactive monomer acrylamide to the crosslinking agent bisacrylamide is 63:1.

[0279] Appropriately, the molar ratio of the inactive monomer PEG bisazide to the crosslinking agent 4-arm PEGyne is 2:1 to 10:1.

[0280] Appropriately, the molar ratio of the inactive monomer PEG bisazide to the crosslinking agent 4-arm PEGyne is 2:1 to 5:1.

[0281] Appropriately, the molar ratio of the inactive monomer PEG bisazide to the crosslinking agent 4-arm PEGyne is 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1.

[0282] Appropriately, the molar ratio of the inactive monomer PEG bisazide to the crosslinking agent 4-arm PEGyne is 2:1.

[0283] Preferred hydrogel

[0284] Suitable, the hydrogel containing the polymer can be formed from a plurality of inactive monomers and a plurality of active monomers, wherein the inactive monomer is acrylamide and the active monomer is polyethylene glycol methacrylamide, wherein each polyethylene glycol methacrylamide monomer contains at least one fluorescein isothiocyanate group capable of covalently binding to a protein, and the (molar) ratio of the inactive monomer to the active monomer is 100:1 to 5:1, and each fluorescein isothiocyanate is linked to the polyethylene glycol methacrylamide via a cleavable bond, wherein the cleavable bond is an o-nitrobenzyl group.

[0285] Appropriately, the hydrogel contains F-NVOC-PEG. 400 - Methacrylamide. "F" refers to fluorescein, and "NVOC" refers to 4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy)butyric acid.

[0286] Appropriately, the hydrogel contains F-NVOC-PEG. 3400 - Methacrylamide. "F" refers to fluorescein, and "NVOC" refers to 4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy)butyric acid.

[0287] Appropriately, the hydrogel contains F-NVOC-allylamide. "F" refers to fluorescein, and "NVOC" refers to 4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy)butyric acid.

[0288] Appropriately, the hydrogel contains FITC-NVOC-PEG. 3400 - Methacrylamide. "FITC" refers to fluorescein isothiocyanate. "NVOC" refers to 4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy)butyric acid.

[0289] In a particularly preferred embodiment, the hydrogel comprises a polymer, wherein the polymer is a cross-linked FITC-NVOC-PEG. 3400 -Methacrylamide / acrylamide copolymer, wherein the crosslinking agent is bisacrylamide.

[0290] Appropriately, the hydrogel contains acrylamide.

[0291] Appropriately, the polymer is FITC-NVOC-PEG. 3400 - Methacrylamide / acrylamide copolymer. "FITC" refers to fluorescein isothiocyanate. "NVOC" refers to 4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy)butyric acid.

[0292] Appropriately, the polymer is a cross-linked FITC-NVOC-PEG. 3400 - Methacrylamide / acrylamide copolymer. "FITC" refers to fluorescein isothiocyanate. "NVOC" refers to 4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy)butyric acid.

[0293] In a particularly preferred embodiment, the polymer is a cross-linked FITC-NVOC-PEG. 3400 - Methacrylamide / acrylamide copolymer, wherein the crosslinking agent is bisacrylamide. "FITC" refers to fluorescein isothiocyanate. "NVOC" refers to 4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy)butyric acid.

[0294] Alternatively, the polymer-containing hydrogel can be formed from multiple inactive monomers and multiple active monomers, wherein the inactive monomer is PEG bis(azide) and the active monomer is FITC-NVOC-PEG. 1000 - an azide, wherein each polyethylene glycol monomer contains at least one fluorescein isothiocyanate group capable of covalently binding to a protein, and the (molar) ratio of inactive monomer to active monomer is 100:1 to 5:1, and each fluorescein isothiocyanate is linked to the polyethylene glycol via a cleavable bond, wherein the cleavable bond is an o-nitrobenzyl group.

[0295] In a particularly preferred embodiment, the hydrogel comprises a polymer, wherein the polymer is a cross-linked FITC-NVOC-PEG. 1000 Azide / PEG bis-azide copolymer, wherein the crosslinking agent is 4-arm PEGyne.

[0296] Appropriately, the polymer is FITC-NVOC-PEG. 1000 - Azide / PEG bis-azide copolymer. "FITC" refers to fluorescein isothiocyanate. "NVOC" refers to 4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy)butyric acid.

[0297] Appropriately, the polymer is a cross-linked FITC-NVOC-PEG. 1000 - Azide / PEG bis-azide copolymer. "FITC" refers to fluorescein isothiocyanate. "NVOC" refers to 4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy)butyric acid.

[0298] In a particularly preferred embodiment, the polymer is cross-linked FITC-NVOC-PEG. 1000 - Azide / PEG bis-azide copolymer, wherein the crosslinking agent is 4-arm PEGyne. "FITC" refers to fluorescein isothiocyanate. "NVOC" refers to 4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy)butyric acid.

[0299] Pharmaceutical preparations and oral preparations

[0300] In one aspect, a formulation comprising a core and a shell is provided, wherein the core comprises a hydrogel of any of the foregoing aspects and embodiments, and the shell comprises a hydrogel with a pore size of less than 30 nm.

[0301] As used in this article, the "core" is basically surrounded by a shell.

[0302] Suitably, the core comprises a hydrogel of any of the foregoing aspects and embodiments. Therefore, the hydrogel comprises a polymer containing a plurality of inactive monomers and a plurality of active monomers polymerized together. The active monomers contain fluorophores bound thereto via cleavable bonds.

[0303] As used herein, "shell" comprises a hydrogel, and suitably, the hydrogel comprises a polymer. Suitably, the hydrogel forming the shell can be a different hydrogel from the core. Suitably, the shell can be formed from any hydrogel with a pore size of less than 30 nm.

[0304] Suitable, the shell comprises a polymer formed from polyethylene glycol, acrylamide, N-isopropylacrylamide, methacrylamide and / or methacrylate.

[0305] Appropriately, the formulation is a core-shell hydrogel.

[0306] Appropriately, the formulation has a core-shell structure.

[0307] As used herein, “pore size” characterizes the size of the openings in the hydrogel described herein. Those skilled in the art will understand that pore size depends on the monomer used to produce the polymer; that is, methacrylamide produces different pore sizes compared to N-isopropylacrylamide, and thus different pore sizes. Those skilled in the art will also understand that different concentrations of a given monomer can produce different pore sizes in the resulting hydrogel. For example, higher concentrations of methacrylamide produce hydrogels with smaller pore sizes compared to lower concentrations producing hydrogels with larger pore sizes.

[0308] Appropriately, 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, and less than 2 nm.

[0309] Appropriately, the pore size of the hydrogel is less than 7 nm, less than 6 nm, less than 5 nm, less than 4 nm, or less than 3 nm.

[0310] Appropriately, the pore sizes of the hydrogel are 4 nm, 3.9 nm, 3.8 nm, 3.7 nm, 3.6 nm, and 3.5 nm.

[0311] Suitablely, the pore size of the hydrogel is adapted to the size of the protein to be detected. A non-limiting example of a protein to be detected is cardiac troponin. Suitablely, 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).

[0312] Suitable for use, the pore size of the shell 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, and less than 2 nm. Therefore, the pore size of the shell hydrogel is controlled to limit the uptake of large amounts of larger proteins from the sample. A non-limiting example of such larger proteins is mucin.

[0313] Appropriately, the pore size of the hydrogel in the shell is less than 7 nm, less than 6 nm, less than 5 nm, less than 4 nm, or less than 3 nm.

[0314] Appropriately, the pore size of the hydrogel in the shell is 4 nm, 3.9 nm, 3.8 nm, 3.7 nm, 3.6 nm, or 3.5 nm.

[0315] Appropriately, the pore size of the hydrogel is adapted to the size of the protein to be detected. A non-limiting example of a protein to be detected is cardiac troponin T. Appropriately, 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).

[0316] Appropriately, the formulation contains additional pharmaceutically acceptable excipients. Suitable additional excipients are, for example, disintegrants, binders, lubricants, flow aids, and / or surfactants.

[0317] Appropriately, the shell contains additional excipients.

[0318] Appropriately, the core contains additional excipients.

[0319] Appropriately, the shell or core contains additional excipients.

[0320] Appropriately, the shell and core contain additional excipients.

[0321] As used herein, a "disintegrant" is an excipient that promotes dissolution and enhances usability. Suitable disintegrants are, for example, starch, starch derivatives, and cross-linked polymers (e.g., polyvinylpyrrolidone or cross-linked polyvinylpyrrolidone).

[0322] As used herein, a “binder” is an excipient that aggregates hydrogels and other excipients (if present). Binders also improve compressibility. Suitable binders are, for example, cellulose derivatives (e.g., microcrystalline cellulose, methylcellulose, sodium carboxymethylcellulose, hydroxypropyl methylcellulose, hydroxyethylcellulose, and hydroxypropylcellulose). Other binders include povidone, polyvinylpyrrolidone, gelatin, natural gums, starch paste, pregelatinized starch, sucrose, corn syrup, polyethylene glycol, and sodium alginate, calcium ammonium alginate, and polyethylene glycol.

[0323] As used herein, a "lubricant" is an excipient that prevents hydrogel adhesion and reduces friction during the potential compression phase of a hydrogel formulation. Suitable lubricants include vegetable oils, mineral oils, polyethylene glycol, stearates (e.g., calcium stearate, magnesium stearate, and sodium stearoyl fumarate), mineral salts (e.g., talc), organic salts (e.g., sodium benzoate, sodium acetate, and sodium oleate), and polyvinyl alcohol.

[0324] As used in this article, a "flow aid" is an excipient that reduces interparticle friction in a formulation, thereby improving flow. Suitable flow aids include alkali metal stearates (such as magnesium stearate or calcium stearate), silicates (such as magnesium silicate, magnesium trisilicate, anhydrous magnesium silicate, and calcium silicate), starch, mineral salts (such as talc), and colloidal silica.

[0325] As used herein, a "surfactant" is an amphiphilic excipient that reduces the surface tension or interfacial tension between two phases (such as between two liquids or between a liquid and a solid). Suitable surfactants can be ionic, nonionic, and amphoteric. Anionic surfactants include, but are not limited to, sodium lauryl sulfate, sodium laurate, sodium dialkyl sulfosuccinate, sodium stearate, potassium stearate, sodium oleate, deoxycholic acid, sodium deoxycholate, cholic acid, and sodium taurocholate. Nonionic surfactants include, but are not limited to, polyethylene oxide, sorbitol, fatty acid esters, fatty alcohols, glycerides, fatty acid esters of fatty alcohols, and one or more of alcohols.

[0326] In one aspect, an oral formulation comprising a hydrogel according to any one of the foregoing aspects and embodiments is provided, or a formulation according to the foregoing aspects.

[0327] As used herein, "oral formulation" is a formulation suitable for administration / incubation via the oral route. In other words, the oral formulation of the present invention is biocompatible. Oral routes include oral, sublingual, and sublipal routes.

[0328] Appropriately, oral formulations may contain a hydrogel formulated into a shell.

[0329] Appropriately, the hydrogel is formulated into a hydrogel shell.

[0330] Appropriately, the hydrogel is not formulated into a hydrogel shell.

[0331] Suitable oral formulations are pills, tablets, capsules, granules, lozenges, lollipops, or sampling materials. Suitable sampling materials may be, but are not limited to, sheets, beads, round tablets, or nanoparticles. In a non-limiting example, the nanoparticles are formulated into a shell. Suitablely, the shell is a hydrogel shell.

[0332] In one embodiment, the oral formulation is a round tablet. In another embodiment, the oral formulation is a sampling material, which is in the form of a round tablet.

[0333] Appropriately, oral preparations are lozenges, lollipops, or sampling materials.

[0334] Appropriately, oral preparations are lollipops.

[0335] As used herein, a “lollipop” is a hydrogel contained on a stick. The hydrogel has, for example, a disc or a sphere shape. This shape is configured to facilitate the collection of biological samples. Furthermore, the shape is configured so that the hydrogel can take up biological samples and thus proteins in a time- and resource-efficient manner.

[0336] Appropriately, oral formulations containing hydrogels may contain additional excipients. These additional excipients are defined elsewhere in this document.

[0337] Suitable additional excipients are, for example, disintegrants, binders, lubricants, flow aids and / or surfactants as defined elsewhere in this document.

[0338] Other suitable additional excipients for oral formulations include colorants, diluents, buffers, preservatives, flavoring agents, and pharmacologically compatible carriers.

[0339] Appropriately, lozenges may contain coloring agents and / or flavoring agents.

[0340] Appropriately, lollipop formulations may contain coloring agents and / or flavoring agents.

[0341] Sampling device

[0342] In one aspect, a sampling device is provided comprising a hydrogel according to any of the foregoing aspects and embodiments, a formulation comprising any of the foregoing aspects and embodiments, or an oral formulation comprising any of the foregoing aspects and embodiments, wherein the sampling device is a test tube.

[0343] As used herein, a “sampling device” is a device for collecting biological samples. Biological samples are defined elsewhere herein and also include fluid samples (e.g., saliva). Therefore, a sampling device may contain both the hydrogel of the present invention and biological samples.

[0344] Appropriately, the sampling device is configured such that biological samples can be deposited within the sampling device and come into contact with the hydrogel.

[0345] In one example, the sampling device is an oral preparation as defined elsewhere in this document, and the oral preparation is deposited in another sampling device (such as a test tube). Thus, there may be one or more sampling devices; for example, the first sampling device may be an oral preparation as defined elsewhere in this document, and the second sampling device may be a test tube.

[0346] Those skilled in the art know of suitable sampling devices and test tubes and can readily identify such sampling devices and test tubes.

[0347] Appropriately, the sampling device is a test tube.

[0348] In other embodiments, the hydrogel of the present invention may be contained on a substrate (suitably an inert substrate). Suitablely, the hydrogel may be contained in a thin film on the substrate, the thin film being coated on the substrate. Suitablely, a substrate comprising the hydrogel of the present invention is provided. Suitablely, an inert substrate comprising a coating thereon is provided, wherein the coating comprises the hydrogel of the present invention. Suitablely, the coating is a thin film. Such a suitable substrate may be glass or metal. Suitablely, the substrate may be used as a sampling device.

[0349] In other embodiments, the hydrogel of the present invention may be contained in a circular tablet. Suitablely, a circular tablet containing the hydrogel of the present invention is provided. Suitablely, the circular tablet can be used as an oral formulation or sampling device. Suitablely, the diameter of the circular tablet is between 1 mm and 10 mm, suitablely between 2 mm and 8 mm, and suitablely about 6 mm. Suitablely, the height of the circular tablet may be about 1 mm. Suitablely, the circular tablet is easily fitted into the oral cavity of the subject and further provides the benefit of requiring only a smaller buffer volume to release any captured proteins compared to a hydrogel film. The smaller the volume of buffer used to release proteins captured in the hydrogel, the higher the resulting pre-concentration factor.

[0350] Multi-component reagent kit

[0351] As used herein, "multi-component kit" refers to a package of related components, typically one or more hydrogels, formulations, and / or oral formulations of the present invention.

[0352] A multi-component kit is provided for protein labeling and analysis using a hydrogel according to any of the foregoing aspects and embodiments, a formulation according to any of the foregoing aspects and embodiments, an oral formulation according to any of the foregoing aspects and embodiments, or a sampling device according to any of the foregoing aspects and embodiments.

[0353] Therefore, in one aspect, a multi-component kit is provided, the multi-component kit comprising: a. a hydrogel according to any of the foregoing aspects and embodiments, a formulation according to any of the foregoing aspects and embodiments, an oral formulation (optionally, a round tablet) according to any of the foregoing aspects and embodiments, a sampling device according to any of the foregoing aspects and embodiments, a substrate according to any of the foregoing aspects and embodiments; and b. an instruction manual.

[0354] In one aspect, the multi-component kit includes: a. a hydrogel according to any of the foregoing aspects and embodiments; and b. instructions for use.

[0355] In one aspect, the multi-component kit includes: a. a formulation according to any of the foregoing aspects and embodiments; and b. instructions for use.

[0356] In one aspect, the multi-component kit includes: a. an oral formulation according to any of the foregoing aspects and embodiments; and b. instructions for use, optionally wherein the oral formulation may be a round tablet.

[0357] In one aspect, the multi-component kit includes: a. a sampling device according to any of the foregoing aspects and embodiments, and b. an instruction manual.

[0358] In one aspect, the multi-component kit includes: a. a substrate according to any of the foregoing aspects and embodiments; and b. an instruction manual.

[0359] In one aspect, the multi-component kit includes: a. a round tablet according to any of the foregoing aspects and embodiments; and b. instructions for use.

[0360] Suitable, multi-component kits may include other components, such as neutralizing buffers, mouthwashes, and / or tissues. Suitable, multi-component kits may include containers according to one aspect of the invention.

[0361] Appropriately, the sampling device is a test tube.

[0362] biomaterials

[0363] In one aspect, a biomaterial is provided comprising a hydrogel according to any of the foregoing aspects and embodiments, or a formulation comprising any of the foregoing aspects and embodiments.

[0364] As used herein, “biomaterial” is a synthetic material intended for and suitable for biological purposes. These biological purposes include, but are not limited to, labeling proteins and thus cells, or labeling proteins in biological samples (such as biological fluid samples as further defined elsewhere herein).

[0365] Suitablely, the biomaterial contains additional excipients that impart or enhance the desired properties of the biomaterial. Suitablely, additional excipients are disintegrants, binders, lubricants, flow aids, and / or surfactants as defined elsewhere herein.

[0366] Suitable, non-limiting examples of additional excipients are colorants, diluents, buffers, antibiotics, growth factors and / or preservatives.

[0367] Appropriately, additional excipients may be antibiotics, growth factors, and / or preservatives.

[0368] Appropriately, additional excipients are preservatives.

[0369] Appropriately, biological materials are sterile.

[0370] Appropriately, biological materials are biocompatible.

[0371] Appropriately, biological materials are used for cell culture.

[0372] use

[0373] In one aspect, the present invention provides the use of the biomaterials of the present invention in cell culture.

[0374] As used in this article, “cell culture” refers to any application in which cells are cultured, such a cell culture system is also called a tissue culture system, and is used for cell-based assays, for example.

[0375] Therefore, biomaterials are used to label cells in cell culture systems and, for example, in cell-based assays.

[0376] Appropriately, label the proteins expressed on the cell surface.

[0377] Appropriately, proteins expressed on the cell surface are, for example, but not limited to, annexin V.

[0378] Appropriately, biological materials are used in cell culture to label apoptotic cells.

[0379] Appropriately, apoptotic cells express annexin V on their cell surface.

[0380] In an alternative, the present invention provides the use of the hydrogel of the present invention as a cell support scaffold, which simultaneously labels proteins expressed on the cell surface.

[0381] As used herein, the term "scaffold" refers to any material that allows cells to connect and subsequently proliferate and differentiate. As used herein, "connection" refers to the direct or indirect adhesion of cells to a substrate and the adhesion of cells to other cells.

[0382] In one aspect, a hydrogel according to any of the foregoing aspects and embodiments, a formulation or oral formulation (optionally a round tablet) according to any of the foregoing aspects and embodiments, or a sampling device according to any of the foregoing aspects and embodiments, or a substrate according to any of the foregoing aspects and embodiments, is used to concentrate and label proteins in a sample.

[0383] As used herein, “concentrated protein” refers to the covalent capture of a protein in a hydrogel. This covalent capture is facilitated by a reaction between a primary amine in the protein and at least one fluorophore present in the hydrogel capable of covalently binding to the protein. Thus, the protein is concentrated and labeled in a single step.

[0384] In an alternative, "concentrated protein" refers to protein concentration using methods well known to those skilled in the art (such as dialysis, precipitation, chromatography, and the use of a cellulose membrane concentrator). This alternative can be combined with the concentration steps according to the invention.

[0385] As used herein, “labeled protein” refers to the labeling of an active reactive site on a protein or protein fragment. The label is a fluorophore and can therefore be detected directly. Fluorescent labels are detected by exciting suitable molecular adducts, which can be visualized by light excitation absorbed by the dye, or measured using, for example, a standard fluorometer or imaging system.

[0386] Appropriately, the “concentration and labeling of proteins in the sample” occurs in a single step.

[0387] In one aspect, a hydrogel according to any of the foregoing aspects and embodiments, a formulation according to any of the foregoing aspects and embodiments, an oral formulation (optionally a round tablet) according to any of the foregoing aspects and embodiments, a sampling device according to any of the foregoing aspects and embodiments, or a substrate according to any of the foregoing aspects and embodiments is used to concentrate and label proteins in a sample, followed by controlled release of the concentrated and labeled proteins.

[0388] As used in this article, “controlled release” means release at predetermined intervals or release gradually over a period of time.

[0389] In one aspect, a method for diagnosing a disease or symptom is used for a hydrogel, a formulation, an oral formulation (optionally a round tablet), a sampling device, a substrate, or a container according to any of the foregoing aspects and embodiments.

[0390] As used herein, “diagnosis” means any quantitative or semi-quantitative determination of the presence of a disease or symptom or the extent of its progression (prognosis), and should be interpreted freely accordingly.

[0391] As used in this article, “disease” refers to a state caused by a pathophysiological response to external or internal factors.

[0392] As used in this article, "symptom" refers to the disruption of normal or routine functions of the body or a part of the body.

[0393] Methods for concentrating and labeling proteins

[0394] In one aspect, a method for concentrating and labeling proteins in a sample is provided, the method comprising:

[0395] a. Contact the sample with a hydrogel of any of the foregoing aspects and embodiments, a formulation of any of the foregoing aspects and embodiments, an oral formulation (optionally, a round tablet) of any of the foregoing aspects and embodiments, a sampling device of any of the foregoing aspects and embodiments, or a substrate of any of the foregoing aspects and embodiments under suitable conditions to allow proteins in the sample to bind to the hydrogel via fluorophores, thereby obtaining fluorescently labeled proteins bound to the hydrogel.

[0396] As used herein, “contact sample” means bringing a sample into contact with the hydrogel of the present invention or a formulation thereof under suitable conditions to allow proteins in the sample to bind to the hydrogel.

[0397] As used in this article, “suitable conditions” are a temperature of about 15°C to 40°C, a pH of about 4 to 8, and an atmospheric pressure.

[0398] Suitablely, the contact step is performed for an appropriate amount of time to allow proteins in the sample to bind to the hydrogel. Suitablely, the contact step includes an incubation period. Suitablely, the incubation time is 15 to 48 hours. Suitablely, the incubation time is 24 to 48 hours. Suitablely, the incubation time is at least 24 hours, suitablely about 24 hours. Suitablely, the contact step includes contacting the sample with the hydrogel for at least 24 hours to allow proteins in the sample to bind to the hydrogel via fluorophores, thereby obtaining fluorescently labeled proteins bound to the hydrogel.

[0399] Typically, the method described in this paper is an in vitro method that uses a sample already obtained from the object (i.e., the sample is provided for the method, and the steps taken to obtain the sample from the object are not included as part of the method).

[0400] However, in some examples, the method may include the step of providing a biological fluid sample from the object.

[0401] As used herein, “provide” and “obtain” can refer to any means by which a sample is obtained, whether “direct” or “indirect.” Obtaining a sample directly means obtaining it through a process (e.g., by performing physical methods such as extraction). Obtaining a sample indirectly means receiving it from another party or source (e.g., a third-party laboratory that directly obtained the sample).

[0402] The methods described herein include providing biological fluid samples from the subject (e.g., saliva, blood samples such as serum or plasma samples, urine samples). The samples tested in the methods described herein are also referred to as "test samples".

[0403] As used herein, the terms “biological sample,” “test sample,” “sample,” and variations thereof refer to a sample obtained from or derived from an object. For the purposes described herein, a sample is or includes a biological fluid (also referred to herein as a bodily fluid) sample. In alternative examples according to the invention, “biological sample” or “sample” also encompasses environmental samples or samples from food sources. Biological, environmental, and food samples contain proteins of interest. Samples can be fresh, frozen, or preserved, such as those preserved in formalin. Samples according to the invention may contain other compounds (e.g., metabolites of pharmaceuticals), antibiotics, anticoagulants, chemicals (e.g., preservatives, fixatives, or buffers), nutrients, fertilizers, etc. In some cases, the sample originates from a source of contamination.

[0404] Environmental samples involve samples from nature (such as soil or water) and the surrounding environment, including non-natural environments or surroundings, such as the interior of a building (e.g., walls, floors, surfaces, ventilators, drains, conveyor belts, and containers). Food samples are samples from any stage of a food or beverage source. Samples can be raw materials, materials being processed, "in-process samples," or samples from the final food product.

[0405] As used herein, the term "biofluid sample" encompasses saliva samples. The term "biofluid sample" also encompasses other bodily fluids (such as urine or blood samples). It includes all biological fluids and excretions.

[0406] As defined elsewhere herein, the proteins of this invention also encompass low-abundance proteins.

[0407] As used herein, "fluorescently labeled protein" refers to a protein that contains a fluorophore. The fluorophore is chemically linked to the protein, or in other words, bound to the protein.

[0408] Properly, proteins are pre-concentrated using hydrogels.

[0409] Suitably, the hydrogel pre-concentrates the protein at 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, or up to 450. Suitably, the hydrogel pre-concentrates the protein at a pre-concentration factor of 190 to 300. In some embodiments, the hydrogel pre-concentrates the protein at a pre-concentration factor of up to 192. In some embodiments, the hydrogel pre-concentrates the protein at a pre-concentration factor of up to 295.

[0410] As used herein, "pre-concentration factor" refers to an increase in protein concentration before and after contact with the hydrogel. For example, a pre-concentration factor of 2 means doubling the protein concentration before and after contact with the sample. In a non-limiting example, the protein to be concentrated and labeled is additionally fluorophored. Suitably, the protein to be concentrated is streptavidin, and the fluorophore is rhodamine. Suitably, the fluorescence of the labeled protein is used to determine the protein concentration before and after contact with the hydrogel. Thus, a pre-concentration factor is provided in this non-limiting example. Therefore, as defined elsewhere herein, "concentrated protein" refers to a protein covalently captured in the hydrogel, and said protein is concentrated, as demonstrated by the exemplary pre-concentration.

[0411] Appropriately, the protein is streptavidin. Appropriately, the protein is CRP. Appropriately, the protein is IL6. Appropriately, the protein is IL8.

[0412] Appropriately, the protein is rhodamine-streptavidin.

[0413] Appropriately, the fluorophore is fluorescein isothiocyanate.

[0414] Appropriately, upon release from the hydrogel, the protein is labeled 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, or at least 90 fluorophores.

[0415] Appropriately, upon release from the hydrogel, the protein is labeled with up to 5, 6, 7, 8, 9, or 10 fluorophores.

[0416] Appropriately, the protein is labeled with up to 10 fluorophores upon release from the hydrogel.

[0417] Appropriately, upon release from the hydrogel, streptavidin, CRP, IL8, or IL6 is labeled 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, or at least 90 fluorophores.

[0418] Appropriately, the fluorophore is fluorescein isothiocyanate.

[0419] Appropriately, upon release from the hydrogel, streptavidin, CRP, IL8, or IL6 is labeled 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, or at least 90 luciferin molecules.

[0420] Appropriately, upon release from the hydrogel, at least 50 luciferin molecules are labeled with streptavidin, CRP, IL8, or IL6.

[0421] Appropriately, upon release from the hydrogel, streptavidin, CRP, IL8, or IL6 are labeled with 85 luciferin molecules.

[0422] Suitable, the hydrogel can provide a concentration factor of 192 for 0.1 ppm of protein in a sample. Suitable, this is also suitable when using an inert substrate containing the hydrogel.

[0423] Suitablely, the hydrogel can provide a concentration factor of 295 for 0.01 ppm of protein in a sample. Suitablely, when using an oral formulation containing the hydrogel, it is suitable to be a round tablet.

[0424] Suitable, the hydrogel comprises a polymer formed of a plurality of inactive monomers and a plurality of active monomers, wherein the inactive monomer is acrylamide and the active monomer is fluorescein isothiocyanate-NVOC-polyethylene glycol methacrylamide, and the (molar) ratio of the inactive monomer to the active monomer is 100:1 to 5:1, wherein each fluorescein isothiocyanate-NVOC-polyethylene glycol methacrylamide contains 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 linked to the polyethylene glycol methacrylamide via a cleavable bond, wherein the cleavable bond is o-nitrobenzyl, and the hydrogel provides a concentration factor of 192 for 0.1 ppm of protein in the sample. "NVOC" refers to 4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy)butyric acid.

[0425] Suitable, the hydrogel comprises a polymer formed of a plurality of inactive monomers and a plurality of active monomers, wherein the inactive monomer is acrylamide and the active monomer is fluorescein isothiocyanate-NVOC-polyethylene glycol methacrylamide, and the (molar) ratio of the inactive monomer to the active monomer is 100:1 to 5:1, wherein each fluorescein isothiocyanate-NVOC-polyethylene glycol methacrylamide contains at least one fluorescein isothiocyanate capable of covalently binding to streptavidin, CRP, IL8 or IL6, and each fluorescein isothiocyanate is linked to the polyethylene glycol methacrylamide via a cleavable bond, wherein the cleavable bond is o-nitrobenzyl, and the hydrogel provides a concentration factor of 295 for 0.01 ppm of protein in a sample. “NVOC” refers to 4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy)butyric acid.

[0426] Suitable, the hydrogel comprises a polymer formed of a plurality of inactive monomers and a plurality of active monomers, wherein the inactive monomer is a PEG bisazide and the active monomer is fluorescein isothiocyanate-NVOC-polyethylene glycol azide, and the (molar) ratio of the inactive monomer to the active monomer is 100:1 to 5:1, 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 linked to the polyethylene glycol azide via a cleavable bond, wherein the cleavable bond is o-nitrobenzyl, and the hydrogel provides a concentration factor of 295 for 0.01 ppm of protein in the sample. "NVOC" refers to 4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy)butyric acid.

[0427] Methods for detecting proteins

[0428] In one aspect, a method for detecting proteins in a sample is provided, the method including:

[0429] a. Contacting a sample with a hydrogel of any of the foregoing aspects and embodiments, a formulation of any of the foregoing aspects and embodiments, an oral formulation (optionally a round tablet) of any of the foregoing aspects and embodiments, a sampling device of any of the foregoing aspects and embodiments, or a substrate of any of the foregoing aspects and embodiments under suitable conditions to allow proteins in the sample to bind to the hydrogel via fluorophores, or to the sampling device of any of the foregoing aspects and embodiments, thereby obtaining fluorescently labeled proteins bound to the hydrogel;

[0430] b. Exposing the hydrogel from step (a) to a cleavage inducer under suitable conditions to cleave cleavable bonds, thereby releasing the fluorescently labeled protein from the hydrogel; and

[0431] c. Determine the presence of proteins in the sample, where the presence of fluorescence indicates the presence of proteins in the sample.

[0432] "Contact sample" is defined elsewhere in this article.

[0433] "Suitable conditions" are defined elsewhere in this article.

[0434] "Fluorescently labeled protein" is defined elsewhere in this article.

[0435] "Clearance inducer" is defined elsewhere in this document. Suitable cleavage inducers, such as ultraviolet light, reducing agents, or esterases, are also defined elsewhere in this document.

[0436] "Disintegrable bond" is defined elsewhere in this article.

[0437] "Fluorescently labeled protein" is defined elsewhere in this article.

[0438] Appropriately, fluorescently labeled proteins are released from the hydrogel of the present invention.

[0439] Properly, 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%, and at least 99% of the fluorescently labeled proteins are released from the hydrogel of the present invention.

[0440] Suitable, 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%, and at least 99% of the fluorescently labeled protein are released from the hydrogel of the present invention in the first release. Suitable, this is after the first exposure to the lysis inducer.

[0441] In a non-limiting example, the release of fluorescently labeled proteins from the hydrogel of the present invention is repeated using the same hydrogel, resulting in a second release. For example, if approximately 50% of the fluorescently labeled protein is released from the hydrogel of the present invention in the first release, then approximately at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% of the remaining fluorescently labeled protein is suitably released from the hydrogel of the present invention in the second release. Suitably, this is done after the second exposure to the lysis inducer.

[0442] Suitablely, the fluorescently labeled protein is released from the hydrogel of the present invention during a third or fourth release. Suitablely, this is after the third or fourth exposure to the lysis inducer.

[0443] Suitably, the release of one or more fluorescently labeled proteins from the hydrogel of the present invention occurs for 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, and at least about 600 seconds. Suitably, the hydrogel is exposed to the lysis inducer for this duration.

[0444] Suitablely, the release of one or more fluorescently labeled proteins from the hydrogel of the present invention takes less than 10 minutes. Therefore, suitablely, the hydrogel is exposed to the lysis inducer for less than 10 minutes.

[0445] Appropriately, the fluorescently labeled protein is released within 10 minutes. In some embodiments, 50% of the fluorescently labeled protein is released within at least about 100 seconds.

[0446] Appropriately, fluorescently labeled streptavidin, CRP, IL8, or IL6 is released within 10 minutes. Appropriately, 50% of the fluorescently labeled streptavidin, CRP, IL8, or IL6 is released within at least approximately 100 seconds.

[0447] Appropriately, rhodamine-labeled streptavidin, CRP, IL8, or IL6 is released within 10 minutes. In some embodiments, 50% of the rhodamine-labeled streptavidin, CRP, IL8, or IL6 is released within at least about 100 seconds.

[0448] As used herein, "determining the presence of protein in a sample" means performing a assay to determine whether protein is present in the sample. In other words, detecting the presence of protein in a sample.

[0449] As defined elsewhere herein, the proteins of this invention also encompass low-abundance proteins.

[0450] Suitablely, the method detects proteins at concentrations 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, and 0.0020 ppm. Suitablely, the method has limits of detection (LODs) 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, and 0.0020 ppm. Suitablely, the LOD is 0.0020 ppm.

[0451] Appropriately, this method detects proteins at a concentration of 0.1 ppm.

[0452] Appropriately, this method detects proteins at a concentration of 0.01 ppm.

[0453] Appropriately, the protein is streptavidin, and the detection limit of this method is 0.0033 ppm.

[0454] Appropriately, the protein is CRP, and the detection limit of the method is 0.0022 ppm.

[0455] Appropriately, the protein is IL6, and the detection limit of the method is at least 0.005 ppm.

[0456] Appropriately, the protein is IL8, and the detection limit of the method is at least 0.005 ppm.

[0457] Suitablely, the method detects the protein of interest in the presence of interfering substances. Suitablely, in the presence of one or more interfering substances, the method detects the protein of interest down to the lower limit of detection, suitablely reaching the range defined above. Suitablely, the interfering substance can be another protein that is not the protein of interest and can bind to the hydrogel. Suitable common interfering substances can be proteins present in biological samples, such as mucins abundant in saliva. Suitablely, the method detects the protein of interest in the presence of interfering substances at concentrations up to 5 ppm.

[0458] Suitablely, the hydrogel comprises a polymer formed of a plurality of inactive monomers and a plurality of active monomers, wherein the inactive monomer is acrylamide and the active monomer is polyethylene glycol methacrylamide, and the (molar) ratio of the inactive monomer to the active monomer is 100:1 to 5:1, wherein each polyethylene glycol methacrylamide contains at least one fluorescein isothiocyanate capable of covalently binding to a protein, and each fluorescein isothiocyanate is linked to the polyethylene glycol methacrylamide via a cleavable bond, wherein the cleavable bond is o-nitrobenzyl, and wherein the hydrogel is capable of detecting proteins at concentrations 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, and 0.0020 ppm.

[0459] Suitable, the hydrogel comprises a polymer formed of a plurality of inactive monomers and a plurality of active monomers, wherein the inactive monomer is acrylamide and the active monomer is polyethylene glycol methacrylamide, and the (molar) ratio of the inactive monomer to the active monomer is 100:1 to 5:1, wherein each polyethylene glycol methacrylamide contains at least one fluorescein isothiocyanate capable of covalently binding to a protein, and each fluorescein isothiocyanate is linked to the polyethylene glycol methacrylamide via a cleavable bond, wherein the cleavable bond is an o-nitrobenzyl group, and wherein the hydrogel is capable of detecting proteins at a concentration of 0.1 ppm.

[0460] Suitable, the hydrogel comprises a polymer formed of a plurality of inactive monomers and a plurality of active monomers, wherein the inactive monomer is acrylamide and the active monomer is polyethylene glycol methacrylamide, and the (molar) ratio of the inactive monomer to the active monomer is 100:1 to 5:1, wherein each polyethylene glycol methacrylamide contains at least one fluorescein isothiocyanate capable of covalently binding to a protein, and each fluorescein isothiocyanate is linked to the polyethylene glycol methacrylamide via a cleavable bond, wherein the cleavable bond is o-nitrobenzyl detection, wherein the hydrogel is capable of detecting proteins at a concentration of 0.01 ppm.

[0461] Suitablely, the hydrogel comprises a polymer formed of a plurality of inactive monomers and a plurality of active monomers, wherein the inactive monomer is acrylamide and the active monomer is polyethylene glycol methacrylamide, and the (molar) ratio of the inactive monomer to the active monomer is 100:1 to 5:1, wherein each polyethylene glycol methacrylamide contains at least one fluorescein isothiocyanate capable of covalently binding to a protein, and each fluorescein isothiocyanate is linked to the polyethylene glycol methacrylamide via a cleavable bond, wherein the cleavable bond is o-nitrobenzyl detection, wherein the hydrogel is capable of detecting proteins at a concentration of 0.0020 ppm.

[0462] The term "sample" is defined elsewhere in this article.

[0463] Methods for determining protein

[0464] In one aspect, a method is provided for determining the amount of a protein of interest in a sample, the method comprising:

[0465] a. Contact the sample with the hydrogel of any of the foregoing aspects and embodiments, the formulation of any of the foregoing aspects and embodiments, the oral formulation (optionally, a round tablet) of any of the foregoing aspects and embodiments, or the sampling device of any of the foregoing aspects and embodiments, or the substrate of any of the foregoing aspects and embodiments under suitable conditions to allow the protein in the sample to bind to the hydrogel via a fluorophore, thereby obtaining fluorescently labeled protein bound to the hydrogel.

[0466] b. Exposing the hydrogel from step (a) to a cleavage inducer under suitable conditions to cleave cleavable bonds, thereby releasing the fluorescently labeled protein from the hydrogel;

[0467] c. Separate fluorescently labeled proteins;

[0468] d. Contact the fluorescently labeled protein with a binding molecule that can specifically bind to the protein of interest;

[0469] e. Remove any unbound fluorescently labeled proteins; and

[0470] f. Measure the fluorescence level, where the fluorescence level indicates the amount of the protein of interest in the sample.

[0471] "Contact sample" is defined elsewhere in this article.

[0472] The methods described above include the step of determining the fluorescence level of the protein of interest in a sample. Conventional "assay" methods may include sending clinical samples to a commercial laboratory to determine the fluorescence level of the protein of interest in the samples, or using a commercially available assay kit to determine the fluorescence level in the samples. Exemplary kits and suppliers will be readily apparent to those skilled in the art. In various examples, spectrophotometry may be used to determine, detect, and / or quantify the amount of the protein of interest, for example, for point-of-care testing.

[0473] The amount of a protein of interest in a sample can be determined, for example, by measuring the fluorescence level present in the sample. Methods for determining the amount of a specific protein are well known in the art and include both direct and indirect methods.

[0474] The amount of a protein of interest in a sample can also be determined by assessing the level of its activity. Therefore, the fluorescence “level” encompasses both the amount of the protein itself and its activity level.

[0475] As defined elsewhere herein, the proteins of this invention also encompass low-abundance proteins.

[0476] "Fluorescently labeled protein" is defined elsewhere in this article.

[0477] Appropriately, fluorescently labeled proteins contain a large number of fluorophores relative to the protein itself. This allows for the determination of low-abundance proteins. In this document, the term "low-abundance protein" is used synonymously with "protein present at low levels."

[0478] Appropriately, each fluorescently labeled protein is labeled 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, or at least 90 fluorophores.

[0479] Appropriately, the fluorescently labeled protein is streptavidin. Appropriately, the fluorescently labeled protein is CRP. Appropriately, the fluorescently labeled protein is IL6. Appropriately, the fluorescently labeled protein is IL8.

[0480] Appropriately, the fluorescently labeled protein is rhodamine-streptavidin.

[0481] Appropriately, fluorescently labeled proteins are labeled with the fluorophore fluorescein isothiocyanate.

[0482] Appropriately, the fluorescently labeled streptavidin, CRP, IL8 or IL6 label has 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 or at least 90 fluorophores.

[0483] Appropriately, the fluorescently labeled streptavidin, CRP, IL8 or IL6 is labeled 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 or at least 90 luciferin molecules.

[0484] Appropriately, the fluorescently labeled streptavidin, CRP, IL8, or IL6 label contains at least 65 fluorophores. Therefore, the fluorescently labeled streptavidin contains at least 65 fluorophores.

[0485] Appropriately, fluorescently labeled streptavidin, CRP, IL8, or IL6 are labeled with 85 fluorophores. Therefore, fluorescently labeled streptavidin, CRP, IL8, or IL6 contains 85 fluorophores.

[0486] "Cleavage inducer" is defined elsewhere in this document.

[0487] "Suitable conditions" are defined elsewhere in this article.

[0488] As used herein, “isolation of fluorescently labeled proteins” refers to any suitable method for removing or isolating fluorescently labeled proteins from a hydrogel.

[0489] Suitablely, separating fluorescently labeled proteins may include washing the hydrogel. Suitablely, the hydrogel should be washed for a sufficient time to elute the fluorescently labeled proteins from it. Suitablely, washing can be performed with a buffer (such as PBS). Suitablely, washing should be performed for 10 to 30 minutes. Suitablely, washing should be performed for at least 10 minutes.

[0490] As used herein, a “binding molecule” is a small molecule or antibody. In a non-limiting example, a small molecule is biotin. Binding molecules also include non-immunoglobulin binding agents (such as phage display-derived peptide binding agents) and antibody mimics (e.g., avidins, tetramer connexins (CTLD), adnectin (monobodies), anticalin, DARPin (anchor protein), avimers, iMab, microbodies, peptide aptamers, Kunitz domains, aptamers, and avidin). The term “antibody” includes, for example, naturally occurring and non-naturally occurring antibodies, polyclonal and monoclonal antibodies, chimeric antibodies, and fully synthetic antibodies and fragments thereof (such as Fab', F(ab')2, Fv, or Fab fragments), or other antigen-recognizing immunoglobulin fragments. Antibodies that bind to a specific epitope can be produced by methods well known in the art. For example, polyclonal antibodies can be produced by conventional methods used to immunize mammals (e.g., rabbits, mice, rats, sheep, goats). Polyclonal antibodies are then contained in the serum of immunized animals and can be analyzed using standard procedures, such as affinity chromatography, immunoprecipitation, size exclusion chromatography, and ion exchange chromatography. Monoclonal antibodies can be prepared using standard methods for immunizing mammals, followed by isolation of plasma B cells producing the monoclonal antibody of interest and fusion with myeloma cells (see, for example, Mishell et al., 1980). Screening for recognition epitopes can be performed using standard immunoassay methods, including ELISA, radioimmunoassay, immunofluorescence, immunohistochemistry, and Western blotting (Ausubel et al., 1992). In vitro methods for antibody selection (such as antibody phage display) can also be used to generate antibodies (see, for example, Schelmann et al., 2011).

[0491] Suitablely, the binding molecule may be located on a surface. Suitablely, the binding molecule may be located in a container. Therefore, suitablely, the contact step is performed within a container containing the binding molecule. Suitablely, the contact step may include contacting the fluorescently labeled protein with a surface containing the binding molecule capable of specifically binding to the protein of interest. Suitablely, the contact step may include contacting the fluorescently labeled protein with a container containing the binding molecule capable of specifically binding to the protein of interest. Suitablely, the contact step may include contacting the fluorescently labeled protein with a microplate containing the binding molecule capable of specifically binding to the protein of interest.

[0492] Suitable containers can be any commercially available microplate coated with the molecules of interest. For example, biotin-coated microplates (e.g., 15151, Thermo Fisher Scientific).

[0493] However, in a preferred embodiment, the container is the container of the present invention as defined below.

[0494] Suitable, the container includes an inner surface operable to contact a fluorescently labeled protein, wherein the inner surface is coated with a base layer, and a reaction layer is coated on the base layer, the reaction layer comprising a mixture of binding molecules and a blocking agent, wherein the blocking agent comprises a compound without amino groups.

[0495] In one embodiment, the base layer is a polymer containing free amine groups. Suitably, such a polymer is, for example, acrylamide / bisacrylamide copolymerized optionally with aminopropylmethacrylamide, or 4-arm PEG succinimide ester (NHS) copolymerized with PEG-diamine, or chitosan. In one embodiment, the base layer is chitosan. In one embodiment, the binding molecule is a protein (e.g., biotin or antibody) that recognizes and binds to the protein of interest, suitably an antibody or its binding fragment that specifically binds to the protein of interest. In one embodiment, the blocking agent contains PEG-methyl groups. Suitably, the capturing protein and the blocking agent bind to the base layer. Suitably, the reactive layer contains a majority of the reactive protein and a small amount of blocking agent. Suitably, the blocking agent binds to the base layer only if the reactive protein does not bind to the base layer. Suitably, the blocking agent binds to any free amine groups in the base layer. Suitably, the blocking agent suitably prevents free FITC from binding to the base layer of the container during use.

[0496] Suitablely, the container can be any container suitable for carrying out the methods of the invention therein. In one embodiment, the container is a microplate or a microtiter plate.

[0497] Suitablely, the container of the present invention reduces the background signal by 110 times compared to commercially available microplates or microtiter plates. Suitablely, the container of the present invention reduces the background signal caused by free fluorophores (especially free FITC) directly bound to the container.

[0498] In one embodiment, the binding molecule is biotin. In one embodiment, the binding molecule is an anti-CRP antibody or a binding fragment thereof. In one embodiment, the binding molecule is an anti-IL8 antibody or a binding fragment thereof. In one embodiment, the binding molecule is an anti-IL6 antibody or a binding fragment thereof. As used herein, “removal of any unbound fluorescently labeled protein” means any suitable method, such as removing the unbound fluorescently labeled protein by one or more washing steps comprising, for example, a buffer solution.

[0499] Appropriately, removal of any unbound fluorescently labeled proteins includes washing, appropriately washing the aforementioned container, and appropriately washing the microplate as described above.

[0500] For example, the fluorescence level in a sample can be determined (e.g., measured) by any suitable method and material well known in the art, including processes such as fluorescence spectrophotometry, protein microarrays, immunoprecipitation, immunofluorescence, Western blot analysis, lateral flow (using, for example, a lateral flow device (LFD)), utilizing membrane-bound antibodies specific to protein biomarkers. In a non-limiting example, the fluorescence level in the sample is determined by fluorescence spectrophotometry.

[0501] Appropriately, proteins of interest are biomarkers.

[0502] Biomarkers are organic biological molecules (such as proteins, polypeptides, peptides and their isoforms, and immunologically detectable fragments) that differ significantly between samples taken from subjects with and without the disease. A difference in biomarker is considered statistically significant if the mean or median level of the biomarker across different groups is calculated to be statistically significant. Common tests for statistical significance include t-tests (e.g., Student's t-test), analysis of variance (ANOVA), Kruskal-Wallis test, Wilcoxon test, Mann-Whitney test, receiver operating characteristics (ROC curve), precision, and odds ratios. Biomarkers, alone or in combination, provide a measure of the relative risk of an individual belonging to one phenotypic state or another. Therefore, they can be used as biomarkers for disease (diagnosis), the therapeutic effect of drugs, and drug toxicity.

[0503] Typically, the biomarkers referred to in this article are measured at the protein level.

[0504] Therefore, according to the present invention, a biomarker can be any biomarker that can be detected or determined at the protein level.

[0505] Therefore, according to the present invention, the biomarker is a protein biomarker. Suitablely, the biomarker can be a marker of disease. Suitablely, the biomarker can be an inflammatory marker. Suitablely, an inflammatory biomarker. Therefore, these inflammatory biomarkers generally indicate disease. Suitablely, the biomarker can be a chemokine or a cytokine.

[0506] Appropriately, biomarkers include cardiac troponin, creatine kinase, creatine kinase-MB, myoglobin, IL-1, IL-2, IL-3, IL-4, 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), prostate acid phosphatase (PAP), and CA. 125. Carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), human chorionic gonadotropin (HCG), CA19-9, CA15-3, CA27-29, lactate dehydrogenase (LDH), neuron-specific enolase (NSE), and C-reactive protein (CRP).

[0507] Appropriately, biomarkers are IL-6, IL-8, cardiac troponin, or CRP.

[0508] Appropriately, cardiac troponin is troponin C (TnC), troponin T (TnT), or troponin I (TnI). Appropriately, cardiac troponin is troponin T (TnT).

[0509] The term "sample" is defined elsewhere in this article.

[0510] Methods for determining whether a person has a disease or condition

[0511] In one aspect, a method is provided for determining whether an object suffers from a disease or ailment, the method comprising:

[0512] a. Contact a sample from the subject with a hydrogel of any of the foregoing aspects and embodiments, a formulation of any of the foregoing aspects and embodiments, an oral formulation (optionally a round tablet) of any of the foregoing aspects and embodiments, a sampling device according to any of the foregoing aspects and embodiments, or a substrate of any of the foregoing aspects and embodiments under suitable conditions to allow proteins in the sample to bind to the hydrogel via fluorophores, thereby obtaining fluorescently labeled proteins bound to the hydrogel.

[0513] b. Under suitable conditions, the hydrogel from step (a) is exposed to a cleavage inducer to cleave cleavable bonds, thereby releasing the fluorescently labeled protein from the hydrogel;

[0514] c. Separate fluorescently labeled proteins;

[0515] d. To bring fluorescently labeled proteins into contact with binding molecules that can specifically bind to protein biomarkers of diseases or symptoms;

[0516] e. Remove any unbound fluorescently labeled proteins;

[0517] f. Detecting the presence of fluorescence or measuring the level of fluorescence, wherein the presence of fluorescence indicates the presence of a protein biomarker in the sample, or wherein the level of fluorescence indicates the level of a protein biomarker in the sample.

[0518] g. Based on f. determining that the object suffers from a disease or condition, wherein the presence or level of fluorescence indicates the disease or condition.

[0519] "Contact sample" is defined elsewhere in this article.

[0520] "Fluorescently labeled protein" is defined elsewhere in this article.

[0521] "Cleavage inducer" is defined elsewhere in this document.

[0522] "Suitable conditions" are defined elsewhere in this article.

[0523] "Separation of fluorescently labeled proteins" is defined elsewhere in this article.

[0524] "Binding molecules" are defined elsewhere in this article.

[0525] "Removal of any unbound fluorescently labeled proteins" is defined elsewhere in this document.

[0526] As defined elsewhere herein, the proteins of this invention also encompass low-abundance proteins.

[0527] As used herein, "detecting the presence of fluorescence" means performing a measurement to determine whether fluorescence is present, and whether a protein is present in the sample. Appropriately, the presence of fluorescence indicates the presence of a protein biomarker in the sample. In a non-limiting example, the mere presence of a protein biomarker in the sample indicates a disease or condition. This means that if such a protein biomarker is present in the sample, the subject has a disease or condition.

[0528] "Measurement of fluorescence level" is defined elsewhere herein. Appropriately, fluorescence level indicates the level of a protein biomarker in a sample. In a non-limiting example, the level of a protein biomarker in a sample indicates a disease or condition. Thus, if a defined level or threshold of said protein biomarker is present in a sample, the subject has a disease or condition. Those skilled in the art understand such thresholds and suitable reference ranges. Those skilled in the art also understand that such thresholds and suitable reference ranges may depend on the specific assay used. In a non-limiting example, fluorescence level indicates the level of the protein biomarker IL-6, IL-8, or cardiac troponin. Appropriately, fluorescence level indicates the level of the protein biomarker troponin C (TnC), troponin T (TnT), or troponin I (TnI). Appropriately, fluorescence level indicates the level of the protein biomarker troponin T (TnT).

[0529] "Disease" and "symptom" are defined elsewhere in this article.

[0530] Appropriately, the disease or ailment is cancer or cardiovascular disease.

[0531] Examples of cancers include, but are not limited to, lung cancer (e.g., bronchial carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), lung adenocarcinoma); kidney cancer (e.g., nephroblastoma, also known as Wilms' tumor, renal cell carcinoma); acoustic neuroma; acute myeloid leukemia; adenocarcinoma; adrenal carcinoma; anal cancer; angiosarcoma (e.g., lymphangiosarcoma, lymphangioendothelial sarcoma, angiosarcoma); appendix cancer; benign monoclonal gammopathy; cholangiocarcinoma (e.g., cholangiocarcinoma); bladder cancer; breast cancer (e.g., breast adenocarcinoma, breast papillary carcinoma, breast cancer, breast medullary carcinoma); brain cancer (e.g., meningioma, glioblastoma, glioma (e.g., astrocytoma, oligodendroglioma, medulloblastoma); bronchial carcinoma; carcinoid tumor; cervical cancer (e.g., cervical adenocarcinoma); choroidal carcinoma; craniopharyngioma; colorectal cancer (e.g., (e.g., colon cancer, rectal cancer, colorectal adenocarcinoma); connective tissue cancer; epithelial cancer; ependymoma; endothelial sarcoma (e.g., Kaposi's sarcoma, multiple idiopathic hemorrhagic sarcoma); endometrial cancer (e.g., uterine cancer, uterine sarcoma); esophageal cancer (e.g., esophageal adenocarcinoma, Barrett's adenocarcinoma); Ewing's sarcoma; ocular cancer (e.g., intraocular melanoma, retinoblastoma); familial hypereosinophilia; gallbladder cancer; gastric cancer (e.g., gastric adenocarcinoma); gastrointestinal stromal tumor (GIST); germ cell cancer; head and neck cancer (e.g., head and neck squamous cell carcinoma); oral cancer (e.g.) Examples include: oral squamous cell carcinoma; laryngeal cancer (e.g., laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer); heavy chain diseases (e.g., alpha chain disease, gamma chain disease, μ chain disease); angioblastoma; hypopharyngeal cancer; inflammatory myofibroblastoma; immune cell amyloidosis; liver cancer (e.g., hepatocellular carcinoma (HCC), malignant hepatocellular carcinoma); leiomyosarcoma (LMS); mastocytosis (e.g., systemic mastocytosis); muscle cancer; myelodysplastic syndrome (MDS); mesothelioma; myeloproliferative disorders (MPD) (e.g., polycythemia vera (PV), essential thrombocythemia (ET), idiopathic myeloid metaplasia (AMM)). Named as myelofibrosis (MF), chronic idiopathic myelofibrosis, chronic myeloid leukemia (CML), chronic neutrophilic leukemia (CNL), hypereosinophilic syndrome (HES); neuroblastoma; neurofibroma (e.g., neurofibromatosis type 1 or 2 (NF), schwannoma); neuroendocrine carcinoma (e.g., gastrointestinal pancreatic neuroendocrine tumor (GEP-NET), carcinoid tumor); 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 tumor (IPMN), islet cell tumor);Penile cancer (e.g., Paget's disease of the penis and scrotum) Diseases including: pineal gland tumors; primitive neuroectodermal tumors (PNT); plasmacytoma formation; paraneoplastic syndromes; intraepithelial tumors; 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 sarcomas (e.g., malignant fibrous histiocytoma (MFH), liposarcoma, malignant peripheral nerve sheath tumor (MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma); sebaceous gland cancer; small intestine cancer; sweat gland cancer; synovial tumors; testicular cancer (e.g., seminoma, embryonal testis); thyroid cancer (e.g., papillary thyroid carcinoma, papillary thyroid carcinoma (PTC), medullary thyroid carcinoma); urethral cancer; uveal melanoma; vaginal cancer; and vulvar cancer (e.g., vulvar Paget's disease).

[0532] Examples of cardiovascular diseases include, but are not limited to, coronary artery disease (such as angina pectoris, heart attack), stroke, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, congenital heart disease, valvular heart disease, carditis, aortic aneurysm, peripheral artery disease, thromboembolic disease, and venous thrombosis.

[0533] As used herein, “the presence of fluorescence” means the presence of fluorescence and may optionally include determining the level of fluorescence, such as low, moderate, or high levels of fluorescence. The presence or level of fluorescence in a sample can be determined (e.g., measured) by any suitable methods and materials well known in the art as defined elsewhere herein.

[0534] The term "sample" is defined elsewhere in this article.

[0535] Example

[0536] 1. Experimental Methods

[0537] 1.1 Materials and Equipment

[0538] Allyltrichlorosilane (95%), Acrylamide / bisacrylamide (40% solution, 29:1), Ammonium persulfate (≥98%), Ethanol (≥98%, Supelco), fluorescein, fluorescein isothiocyanate isomer I (FITC) (≥90%), Poly(ethylene glycol) (PEG) bis(amine) (average Mn 400 to 3400), 4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy)butyric acid (NVOC), (1 -[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) (97%), N,N-diisopropylethylamine (DIPEA) (98%), triethylamine (TEA) (99%), methacrylamide chloride (97%), phosphate-buffered saline (PBS, 10 mM, pH 7.4), phosgene solution (15 wt.% toluene solution), silica gel (high purity, pore size 60). Sodium sulfate, methanol, ethyl acetate, hexane, DMSO-d6 (99.9%, MagniSolv), CDCl3 (99.9%, MagniSolv), and acetone-d6 (99.9%, MagniSolv) were purchased from Merck. Decon 90 was purchased from Fisher Scientific. The remaining solvents were purchased from Merck and prepared before use. Tetrahydrofuran (THF) and toluene were distilled from sodium under nitrogen using benzophenone as an indicator. Dichloromethane (DCM), dimethylformamide (DMF), and acetonitrile were distilled from calcium hydride under nitrogen. Streptavidin labeled with rhodamine (S6366) was purchased from Thermo Fisher.

[0539] Thin-layer chromatography was performed on Macherey Nagel aluminum TLC plates pre-coated with 0.20 mm silica gel 60 and UV 254 fluorescent indicator. Visualization was performed on silica with iodine, followed by heating and UV irradiation (λ = 254 nm). PEG 3400 monomer was purified using a Slide-A-Lyzer Dialysis Cascade (MWCO 2K, Thermo Fisher). Pierce™ biotin-coated plates (clear, 8-well strips, Thermo Scientific) were used for protein release studies. Microscope slides (1 mm thick, low-iron standard) were purchased from VWR (Leicestershire, UK).

[0540] Obtained at 25℃ 1 H and 13C10 NMR spectra were recorded on 300 MHz Varian VNMRS, 400 MHz Varian Unity Inova, or 600 MHz Varian Unity Inova. Chemical shifts (δ) were reported in parts per million (ppm), and splitting modes were specified as s (singleton), d (doublet), dd (doublet), t (triplet), q (quartet), m (multiplet), and brs (broad singlet). Coupling constants (J) were expressed in Hertz (Hz). 1 H and 13 C NMR spectra reference residual solvent signals: DMSO-d6 (2.50 ppm or 39.52 ppm) and CDCl3 (7.26 ppm or 77.16 ppm).

[0541] Using an internally constructed UV LED (Nichia NVSU 233 A-U365, RS Components) for 365nm radiation, the radiant flux at 1A forward current was 1030mW. Extended to a diameter of 25mm, the radiant flux was approximately 210mW / cm². -2 Flux density. The LED and the DC-DC converter assembly driving the LED are mounted in a 60×60mm... 2 On the aluminum core PCB, the aluminum core PCB is then mounted to a 60×60mm size using thermal paste. 2 On the heatsink and fan (Thermo Electric Devices TDEX 6015 / TH12G, RS Components). The LED driver and fan are powered by a 12V 1A DC power adapter.

[0542] UV-Vis spectroscopy measurements were performed at 25 °C on a Jenway 6715 UV-Vis spectrometer. (Using Vanquish...) TM High-performance liquid chromatography (HPLC) was performed using an Agilent 1260 Infinity multi-wavelength absorbance detector coupled with a C18 column (1.8 µm particle size, 3 mm inner diameter, and 50 mm length). The fluorescence intensity (fluorescein, λ) of the excited samples was determined using a CLARIOstar Plus microplate reader. 激发 = 472nm, measured emission wavelength = 500nm to 620nm; Rhodamine, λ 激发 = 540nm, measured emission wavelength = 570nm to 670nm).

[0543] 1.2 Monomer Synthesis and Purification

[0544] Used in the synthesis of F-NVOC-allylamide, F-NVOC-PEG400-methacrylamide, and F-NVOC 3400 - Methacrylamide and FITC-NVOC 3400 The reaction scheme for methylacrylamide is as follows: Figure 2 As shown. The reaction scheme is adapted from Landfester and Klinger. 25 And our previous work 26,27 The reported procedures. Detailed information on the reaction and purification conditions of the monomers, as well as the characteristic shifts in their NMR spectra, is provided in SI.

[0545] Regarding F-NVOC-allylamide and NVOC-PEG 400 - Methacrylamide monomer, F-NVOC-PEG 3400 - Methacrylamide and FITC-NVOC-PEG 3400 Further details regarding the synthesis and purification of the methacrylamide monomer are provided below:

[0546] F-NVOC-Allylamide

[0547] NVOC (300 mg, 1.00 mmol, 1 equivalent) and anhydrous DMF (5 mL) were added to a 25 mL round-bottom flask (RBF) purged with nitrogen and dried in an oven. Allylamine (75 μL, 1.00 mmol, 1 equivalent), HATU (381 mg, 1.00 mmol, 1 equivalent), and DIPEA (873 μL, 5.00 mmol, 5 equivalent) were added to the solution. The reaction was stirred for 18 h. The next day, the solution was diluted with ethyl acetate (50 mL) and transferred to an extraction vessel. The organic phase was washed three times with DI water (50 mL). Finally, the organic phase was collected, dried over sodium sulfate, filtered, and concentrated under vacuum. The crude product was purified by rapid chromatography using a 5% methanol-DCM solution as the eluent. The expected product, N-allyl-4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy)butyramide (NVOC-allylamide), was obtained as a light yellow solid (322 mg, 95%).

[0548] In a 25 mL round-bottom flask purged with nitrogen, oven-dried, and wrapped in aluminum foil, NVOC-allylamide (135 mg, 0.40 mmol, 1 equivalent) was dissolved in anhydrous DCM (5 mL). The mixture was cooled to 0 °C, and phosgene solution (455 mL, 0.60 mmol, 1.5 equivalent) (15 wt.% toluene solution) was added dropwise over 20 min, followed by stirring at 0 °C for 1 h. In another dry, dark-colored, double-necked round-bottom flask, sodium fluorescein (450 mg, 1.20 mmol, 2 equivalent) and TEA (83 μL, 0.60 mmol, 1.5 equivalent) were dissolved in cooled anhydrous DCM (5 mL). The first reaction mixture was added dropwise to this fluorescein-containing flask over 30 min under inert conditions. The reaction was carried out in an ice bath for 2 h, and then the reactants were cooled to room temperature overnight. The reaction vessel was reduced in volume by purging with nitrogen, and then purified by rapid chromatography (5% to 40% ethyl acetate:hexane) in a dark fume hood to give the product as a yellow solid (F-NVOC-allylamide, 225 mg).

[0549] 1 H NMR (400 MHz, DMSO-d6): δ 8.03 (t, J = 5.7 Hz, 1H), 7.52 (s, 1H), 7.36 (s, 1H), 5.79 (ddt, J = 17.2, 10.4, 5.3 Hz, 1H), 5.47 (d, J = 4.4 Hz,1H), 5.26 (qd, J = 6.2, 4.3 Hz, 1H), 5.11 (dq, J = 17.2, 1.8 Hz, 1H), 5.03(dq, J = 10.2, 1.6 Hz, 1H), 4.04 (t, J = 6.5 Hz, 2H), 3.90 (s, 3H), 3.69 (tt,J = 5.5, 1.7 Hz, 2H), 2.69 (s, 2H), 2.28 (t, 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) δ 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.

[0550] F-NVOC-PEG 400 -Methacrylamide monomer

[0551] First, PEG 400 Bis(amine) (500 mg, 1 equivalent) was added to anhydrous DCM (20 mL). The mixture was then cooled to 0 °C and TEA (15 μL, 0.1 mmol, 1.2 equivalent) was added dropwise, while the mixture was stirred under inert conditions for 1 h. Subsequently, methacryloyl chloride (12 μL, 0.36 mmol, 0.6 equivalent) was added dropwise. The mixture was stirred for 1 h and purified by rapid chromatography to obtain amine-PEG. 400 -Methacrylamide (M n = 504g.mol -1 The yield was 80%.

[0552] A suspension of NVOC (150 mg, 0.50 mmol, 1 equivalent), HATU (229 mg, 0.70 mmol, 1.4 equivalent), and 183 μL of DIPEA (1.05 mmol, 2.1 equivalent) in 10 mL of DMF was stirred while cooled in an ice bath. Amine-PEG was then added. 400 1-Methacrylamide (281 mg, 0.70 mmol, 1.4 equivalences) was dissolved in a minimal amount of DMF and added dropwise to a solution of NVOC, HATU, and DIPEA. The reaction mixture was warmed to room temperature and stirred for 18 h. After the reaction was complete, the mixture was treated with water (20 mL) and extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, and concentrated to give a crude product, which was purified by column chromatography (100% ethyl acetate) to obtain NVOC-PEG. 400 1-Methacrylamide, a crystalline oil, with a yield of 20%.

[0553] The oily substance was then redissolved in anhydrous DCM (20 mL) in a dark RBF and stirred at 0 °C under a constant nitrogen flow for 30 min. Over 20 min, 4 mL of cooled phosgene solution (15 wt% toluene, 1.5 equivalents) was added dropwise to the RBF. The mixture was stirred in the dark and under inert conditions for 30 min. For another dried dark RBF, FITC (291 mg, 0.75 mmol) and TEA (60 μL, 0.41 mmol) were dissolved in cooled anhydrous DCM (5 mL) and stirred for 30 min. Over 30 min, NVOC-PEG was added dropwise to the RBF. 400 -Methacrylamide solution. The mixture was stirred at room temperature for 18 h. The solvent was evaporated by purging with nitrogen in a fume hood, and the residue was purified by rapid chromatography to yield the target monomer (F-NVOC-PEG). 400 -Methacrylamide, 169 mg, 35%.

[0554] 1 H NMR (400 MHz, DMSO-d6) δ 8.01 – 7.88 (m), 7.61 (s), 7.27 (t, J =5.7 Hz), 7.23 – 7.13 (m), 6.58 (d, J = 8.9 Hz), 6.37 (s), 5.66 (t, J = 1.3Hz), 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 (t, J = 6.1 Hz), 3.07 (q, J = 5.9 Hz), 2.43 (q, J =7.1 Hz), 2.10 (d, J = 1.5 Hz), 1.99 (t, J = 6.5 Hz), 1.85 (t, 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(t, J = 7.1 Hz). 13 C NMR (400 MHz, DMSO-d6) δ 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.

[0555] F-NVOC-PEG 3400 - Methacrylamide and FITC-NVOC-PEG 3400 -Methacrylamide monomer

[0556] By following the same procedure as described above, PEG 3400 Amine-PEG prepared by reacting bis(amine) with methacryloyl chloride 3400 -Methacrylamide (M n = 3604 g·mol -1 ).

[0557] Amine-PEG 3400 1-Methacrylamide (3 g, 1 equivalent) was dissolved in 2 mL of DMF. A suspension of NVOC (399 mg, 1.33 mmol, 1.5 equivalent), HATU (229 mg, 0.70 mmol, 1.4 equivalent), and 183 μL of DIPEA (1.05 mmol, 2.1 equivalent) in 10 mL of DMF was stirred while cooled in an ice bath. Amine-PEG was then added. 3400 A solution of methacrylamide was added dropwise to a solution of NVOC, HATU, and DIPEA. The reaction mixture was warmed to room temperature and stirred for 18 hours. The product was NVOC-PEG. 3400 1-Methacrylamide was purified by immediate precipitation in diethyl ether, redissolving in DCM, and finally precipitating in diethyl ether. The resulting precipitate was dried under vacuum at 40°C.

[0558] The precipitate was separated into two fractions and reacted with fluorescein or FITC using the same procedure described above. The monomer was purified by dialysis and lyophilized to obtain a viscous, off-white powder (F-NVOC-PEG). 3400 - Methacrylamide, 680 mg, 59%; FITC-NVOC-PEG 3400 1-Methacrylamide, 682 mg, 60%).

[0559] 1H NMR (400 MHz, DMSO-d6) δ 7.91 (t, J = 5.6 Hz), 7.24 (t, J = 5.7Hz), 5.65 (t, 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 (t, J = 6.1 Hz), 3.07 (q, J = 6.1 Hz), 2.54 (s), 1.84 (t, J= 1.2 Hz), 1.63 (s), 1.36 (d, J = 10.2 Hz). 13 C NMR (400 MHz, DMSO-d6) δ168.14, 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.

[0560] 1.3 Preparation of hydrogel films

[0561] Cut the glass microscope slide into pieces of 25.4 ± 0.5 mm. 2 The glass cube was then ultrasonically washed for 30 minutes each in Decon 90, water, and ethanol. The cube was then immersed in a 0.5% v:v allyltrichlorosilane toluene solution for 30 minutes, washed with toluene, and dried before use. It contains F-NVOC-allylamide dissolved in 250 μL DMSO or (F or FITC)-NVOC-PEG dissolved in 250 μL deionized water. 400 or 3400 A 1 mL solution of 1-methacrylamide, 40% w:v acrylamide / bisacrylamide (calculated to produce a 10 wt.% gel), 1.25 µL TEMED, and 12.5 µL 10% w:v APS was prepared in water free of nitrogen (N2). The concentration of NVOC monomers was 5%, 10%, or 15% of the total molar concentration of monomers in the precursor solution. The precursor solution was cast in the dark at room temperature between a glass cube and a plastic cap, separated by a 175 μm thick spacer (939-837-76, Goodfellow). After 15 min, the plastic cap was removed, and the hydrogel film deposited on the glass substrate was immersed in PBS overnight to remove any residual unreacted monomers.

[0562] 1.4 Characterization of monomers and hydrogel films

[0563] The UV-Vis absorption spectra of the monomer at different concentrations were determined to determine the molar extinction coefficient of the monomer. The monomer stock solution was irradiated with light at 365 nm, and samples were taken at fixed time intervals, followed by analysis by UV-Vis spectroscopy and HPLC. For HPLC, a 1 mg / mL monomer stock solution was prepared in acetonitrile, and the collected sample solutions were chromatographically analyzed using a solvent gradient of 90 / 10 to 10 / 90 acetonitrile / water. Chromatograms were recorded at 491 nm. The HPLC data were used to determine the phototriggered release kinetics of fluorescein from the monomer.

[0564] The UV-Vis absorption spectra of the hydrogel films deposited on glass slides in different regions (sample 8 in Table 1) were measured to determine differences between areas. Similarly, the absorbance of the precursor solution and the hydrogel films (see Table 1) was recorded at a wavelength of approximately 490 nm. Using these absorbance values ​​and given the path length of the cuvette containing the precursor solution and the thickness of the film, our inventors estimated the molar percentage of fluorescein-containing monomers incorporated into the hydrogel films. To determine the phototriggered release kinetics of fluorescein from the hydrogel films, the films were exposed to 365 nm light for a selected duration, and the UV-Vis spectra of the films were measured after each radiation time interval.

[0565] Table 1. Summary of the percentage of light-labile monomers incorporated into the hydrogel film (where the total concentration of monomers in the precursor solution is 10% w:v).

[0566]

[0567] 1.5 Protein Research

[0568] Using FITC-NVOC-PEG 3400 Functionalized hydrogels prepared by copolymerization of methacrylamide and acrylamide / bisacrylamide were used for protein studies. FITC-NVOC-PEG 3400 The molar fraction of methylacrylamide was 10%, and the total monomer concentration in the precursor solution was 10% w:v. Hydrogel films prepared by polymerizing 10% w:v acrylamide / bisacrylamide were used as negative controls. UV-Vis and fluorescence emission spectra of PBS solutions with different concentrations of rhodamine-streptavidin (RS) were measured. FITC-NVOC-PEG was used for both non-FITC-NVOC-PEG and FITC-NVOC-PEG solutions. 3400 Hydrogel films containing methacrylamide / bisacrylamide were immersed overnight in 10 mL of a stock solution of 0.01 ppm or 0.1 ppm RS in PBS. Fluorescence emission spectra of PBS were measured before and after incubation with each type of hydrogel film. The data were used to determine the protein pre-concentration factor.

[0569] The hydrogel film was washed overnight in fresh PBS at room temperature in the dark to remove any unbound RS. The film was then immersed in 3 mL of PBS and exposed to UV radiation for short pulses between 1 and 10 min. After each irradiation, the inventors waited 30 min to allow the released RS to diffuse out of the hydrogel film before collecting the PBS. The hydrogel film was then immersed in 3 mL of fresh PBS, and the above process was repeated. The phototriggered release kinetics of RS were investigated by monitoring the change in fluorescence emission of the corresponding rhodamine over irradiation time. All measurements were performed in triplicate. To verify whether the released RS were simultaneously labeled with rhodamine and fluorescein, 200 µL of PBS solution collected after UV exposure was pipetted into the wells of a biotin-coated microtiter plate and incubated for 30 min. The wells were then washed with 200 µL of PBS, and fluorescence emission spectra were measured at excitation wavelengths of 470 nm and 540 nm, corresponding to fluorescein and rhodamine, respectively.

[0570] 2. Results

[0571] 2.1 Monomer

[0572] The chemical structure of the synthetic monomer is in Figure 3 Provided by China. Figure 9 The peak absorption wavelength of F-NVOC-allylamide dissolved in DMSO is shown at 519 nm, with a shoulder peak at approximately 490 nm, indicating aggregation. The fact that the DMSO solution of F-NVOC-allylamide is predominantly non-fluorescent further supports aggregation. Conversely, as... Figure 10 and Figure 11 As shown, F-NVOC-PEG 400 - Methacrylamide and F-NVOC-PEG 3400 The methylacrylamide monomer is water-soluble and exhibits absorption at wavelengths of approximately 490 nm and 365 nm, which is attributed to the fluorescein and NVOC groups.

[0573] Photolysis occurs when the radiation wavelength overlaps with the absorption band of a photoinstantaneous group (NVOC in this case). 28-30 Therefore, the monomer solution was irradiated with 365 nm ultraviolet light. Typical ultraviolet-visible spectra of the monomer at different irradiation times (see...). Figure 12 The results showed a red shift in the peak attributed to the NVOC group, indicating the formation of nitrosobenzaldehyde, thus confirming the success of the photoreaction. The absorbance at approximately 490 nm remained constant with irradiation time because both the released fluorescein and the fluorescein bound to the monomer remained in the same solution. Therefore, the released fluorescein and the monomer were separated using HPLC, and the resulting chromatogram was obtained at [insert chromatogram here]. Figure 4 (a) and Figure 13and Figure 14 Provided in [the text]. HPLC determination was performed in acetonitrile at different irradiation times. The monomer disappearance rate ([M]) was monitored. t To study the phototriggered release kinetics of luciferin. ln[M]0 / [M] t Plotting of relative radiation time (see) Figure 4 (b) shows excellent linearity, indicating the expected first-order kinetics. Furthermore, Figure 4 The rate plot provided in (b) shows that the kinetics of fluorescein release from the phototriggered PEG-methacrylamide monomer are faster than those from the allylamide monomer.

[0574] 2.2 Hydrogel Film

[0575] Hydrogels were prepared by free radical copolymerization of acrylamide / bisacrylamide with different monomers. The compositions of the hydrogels studied in this work are summarized in Table 1. The hydrogels were cast onto glass slides for support and the slides were placed in 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 intensity of fluorescein in the hydrogel. Figure 5 (c) shows the UV-Vis absorption spectra at four different corners of the slide, indicating that the film is not uniform. These spectra were obtained by changing the position of the slide relative to the spectrophotometer beam. Figure 5 (d) presents the monomer incorporation factor for NVOC-containing monomers under different types and molar ratios. The incorporation factor is defined as the incorporation factor of the hydrogel (m 水凝胶 ) and precursor solution (m 前体 The molar concentration ratio of NVOC monomers in the sample was determined. m was estimated based on the absorbance of the hydrogel and precursor solution at approximately 490 nm and using the molar extinction coefficient of the fluorescein. 水凝胶 and m 前体 ( Figure 15 ).like Figure 5 As shown in (d), PEG-based methacrylamide monomers exhibit an increased monomer incorporation factor compared to allylamide monomers. This is attributed to the monomer reactivity of PEG-based monomers (methacrylamide > allylamide). 31 And increased water solubility. As the molecular weight of PEG increases, the monomer incorporation amount decreases slightly.

[0576] Next, we will investigate the light-induced release of luciferin from the hydrogel. Figure 6 (a) shows that the absorption band of fluorescein in the hydrogel film of slide 8 (Table 1) decreases with increasing irradiation time. Figure 6(b) shows the first-order reaction rate plot of the decrease in fluorescein concentration in the hydrogel, indicating that the release is first-order with irradiation lasting up to 30 min. Similar results were obtained for other prepared hydrogels, showing first-order release kinetics with irradiation lasting up to 30 min. This indicates that once fluorescein is released, it diffuses rapidly out of the hydrogel, but the nonlinearity after about 30 min of irradiation suggests that a small amount of irreversibly bound fluorescein may remain in the hydrogel.

[0577] In summary, F-NVOC-PEG 3400 -Methacrylamide can be produced in high yields (59%, compared to F-NVOC-PEG). 400 The preparation of methylacrylamide (only 35%) does not require purification by rapid chromatography. Furthermore, in F-NVOC-PEG... 3400 The release kinetics of fluorescein are fastest in methacrylamide solution. Finally, for F-NVOC-PEG... 3400 -Methacrylamide, relative to the precursor solution, showed a higher percentage of monomer incorporated into the hydrogel than F-NVOC-allylamide (approximately 32% vs. 65%, respectively). This is in consideration of F-NVOC-PEG. 3400 - Methacrylamide provides these benefits using derivatives of this monomer (FITC-NVOC-PEG) 3400 1-Methacrylamide (with isothiocyanate groups that can react with primary amines in proteins) is used for protein research.

[0578] 2.3 Protein Research

[0579] Rhodamine-streptavidin (RS) was selected as an example protein to study the pre-concentration, labeling, and release of proteins, which were then quantified using fluorescence in biotin-coated microtiter plates. The streptavidin used in this work was labeled with rhodamine, allowing the fluorescence of rhodamine to be used to determine protein concentrations before and after hydrogel incubation. This, in turn, provides the pre-concentration factor. Streptavidin was chosen because its binding to biotin is very strong and selective, making it easily captured in biotinylated microtiter plates after release from the hydrogel.

[0580] 2.3.1 Protein capture and pre-concentration

[0581] Composed of acrylamide / bisacrylamide and FITC-NVOC-PEG 3400 Functionalized hydrogels prepared by copolymerization of methacrylamide monomers can capture proteins because the isothiocyanate groups react with terminal and primary amines in proteins. 32This was confirmed by immersing the formed hydrogel in 10 mL of 0.10 ppm and 0.01 ppm (1.66 nM and 0.166 nM, respectively) PBS RS solution. Figure 8 (a) and Figure 8 As shown in (b), most RS were lost from solution after overnight incubation with the functionalized hydrogel. In contrast, the control experiment with the unfunctionalized polyacrylamide hydrogel resulted in no significant loss of RS in solution. The negligible reduction in fluorescence of the stock solution exposed to the unfunctionalized hydrogel indicates that the RS conjugates do not bind significantly in the absence of the FITC moiety.

[0582] Six functionalized hydrogels were prepared; three were incubated with 0.1 ppm RS solution, and the remaining three were incubated with 0.01 ppm RS solution. As summarized in Table 2, the percentages of RS loaded in the functionalized hydrogels for 0.1 ppm and 0.01 ppm protein solutions were 77 ± 8% and 94 ± 6%, respectively. This result is comparable to that of the photoresponsive hydrogels prepared by Klinger and Landfester. 25 The difference is significant; in their study, protein was trapped within the hydrogel due to the reduced pore size caused by electrostatic interactions, with a reported protein loading of 53.2 ± 7.4%. Subsequently, the concentration of RS in the functionalized hydrogel was estimated, and the results are provided in Table 2. The concentration ratio of RS in the hydrogel and the stock solution provides a pre-concentration, such as... Figure 8 As shown in (c), the pre-concentration estimates for 0.1 ppm and 0.01 ppm RS solutions are 192 and 236, respectively.

[0583] Table 2. By making FITC-NVOC-PEG 3400 Summary of RS loading in hydrogels prepared by copolymerization of methacrylamide and acrylamide / bisacrylamide (FITC-NVOC-PEG) 3400 The molar ratio of methylacrylamide was 10%, the volume of the RS stock solution was 10 mL, the volume of the hydrogel was estimated to be 0.04 mL, and ‡: determined by the change in fluorescence emission intensity).

[0584]

[0585] 2.3.2 Protein Release and Detection

[0586] After demonstrating the successful loading of model proteins into the prepared hydrogels, the photodegradation release of proteins from the hydrogels was investigated. The loaded hydrogels were exposed to short pulses of UV light for 1 to 10 minutes while immersed in 3 mL of PBS, and then held in PBS for 30 minutes to allow the released RS to diffuse out of the hydrogel. The PBS was then collected. This process was repeated until no additional RS was detectable in the supernatant PBS.

[0587] The fluorescence emission spectrum of PBS collected after each irradiation was in Figure 8 (a) shows the result. The function of the cumulative RS release as a function of radiation time is shown in Figure 8 As shown in (b). Figure 8 (b) The experimental data shown show a poor fit when fitted to a single exponential rise (r = 0.966), but a good fit when fitted to a double exponential rise to its maximum, with a correlation coefficient of 0.9995, indicating that two processes occurred during the photodecomposition and decomposition of the captured RS. The first rapid process accounted for approximately 54% of the released RS, with a time constant of 1.124 min. -1 The second, slower process accounts for approximately 46% of the released RS, with a time constant of 0.075 min. -1 Thus, the inventors showed that 50% of the RS was released within approximately 1.68 minutes or approximately 100 seconds. The origin of these two processes is currently unclear, but it is likely a result of the released RS slowly diffusing from the deeper layers of the hydrogel.

[0588] To verify whether the released streptavidin was labeled with fluorescein, one of the PBS solutions containing labeled proteins collected from a hydrogel incubated with 0.01 ppm streptavidin and exposed to UV light was partitioned into biotin-coated wells of a microtiter plate. The released streptavidin was allowed to bind to biotin for 30 min, followed by buffer washing. This process allows removal of any fluorescein dye, which is not bound to the protein but is released when the hydrogel is irradiated with UV light. The wells of the microtiter plate were irradiated with 470 nm light, followed by 540 nm light. The resulting fluorescence spectra (see [link to sample]). Figure 8 (c) shows the emission peaks corresponding to fluorescein and rhodamine, confirming that the released RS successfully labeled fluorescein. Figure 15 and Figure 16 The results show that, in RS, the molar emission coefficient of fluorescein at 514 nm is approximately 10 times higher than that of rhodamine at 590 nm. Based on this, and considering... Figure 5(c) Regarding the fluorescence emission intensity at 514 nm and 590 nm, the inventors assessed that for each rhodamine molecule, RS was labeled with approximately 17 luciferin molecules. Since each streptavidin was labeled with 5 rhodamine molecules, the inventors estimated that 85 luciferin molecules were attached to each streptavidin. One possible explanation for this observation is that FITC is PEGylated. 3400 The separation of the spacer arms from the main chain of the functionalized hydrogel makes it easier for many FITC molecules to react with each RS molecule loaded in the hydrogel. Labeling RS with a large amount of fluorescein is beneficial for the determination of low-abundance proteins.

[0589] 2.3.3. Conclusion

[0590] This work demonstrates a hydrogel for the pre-concentration, labeling, and controlled release of proteins, followed by detection of proteins via fluorescence. The hydrogel is formed by copolymerizing acrylamide / bisacrylamide with a designed monomer. In this example, the designed monomer comprises fluorescein (F) or fluorescein isothiocyanate (FITC), which is linked to a polymerizable group via a photolytically cleavable bond achieved using an o-nitrobenzyl (NVOC) group. The monomer polymerizable group is allylamide or methacrylamide. A polyethylene glycol (PEG) spacer arm may or may not be present between the NVOC and the polymerizable group. The inventors demonstrate that a 3400 g mol content in the hydrogel... -1 Methacrylamide monomer with molecular weight PEG spacer arm (F-NVOC-PEG) 3400 The incorporation rate of methylacrylamide (MAMA) is approximately 65%, which is twice that of allyl amide monomers without PEG. F-NVOC-PEG 3400 Other benefits offered by methylacrylamide include its water solubility, ease of preparation and high yield, and rapid phototriggered release kinetics.

[0591] Proteins are captured by reacting amines on proteins with isothiocyanate groups via FITC substitution of fluorescein in monomers and subsequently in a hydrogel. For exemplary proteins (streptavidin) at concentrations of 0.1 ppm and 0.01 ppm, the designed hydrogels provide pre-concentration factors of 192 and 236, respectively. Once pre-concentrated, the proteins are released by UV radiation, leaving free protein labeled with fluorescein. The inventors demonstrate that 50% of the streptavidin is released from our hydrogel within approximately 100 seconds, and each protein molecule is labeled with 85 fluorescein molecules. Labeling proteins with a large number of fluorophores allows for the determination of proteins present at low levels. Finally, the designed hydrogel, when combined with biotin-based capture of released streptavidin and fluorescence detection, allows for the detection of proteins at least 0.01 ppm (or approximately 166 pM).

[0592] This hydrogel shows promise for protein pre-concentration, labeling, and on-demand release. When combined with selective capture and fluorescence detection of labeled proteins, the hydrogel offers the potential to measure low-abundance proteins, thereby enabling early disease detection.

[0593] 3. Further polyacrylamide hydrogel experiments

[0594] Further examples were provided to optimize variables such as the length of the PEG chain of the active monomer, the ratio of active to inactive monomers, the incubation time with the protein solution, and the washing time for releasing proteins from the hydrogel after exposure to 365 nm light. Similarly, experimental protocols were developed to immobilize recognition elements (e.g., biotin and antibodies) in the wells of microtiter plates. The internally developed microtiter plates exhibited lower nonspecific adsorption of free FITC released from the hydrogel compared to commercially available biotin-coated microtiter plates. Furthermore, the polyacrylamide hydrogels of this invention were fabricated into films on glass substrates as previously described, but also into circular tablets, and were shown to be able to be stored in buffer solution for at least 7 days without any adverse effects.

[0595] Further examples demonstrate that the polyacrylamide hydrogel of the present invention can pre-concentrate proteins by approximately 295-fold. Proteins captured by the hydrogel can be released by irradiation with 365 nm light within <10 min. The released proteins can be specifically detected and quantified using microtiter plates coated with recognition elements (e.g., biotin, anti-CRP, and anti-IL8). Specific detection of proteins (e.g., streptavidin, CRP, and IL8) has been demonstrated, and calibration curves for streptavidin and CRP have been developed to show that they can be quantified. The limits of detection (LODs) for streptavidin and CRP are 0.0033 ppm (or 60 pM) and 0.0022 ppm (or 19 pM), respectively. These LODs for proteins obtained using the hydrogel of the present invention are comparable to those of commercially available colorimetric ELISA kits [Analyst, 2014, 139, 439]. The present invention offers the additional advantage that the hydrogel does not require a secondary antibody, enzyme labeling, and substrate, nor multiple addition / washing steps.

[0596] The further examples are described in detail below. Unless otherwise stated, the materials and methods used are the same as those described in Section 1 above.

[0597] 3.1 Monomer Synthesis and Purification

[0598] As mentioned in Section 1.2 above and Figure 2 The preparation of F-NVOC-allylamide and F-NVOC-PEG is described. 400 - Methacrylamide, F-NVOC-PEG 3400 - Methacrylamide and FITC-NVOC-PEG3400 1-Methacrylamide monomer.

[0599] 3.2 Composition of the precursor solution used to prepare the hydrogel

[0600] Use a commercially available 40% (w:v) acrylamide:bisacrylamide solution as a stock solution. The weight ratio of acrylamide to bisacrylamide is 29:1. Mix the required amount of acrylamide:bisacrylamide stock solution with the active monomer (see Table 1.1 for details) and add ultrapure water. The total concentration of crosslinking agent and monomer in the precursor solution is 5% (w:v). Add the required amounts of APS and TEMED shown in Table 1.1 and mix the solution thoroughly. This solution is used to prepare films or discs on glass slides using the procedure described below.

[0601] If the total concentration of crosslinking agent and monomer is reduced to 2.5% (w:v), the solution becomes viscous after the addition of APS and TEMED, but no hydrogel forms. If the total concentration of crosslinking agent and monomer is increased to 10% (w:v), a hydrogel forms after the addition of APS and TEMED. Therefore, we investigated hydrogels prepared from 5% and 10% (w:v) precursor solutions.

[0602]

[0603] Table 3: Composition of 1 mL precursor solution used to prepare polyacrylamide hydrogel (total concentration of crosslinking agent and monomer is 5% (w:v))

[0604] 3.3 Hydrogel films and round tablets

[0605] Hydrogel films were prepared using a method similar to that used in Section 1.3. Hydrogel discs were prepared as follows: 2 mL of the selected precursor solution (see Table 3) was poured into a mold to form a 1.2 mm thick hydrogel sheet. Discs were punched out of the hydrogel sheet using a cork punch. The discs had a diameter of 6 mm and a thickness of 1.2 mm. The discs were washed with 5 mL of PBS (20°C, overnight) and stored in PBS in the dark until use.

[0606] 3.4 Biotin-coated microtiter plates

[0607] Some studies were conducted using commercially available biotin-coated microtiter plates (15151, Thermo Fisher Scientific). The procedure for preparing the internally biotin-coated microtiter plates was as follows: 1.5% chitosan was prepared by dissolving 0.015 g of chitosan in 1 mL of 0.1 M acetic acid. The solution was stirred overnight. 300 µL of the solution was pipetted into 48-well microtiter plates and dried in an oven at 75 °C for 2 h. 200 µL of 10 mM PBS (pH 7.4) was pipetted into each well, removed, and this process was repeated five times. Then, 200 µL of 20 mg / mL NHS-PEG-biotin PBS solution was added to each well and allowed to react for 3 h. Subsequently, 200 µL of 20 mg / mL or 200 mg / mL NHS-PEG-methyl PBS solution was added to each well and allowed to react for 3 h. The NHS groups react with the amines in chitosan, thus the procedure allows biotin fixation via amines deposited in the chitosan within the pores, followed by blocking of any remaining amine groups in the chitosan with PEG-methyl groups. Finally, the pores are washed with PBS to remove any unreacted NHS-PEG-biotin and NHS-PEG-methyl groups.

[0608] 3.5 Preliminary Study

[0609] 3.5.1 Fluorescein release kinetics using monomeric solutions and chromatography

[0610] Studying the release kinetics of FITC is important because it determines the release rate of proteins captured in our hydrogel via the isothiocyanate groups of FITC. We investigated the release kinetics of FITC by: (1) exposing monomer solutions to 365 nm light for different durations and analyzing the solutions using liquid chromatography; and (2) exposing hydrogel discs to 365 nm light for different durations and analyzing them using fluorescence spectroscopy. Details are provided below.

[0611] A stock solution of 1 mg / mL active monomer was prepared in acetonitrile (MeCN) and exposed to 365 nm light for a selected duration. Then, 20 µL of the solution was collected. This procedure was repeated to expose the solution for up to 30 min. All collected solutions were analyzed sequentially using reversed-phase high-performance liquid chromatography (RP-HPLC). RP-HPLC was used because after exposure of the active monomer solution to 365 nm light, the active monomer contains both released fluorescein and fluorescein bound to the monomer. Since the released fluorescein and the fluorescein bound to the monomer absorb at the same wavelength, they must be separated to determine their respective amounts.

[0612] For RP-HPLC, a C18 column was used as the stationary phase. The mobile phase gradient was varied from 10 / 90 to 90 / 10 MeCN / H2O from 0 min to 10 min. A UV detector was used, and absorbance was recorded at 491 nm. Active monomer variants (F-NVOC-allylamide, F-NVOC-PEG) were also used. 400 - Methacrylamide and F-NVOC-PEG 3400 The obtained chromatogram of (-methacrylamide) is in Figure 13 and Figure 4 Provided in (a). The peaks corresponding to the released fluorescein and the fluorescein bound to the monomer are labeled F and M, respectively. The disappearance rate of the fluorescein bound to the monomer ([M]) is monitored. t To study the phototriggered release kinetics of luciferin. ln[M]0 / [M] t Plotting of relative exposure time (see) Figure 4 (b) shows excellent linearity, indicating the expected first-order kinetics. Furthermore, Figure 4 The rate plot provided in (b) shows that the kinetics of phototriggered release of fluorescein from the PEG-methacrylamide monomer are faster than those from the allylamide monomer. Fluorescein release from F-NVOC-allylamide and F-NVOC-PEG... 400 - Methacrylamide and F-NVOC-PEG 3400 The time constants for the release kinetics of 1,4-methacrylamide were 9.1 min, 6.8 min, and 4.8 min, respectively.

[0613] 3.5.2 Fluorescein release kinetics, absorbance, and fluorescence spectroscopy using hydrogel films

[0614] A hydrogel film containing a 1:10 molar ratio of active to inactive monomers was deposited on a glass slide. The total concentration of crosslinking agent and monomers was 10% (w:v). The hydrogel film was immersed in fresh PBS and exposed to 365 nm light for a selected time. The gel was left in PBS for 10 min, and then the absorbance of the hydrogel film was measured. This process was repeated to expose the hydrogel discs for up to 60 min.

[0615] Figure 6 (a) shows the decrease in the peak absorbance of fluorescein in the hydrogel film (at approximately 490 nm) with increasing exposure time to 365 nm light. A plot of peak absorbance relative to exposure time (see [link to plot]). Figure 6 (The illustration in (a)) shows the exponential decay to a minimum. Figure 6(b) provides a semi-logarithmic plot of the decrease in fluorescein concentration in the hydrogel relative to exposure time, indicating that the release is first-order with an exposure duration of up to 30 min. Similar results were obtained for other prepared hydrogels, showing first-order release kinetics with exposure durations up to 30 min. This indicates that once fluorescein is released, it diffuses rapidly out of the hydrogel, but the nonlinearity after approximately 30 min of exposure suggests that a small amount of irreversibly bound fluorescein may remain in the hydrogel. Fluorescein was extracted from F-NVOC-PEG-containing hydrogels. 3400 The time constant for the release kinetics of 1,4-methacrylamide in hydrogels is 10.6 min.

[0616] 3.5.3 Effect of PEG length in reactive monomers

[0617] F-NVOC-PEG 3400 -Methacrylamide can be produced in high yield (59%, compared to F-NVOC-PEG). 400 The preparation of methylacrylamide (at only 35%) does not require purification by cumbersome methods (rapid chromatography). Furthermore, as... Figure 4 As shown in (b), fluorescein in F-NVOC-PEG 3400 The release kinetics of methylacrylamide were fastest in solution. Finally, F-NVOC-PEG showed the fastest release kinetics relative to the precursor solution. 3400 -Methacrylamide incorporated into the hydrogel has a higher percentage of monomers than F-NVOC-allylamide (65% and approximately 32%, respectively, see Table 4 and ...). Figure 5 d). Considering F-NVOC-PEG 3400 These benefits are provided by methacrylamide, using (F or FITC)-NVOC-PEG. 3400 -Methacrylamide is used for the remaining work.

[0618]

[0619] Table 4: Summary of the percentage of light-labile monomers incorporated into hydrogel films (where the total concentration of monomers in the precursor solution is 10% w:v).

[0620] 3.5.4 Reproducibility and storage stability of hydrogels

[0621] Hydrogel films prepared using a 10% (w:v) precursor solution were washed overnight in 10 mL PBS to remove any monomers not incorporated into the hydrogel. A comparison of the absorption spectra of the four films after overnight washing in PBS was conducted. Figure 17 (a) indicates that the regenerability of the films is excellent. The four films were stored in PBS for different durations (1 day, 3 days, 5 days and 7 days), and their absorption spectra were then measured. Figure 17 (a) and Figure 17 The comparison in (b) shows that the absorbance spectrum of the film did not change significantly, indicating that the film has a storage stability of at least 7 days in PBS.

[0622] Similar experiments were also conducted on hydrogel films prepared using a 5% (w:v) precursor solution. Results were... Figure 18 The results showed that the membranes exhibited excellent regenerability and could be stored in PBS for at least 7 days. Figure 17 and Figure 18 The comparison highlights that the peak absorbance of the hydrogel film prepared using the 10% (w:v) precursor solution is significantly lower than that of the hydrogel film prepared using the 5% (w:v) precursor solution by about two times. This indicates that F-NVOC-PEG... 3400 - Methacrylamide is better incorporated into films prepared using a 5% (w:v) precursor solution. Furthermore, hydrogels prepared with a 5% (w:v) precursor solution are expected to have a less dense cohesion than hydrogels prepared using a 10% (w:v) precursor solution. For these reasons, the remaining work was carried out using hydrogels prepared with a 5% (w:v) precursor solution.

[0623] 3.5.5 Hydrogel Forms - Thin Films vs. Discs

[0624] Hydrogel films deposited on glass slides are not ideal for placement in the mouth. Therefore, we fabricated hydrogel discs of similar size to Mentos. For this purpose, we fabricated discs approximately 25 × 25 mm in size with a plastic base and a depth of 1.2 mm. 2 The cubic cavity is secured by a top plastic section with two through-holes for the fluid inlet and outlet. The top and bottom plastic sections are clamped together with screws, and the precursor solution is then introduced into the cavity through the inlet in the top plastic section. The precursor solution is left in the cavity to polymerize, and discs with a diameter of 6 mm are punched out using a cork drill. The resulting discs have sufficient mechanical strength to allow them to be easily transferred from one solution to another using a scraper. Furthermore, using discs provides the added benefit of releasing proteins captured in the hydrogel in a smaller volume of buffer (200 μL for discs vs. 3 mL for films). The smaller the volume of buffer used to release the proteins captured in our hydrogel, the higher the resulting pre-concentration factor. Therefore, hydrogel discs are used for the remaining work.

[0625] 3.5.6 Pre-concentration factor

[0626] To determine the pre-concentration factor, we prepared 10 mL of approximately 0.005 ppm RS solution and measured its fluorescence spectrum (black trace). Figure 19(a)). Then, we immersed the hydrogel discs in an unstirred RS solution for 24 hours and measured the fluorescence spectrum of the solution (red trace, Figure 19 (a)). Using the calibration curve of RS (in Figure 19 (b) As shown, we converted the peak fluorescence intensity of the RS solution before and after incubation with the hydrogel discs to determine the RS captured by the hydrogel discs. We found that 85.6% of the RS was captured by our hydrogel after 24 hours of incubation. Since the volume of the hydrogel discs is much smaller than that of the RS solution, the concentration of RS in the hydrogel discs was determined to be 1.2615 ppm (compared to 0.00428 ppm in the solution). Therefore, the pre-concentration factor was approximately 295. The protein was released by exposing the hydrogel discs to 365 nm light while immersing them in 200 μL of PBS. Therefore, the pre-concentration factor for protein release was approximately 50.

[0627] 3.6 Optimization Research

[0628] 3.6.1 Effect of the ratio of active to inactive monomers in hydrogels

[0629] Hydrogels were prepared using a 5% (w:v) precursor solution. The molar ratio of active to inactive monomers varied between 1:10, 1:20, 1:40, and 1:100. Hydrogel discs were incubated in 10 mL of 0.005 ppm RS in PBS for 24 hours, washed overnight in PBS, and then exposed to 365 nm light for a total of 60 min. After each exposure, the supernatant was collected, and the fluorescence at the peak wavelength was plotted relative to the exposure time. Figure 20 The time constants for protein release kinetics do not follow a pattern. For active to inactive monomer ratios of 1:10, 1:20, 1:40, and 1:100, the time constants are 2.97 min, 2.03 min, 2.73 min, and 2.03 min, respectively. However, the signal at a monomer ratio of 1:40 is much higher than that at 1:10, 1:20, or 1:100. This is because at lower monomer ratios, proteins diffused into the hydrogel interior are captured by multiple FITC groups, all of which must be broken to release the labeled protein. For a monomer ratio of 1:100, the lower concentration of FITC means that less protein is captured, but the protein is released rapidly because fewer bonds need to be broken. Conversely, proteins on the hydrogel surface will be captured by less FITC, allowing for rapid release, but at a much lower concentration.

[0630] 3.6.2 Effect of hydrogel incubation time in protein solution

[0631] Based on previous experiments, a molar ratio of 1:40 for active and inactive monomers was selected for subsequent work. 10 mL of 0.1 ppm RS was incubated on hydrogel discs for 1 h, 15 h, 24 h, and 48 h. The hydrogel discs were then exposed to 365 nm light for a total of 60 min. Results were obtained in… Figure 21 In practice, for 0.1 ppm RS, the signal after 48 hours was only slightly higher than after 24 hours, therefore, for subsequent work, the maximum incubation time was limited to 24 hours. Similar studies were conducted with 0.01 ppm and 0.005 ppm RS solutions, but the maximum incubation time was limited to 24 hours. After the selected incubation time, proteins were released from the hydrogel by exposure to 365 nm light, and the fluorescence spectra of the supernatant were measured. After 24 hours of incubation in 0.01 ppm and 0.005 ppm RS solutions, the peak fluorescence of the hydrogel discs compared to the exposure time was plotted in [the graphs]. Figure 22 and Figure 23 It is displayed in the middle. Similar to Figure 21 , Figure 23 It was clearly shown that the fluorescence signal increased significantly as the incubation time of the hydrogel discs in RS solution increased from 15 h to 24 h. Therefore, all subsequent operations were performed within the 24-hour incubation period.

[0632] also, Figures 21 to 23 The comparison showed that, for the selected incubation time, the fluorescence signal increased with the concentration of the RS solution. This is promising because it means that our hydrogel can be used for quantitative determination.

[0633] 3.6.3 Commercially prepared biotin-coated microtiter plates compared to in-house prepared ones

[0634] Next, we investigated the potential of PEG hydrogels for determining proteins not pre-labeled with rhodamine. Upon incubation with a protein solution, exposure of the hydrogel to 365 nm light released both free FITC and FITC-labeled proteins. Therefore, to determine protein concentration, free FITC must be removed. One way to do this is to use microtiter plates with wells coated with the recognition element to capture the protein (e.g., biotin-coated microtiter plates for capturing streptavidin). We used commercially available biotin-coated microtiter plates and performed a control experiment where hydrogel discs with a monomer molar ratio of 1:40 were washed overnight in PBS and then exposed to 365 nm light for up to 60 min, with consecutive 30-minute exposures. Released FITC was collected in 200 µL of PBS. 200 µL of sample was pipetteed into the wells of the commercially available biotinylated microtiter plate, allowed to incubate for 30 min, and then rinsed with PBS. The fluorescence spectrum of each well was recorded using a microtiter plate reader. Figure 24The peak fluorescence intensity is shown as a function of exposure time. Released FITC reacted strongly with the biotinylated plate, producing a very high fluorescence background. For this reason, we developed our own low-FITC-binding biotinylated microtiter plate. 200 µL of a 1.5% solution of chitosan in 0.1 M acetic acid was pipetted into the wells of the microtiter plate and dried at 50 °C for 2 h. Each well was then treated with 200 µL of 25 mg / mL NHS-PEG-Biotin in PBS, followed by treatment with 200 µL of 25 mg / mL or 250 mg / mL NHS-PEG-Methyl in PBS to block any unreacted amines. Subsequently, the same procedure as with commercial plates was followed for direct comparison. The results of FITC release were shown in... Figure 25 The results show that, compared to commercially available biotinylated microtiter plates, higher concentrations of the blocking agent reduced the FITC signal by 110 times.

[0635] 3.6.4 Effect of washing time after exposure to 365nm light

[0636] Polyacrylamide hydrogel discs with a molar ratio of active monomer to inactive monomer of 1:40 were incubated in 10 mL of 0.005 ppm streptavidin in PBS for 24 hours, followed by washing in PBS overnight. The discs were then exposed to 365 nm light for a total of 60 min. After each exposure, the discs were washed with 200 µL PBS for 10 min or 30 min to determine whether the washing time had any significant effect on the fluorescence signal of the released streptavidin. Figure 26 The washing time showed no significant effect on the streptavidin fluorescence signal. This means that the released protein can diffuse rapidly out of the hydrogel, so a washing time of 10 minutes is sufficient.

[0637] 3.7 Protein determination

[0638] 3.7.1 Determination of streptavidin

[0639] 10 mL of unlabeled streptavidin solutions at concentrations of 0 ppm, 0.0025 ppm, 0.005 ppm, 0.0075 ppm, and 0.01 ppm in PBS were incubated on hydrogel discs at a molar ratio of active to inactive monomer of 1:40 for 24 hours. The discs were then washed in PBS overnight and exposed to 365 nm light in 200 µL PBS for a total of 60 min. The released streptavidin and pre-labeled streptavidin solutions were placed in the wells of a microtiter plate containing biotinylated chitosan hydrogel and allowed to bind for 30 min. The streptavidin solution was then removed, and the chitosan hydrogel was washed with PBS. The fluorescence emission of the bound fluorescein-labeled streptavidin was then measured at an excitation wavelength of 470 nm using a microtiter plate reader. Figure 27 The results were summarized as a function of exposure time, showing that the fluorescence signal increases with increasing streptavidin concentration. At a 60-minute exposure time, the streptavidin calibration curve showed... Figure 27 (b) Display. Based on the calibration curve, the limit of detection (LOD) for streptavidin is 0.0033 ppm or 60 pM.

[0640] 3.7.2 Determination of C-Reactive Protein (CRP)

[0641] Using the same method as described above, CRP was captured, labeled, and released using a hydrogel with an active monomer to inactive monomer molar ratio of 1:40. Anti-CRP antibodies were immobilized onto chitosan in the wells of a microtiter plate using a procedure similar to that used for biotinylated wells. 1.5% chitosan was prepared by dissolving 0.015 g of chitosan in 1 mL of 0.1 M acetic acid. The solution was stirred overnight. 300 µL of the solution was pipetted into 48-well microtiter plates and oven-dried at 75 °C for 2 h. 200 µL of 10 mM PBS (pH 7.4) was pipetted into each well, removed, and this process was repeated five times. Then, 200 µL of a PBS solution of 20 mg / mL NHS-PEG-NHS was added to each well and allowed to react for 2 h. Subsequently, 200 µL of a PBS solution of 200 mg / mL NHS-PEG-methyl was added to each well and allowed to react for 2 h. The wells were washed with PBS to remove unreacted material, followed by adding 200 μL of 0.005 ppm anti-CRP PBS solution to each well and incubating for 2 h. The amines on the anti-CRP reacted with the free NHS groups bound to the chitosan to immobilize the antibody. Finally, the wells were washed with PBS to remove any unreacted NHS-PEG-biotin and NHS-PEG-methyl groups. Figure 28The results show that the fluorescence signal from the CRP-resistant microtiter plate is a function of exposure time for PBS solutions with initial CRP concentrations of 0 ppm, 0.0025 ppm, 0.005 ppm, 0.0075 ppm, and 0.01 ppm. The fluorescence signal increases with increasing CRP concentration. At a 60-minute exposure time, the CRP calibration curve shows... Figure 28 (b) Display. According to the calibration curve, the LOD of CRP is 0.0022 ppm or 19 pM.

[0642] 3.7.3 Measurement of interleukin-8 (IL8)

[0643] To demonstrate that our hydrogel is suitable for pre-concentrating and labeling any protein, we conducted experiments in which the same method as described above for anti-CRP was used, but with 0.005 ppm of anti-IL8, and IL8 was captured, labeled and released using a hydrogel with an active to inactive monomer molar ratio of 1:40. Figure 29 The fluorescence signal of an anti-IL8 coated microtiter plate as a function of light exposure time for IL8 at an initial concentration of 0.005 ppm in PBS is shown, confirming that IL8 can be measured.

[0644] 4. Poly(ethylene glycol) (PEG) hydrogel experiment

[0645] Further experiments were conducted to demonstrate the protein-binding function, and it was shown that poly(ethylene glycol) (PEG) can be used for protein concentration and labeling in addition to polyacrylamide hydrogels. For both hydrogels, the optimal values ​​for variables such as the ratio of active to inactive monomers and protein capture time were similar. Furthermore, PEG hydrogels were found to be well-suited for determining low concentrations of proteins in the presence of high concentrations of single and multiple interfering agents. This was demonstrated by determining an exemplary protein, streptavidin, at a concentration of 0.005 ppm in the presence of 0.5 ppm and 5 ppm of mucin. Additionally, it was demonstrated that IL6 as low as 0.005 ppm could be determined in synthetic saliva containing multiple interfering agents.

[0646] The experiments described below demonstrate that the functionality of binding to proteins, as well as the additional concentration and labeling of the proteins of this invention, can also be achieved using hydrogels composed of different types of monomers and cross-linking agents (e.g., using poly(ethylene glycol) (PEG)-based hydrogels). In this case, the cross-linking agent is a 4-arm PEGyne, the inactive monomer is a PEG bis(azide) compound, and the active monomer is a FITC-NVOC-PEG compound. 1000 -Azides. For example... Figure 30 As shown, the crosslinking agent was mixed with active and inactive monomers in the presence of copper sulfate (CuSO4) and sodium ascorbate to trigger the formation of PEG hydrogels.

[0647] The further examples are described in detail below. Unless otherwise stated, the materials and methods used are the same as those described in Section 1 above.

[0648] 4.1 Synthesis of Monomers

[0649] Synthesis of 4-arm PEGyne (crosslinking agent)

[0650] The synthesis of 4-arm PEGyne is adapted from the literature [RSC Advances, 2016, 6, 36568]. Figure 31 As shown, 4-arm PEG 2000 1 g (0.5 mmol, 1 equivalent), propargyl bromide (1.67 mL of 80% toluene solution, 15 mmol, 30 equivalent), and sodium hydroxide (NaOH) granules (0.6 g, 15 mmol, 30 equivalent) were added to 4 mL of toluene and stirred at 50 °C for 24 hours. The mixture was evaporated under vacuum, and the residue was dissolved in 100 mL of water. The solution was extracted with dichloromethane (3 × 50 mL), and the collected organic layer was dried over anhydrous Na₂SO₄ and concentrated. The final product (4-arm PEGyne (molecular weight: approx. 2050 g / mol)) was obtained by precipitation from cooled diethyl ether in a yield of 62% (652 mg, 0.311 mmol).

[0651] MALDI: C 97 H 180 O 44 Na + The measured m / z is 2073, n=11

[0652] 1 H NMR (300 MHz, CDCl3) δ 4.20 (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).

[0653] Synthesis of PEG bis(azide) (inactive monomer)

[0654] The synthesis of PEG bisazides is adapted from the literature [Polymers, 2021, 13, 1403]. For example... Figure 32 As shown, under an argon gas flow, in a double-necked RBF, PEG is... 33505 g (1.493 mmol, 1 equivalent) and triethylamine (TEA) (1.65 mL, 11.94 mmol, 8 equivalents) were dissolved in 25 mL of DCM (5 mL / 1 g polymer). The mixture was cooled in an ice bath, and methanesulfonyl chloride (MsCl, 0.92 mL, 11.94 mmol, 8 equivalents) 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 with 250 mL of ultrapure water (10 mL / 5 mL 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, the organic phase was dried with MgSO4, and then filtered under vacuum using a sintered funnel. The filtrate was concentrated using a rotary evaporator to obtain methanesulfonate-terminated PEG.

[0655] In a round-bottom flask, methanesulfonate-terminated PEG was dissolved in 25 mL of DMF (5 mL / g polymer), and sodium azide (NaN3) (971 mg, 14.93 mmol, 10 equivalents) was added. The mixture was heated to 65 °C for 16 h. After cooling to 20 °C, the mixture was filtered, and insoluble substances were washed with 10 mL of ethanol. The filtrate was concentrated using a rotary evaporator, and the resulting residue was dissolved in ultrapure water (5 mL / g polymer). The aqueous solution was extracted with DCM (3 × 10 mL), and the combined organic extracts were dried (MgSO4) and filtered under vacuum using a sintered funnel. The filtrate (PEG) was... 3350 The diazide (molecular weight: approximately 3416 g / mol) was concentrated using a rotary evaporator to obtain a 52% yield (2.59 g, 0.774 mmol).

[0656] 1 H NMR (400 MHz, CDCl3) δ 3.64 (s, 300H), 3.41 – 3.37 (m, 4H).

[0657] FITC-NVOC-PEG 1000 Synthesis of azides (reactive monomers)

[0658] To prepare the active monomer, the starting materials described below need to be synthesized.

[0659] Synthesis of amine-PEG-azides

[0660] The synthesis of amine-PEG-azides is adapted from the literature [Polymers, 2021, 13, 1403 and ACS Chemical Neuroscience, 2018, 9, 100], see [link to original document]. Figure 33 PEG 10005000 mg (5 mmol, 1 equivalent) and TEA (5.5 mL, 40 mmol, 8 equivalent) were dissolved in 25 mL of DCM (5 mL / g polymer) in RBF packed with a diaphragm and purged with nitrogen. MsCl (3.1 mL, 40 mmol, 8 equivalent) 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 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 filtered under vacuum using a sintered funnel. The filtrate was concentrated using a rotary evaporator. NaN3 (3.251 mg, 50 mmol, 10 equivalent) and 25 mL of DMF were added to the residue and stirred (65 °C, 3 d), then cooled to 20 °C. The mixture was centrifuged (5000 rpm, 3 min), and the precipitate was washed with 10 mL of ethanol, concentrated using a rotary evaporator, dissolved in water (5 mL / g), and extracted with DCM. This process was repeated three times. The combined organic extracts were dried (Na2SO4) and concentrated using a rotary evaporator.

[0661] The polymer obtained by the above procedure was added to a round-bottom flask. Ethyl acetate (50 mL, 10 mL / 1 g polymer) and 1 M HCl (15 mL, 3 mL / 1 g polymer) were then added, and the flask was purged with nitrogen and cooled to 0 °C. Triphenylphosphine (PPh3, 1300 mg, 5 mmol, 1.1 equivalence) 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 (2 × 40 mL). Potassium hydroxide (KOH, 12.5 g, 2.5 g / 1 g polymer) granules were slowly added until dissolved to remove the generated triphenylphosphine oxide (TPPO). The aqueous solution was extracted with DCM (5 × 40 mL), and the combined organic extracts were dried (Na₂SO₄) and filtered under vacuum using a sintered funnel. The filtrate was concentrated using a rotary evaporator and then lyophilized (18 h). The filtrate was amine-PEG. 1000 - Azide (molecular weight: approximately 1055 g / mol), obtained in 66% yield (3.3 g, 3.3 mmol).

[0662] 1 H NMR (400 MHz, CDCl3) δ 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).

[0663] Synthesis of NVOC-PEG-Azide

[0664] like Figure 34 As shown, 4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy)butyric acid (NVOC, 150 mg, 0.5 mmol, 1 equivalent), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU, 228 mg, 0.6 mmol, 1.2 equivalent), and N,N-diisopropylethylamine (DIPEA, 425 μL, 2.5 mmol, 5 equivalent) were added to 5 mL of DMF, stirred at 0 °C, and purged with nitrogen. Amine-PEG-azide (500 mg, 0.5 mmol, 1 equivalent) was dissolved in a minimal amount of DMF and added dropwise to the reaction mixture. The reaction was heated to 20 °C and stirred for 18 h, then water (20 mL) was added and extracted with DCM (3 × 20 mL). The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated using a rotary evaporator to obtain NVOC-PEG. 1000 -Azide (molecular weight: approx. 1337 g / mol), yield 58% (386 mg, 0.288 mmol).

[0665] 1 H NMR (400 MHz, CDCl3) δ 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).

[0666] FITC-NVOC-PEG 1000 Synthesis of azides

[0667] like Figure 35 As shown, NVOC-PEG 1000- Azide (125 mg, 0.093 mmol, 1 equivalent) was dissolved in 0.9 mL of anhydrous THF and cooled to 0 °C. 6.6 mL of 15% phosgene (COCl2, 4.675 mmol, 100 equivalent) was added to the solution and stirred (20 °C, 18 h). Subsequently, nitrogen gas was bubbled into the solution (20 °C, 30 min) to evaporate the THF and unreacted COCl2. The residue was dissolved in anhydrous DCM (0.5 mL). Fibrocyanide (FITC, 36 mg, 0.093 mmol, 1 equivalent) was dissolved in anhydrous DCM (1 mL), and then TEA (28 µL, 0.2 mmol, 1.6 equivalent) was added, and the reaction mixture was stirred (20 °C, 5 min). The first reaction mixture was added dropwise to the second reaction mixture over 30 min and stirred (0 °C to 20 °C, 12 h). DCM was then added to the reaction mixture, which was subsequently washed with NH4Cl and then with NaHCO3. The aqueous solution was extracted with DCM, washed with brine, dried over Na2SO4, filtered, and concentrated using a rotary evaporator at 30°C to obtain FITC-NVOC-PEG. 1000 - Azide (molecular weight: approx. 1752 g / mol), yield 63% (103 mg, 0.0588 mmol)

[0668] 1 H NMR (400 MHz, CDCl3) δ 7.81 (s, 1H), 7.60 (s, 1H), 7.50 (s, 1H), 7.07 (d, J = 18.3 Hz, 1H), 6.84 (d, J = 54.2 Hz, 3H), 6.60 (s, 1H), 1.76 (d, J = 6.4 Hz, 2H).

[0669] 13 C NMR (400 MHz, DMSO-d6) δ 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.

[0670] 4.2 Formation of PEG hydrogel

[0671] Add the 4-arm PEGyne, PEG bisazide, and FITC-NVOC-PEG-azide monomers to the vials in the required amounts (see Table X). Then add ultrapure water. The total concentration of crosslinking agent and monomers in the solution used to prepare the PEG hydrogel is 5% (w:v). Add appropriate volumes of stock solutions of copper sulfate pentahydrate (CuSO4) and sodium ascorbate as described in Table 5. Vortex the reaction mixture and pour it into a mold to form a 1.2 mm thick hydrogel sheet (20 °C, 3 h). Punch discs from the hydrogel sheet using a cork punch. The discs have a diameter of 6 mm and a thickness of 1.2 mm. Wash the discs with 5 mL of 10 mM ethylenediaminetetraacetic acid (EDTA) (20 °C, 6 h) and then with 5 mL of PBS (20 °C, overnight). Store the discs in PBS protected from light until use.

[0672] If the total crosslinking agent and monomer concentration is reduced to 2.5% (w:v), the solution becomes viscous after adding CuSO4 and sodium ascorbate, but no hydrogel forms. If the total crosslinking agent and monomer concentration is increased to 10% (w:v), a hydrogel forms after adding CuSO4 and sodium ascorbate. However, we use hydrogels prepared with a 5% (w:v) crosslinking agent and monomer concentration because these hydrogels are expected to be less tightly bound and therefore have more pores than those prepared with a 10% (w:v) crosslinking agent and monomer concentration.

[0673]

[0674] Table 5: Composition of the 2 mL precursor solution used to prepare PEG hydrogel (total crosslinking agent and monomer concentration: 5% (w:v))

[0675] 4.3 Preliminary Study

[0676] 4.3.1 FITC Release Kinetics

[0677] When exposed to 365nm light, FITC can be released from the active monomer (see [reference]). Figure 36 ).

[0678] Studying the release kinetics of FITC is important because it determines the release rate of proteins captured in our hydrogel via the isothiocyanate groups of FITC. We investigated the release kinetics of FITC by exposing hydrogel discs to 365 nm light for different durations and analyzing the results using fluorescence spectroscopy. Hydrogel discs containing a molar ratio of active to inactive monomers of 1:10 were used. The total crosslinking agent and monomer concentration was 5% (w:v). The hydrogel discs were immersed in 200 μL of fresh PBS and exposed to 365 nm light for selected times. The gels were left in PBS for 10 min, followed by collection of the buffer and measurement of fluorescence. This process was repeated to expose the hydrogel discs for up to 60 min. Figure 37 (a) provides fluorescence spectra of PBS used to immerse gels for different exposure times. The fluorescence intensity at the peak wavelength was then plotted as a function of exposure time to obtain... Figure 37 (b), where the solid line is the best fit of the three parameters exponentially rising to the maximum value (r = 0.9952) to determine the time constant, which is approximately 2 min.

[0679] 4.4 Optimization Research

[0680] To determine the optimal incubation time of the hydrogel in the protein solution and the ratio of active to inactive monomers in the PEG hydrogel, we used streptavidin already labeled with rhodamine (RS). This allowed us to perform these optimization studies by measuring the fluorescence of rhodamine, without relying on FITC-labeled proteins released from our hydrogel.

[0681] 4.4.1 Effect of incubation time of hydrogel in protein solution

[0682] Incubation time is the time required for the protein to be captured by FITC in the hydrogel. Hydrogel discs containing an active monomer and an inactive monomer in a molar ratio of 1:10 were used. The hydrogel discs were incubated in 10 mL of 0.005 ppm RS solution for different durations (1 h, 15 h, and 24 h). Subsequently, the hydrogel discs were washed overnight, then immersed in 200 μL of fresh PBS and exposed to 365 nm light for a given exposure time. The gel was left in PBS for 10 min, then the buffer was collected, and fluorescence was measured. This procedure was repeated to expose the hydrogel discs for a total of up to 60 min. Figure 38 The image provides fluorescence spectra of PBS used to immerse gels for different exposure times. The fluorescence intensity at the peak wavelength is plotted as a function of exposure time, and plotted to obtain... Figure 39 , Figure 39The results show that, as can be expected, the longest protein incubation time results in the largest fluorescence signal from the released labeled protein.

[0683] Therefore, a 24-hour incubation time was used in the protein solution for the remainder of the work. The data were fitted to a three-parameter exponential rise to its maximum (r = 0.9958) to determine the time constant, which was approximately 4 min for 1 h incubation and approximately 1.7 min for both 15 h and 24 h incubation. Overall, the release kinetics of RS from the hydrogel were comparable to those of FITC from the hydrogel, as expected.

[0684] 4.4.2 Effect of the ratio of active to inactive monomers in hydrogels

[0685] The molar ratio of active to inactive monomers varied from 1:10, 1:20, 1:40, and 1:100. Hydrogel discs were incubated in 10 mL of 0.005 ppm RS in PBS solution for 24 hours, washed overnight in PBS, and then exposed to 365 nm light for a total of 60 min. After each exposure, the supernatant was collected, and the fluorescence at the peak wavelength was plotted relative to the exposure time. Figure 40 The results show that the release time constants of RS were similar across hydrogels containing different ratios of inactive to reactive monomers (1.8 min, 1.7 min, 1.7 min, and 2.3 min for reactive to inactive monomer ratios of 1:10, 1:20, 1:40, and 1:100, respectively). However, the signal at a monomer ratio of 1:40 was significantly higher than that at ratios of 1:10, 1:20, or 1:100. This is because at lower monomer ratios, proteins diffusing into the hydrogel interior are captured by multiple FITC groups, all of which must be destroyed to release the labeled protein. Conversely, proteins on the hydrogel surface are captured by fewer FITC groups, allowing for rapid release, but at a much lower concentration.

[0686] 4.5 Protein determination

[0687] Next, we investigated the potential of PEG hydrogels for protein assays. For this purpose, we first used an exemplary protein—streptavidin—and then interleukin-6 (IL6), considered an important biomarker for oral cancer detection. Furthermore, we investigated the effect of interfering agents on the hydrogel's protein assay performance. In the first case, we used mucin as an interfering agent because mucin is abundant in saliva and is therefore a key interfering agent for the assay of salivary protein biomarkers for oral cancer detection. Subsequently, we investigated the applicability of our hydrogel for the assay of IL6 in synthetic saliva. For the selective detection of proteins (i.e., streptavidin and IL6 released from the PEG hydrogel), we used microtiter plates coated with biotin and anti-interleukin-6 (anti-IL6), respectively. Since the proteins are labeled with FITC upon release from the PEG hydrogel, after capturing the selected proteins in the wells of the microtiter plate, these selected proteins were measured using a microtiter plate reader at an excitation wavelength of 470 nm.

[0688] 4.5.1 Determination of streptavidin

[0689] Incubate the hydrogel in one of the following solutions for 24 hours:

[0690] • 0.005 ppm streptavidin (without mucin) (positive control)

[0691] • 0.005 ppm streptavidin containing 0.5 ppm mucin (positive control containing 100× interferon)

[0692] • 0.005 ppm streptavidin containing 5 ppm mucin (positive control containing 1000× interferon)

[0693] • Buffer solution, i.e., streptavidin-free and mucin-free (negative control)

[0694] • 5 ppm mucin (without streptavidin) (negative control, containing 1000× interferon)

[0695] The hydrogel was washed in PBS overnight and then exposed to 365 nm light for a total of 60 min. The supernatant was allowed to incubate in the wells of a biotinylated microtiter plate for 30 min, followed by washing with PBS to remove any unbound material, and the fluorescence of each well was measured. Figure 41 The fluorescence signal of the five solutions as a function of exposure time is shown. The fluorescence intensity of wells treated with the supernatant obtained after exposing the hydrogel to 365 nm light was compared, where the hydrogel was pre-incubated in buffer and 0.005 ppm streptavidin (i.e., Figure 41The black and red lines in the image clearly demonstrate that our hydrogel is well-suited for determining streptavidin at concentrations of at least 0.005 ppm (or approximately 90 pM). The fluorescence intensity of wells treated with the supernatant of the hydrogel, pre-incubated in 5 ppm mucin at a 365 nm exposure, was comparable to that of the buffer (i.e., Figure 41 The blue and red traces in the image indicate that the interfering mucin is undetectable. Finally, the comparison of the supernatants of hydrogels pre-incubated with streptavidin containing mucin at 365 nm exposure is comparable. Figure 41 The black, magenta, and dark yellow traces in the image indicate that our PEG hydrogel is well-suited for determining streptavidin in the presence of interfering substances.

[0696] 4.5.2 Measurement of interleukin-6 (IL6)

[0697] Incubate the hydrogel in one of the following solutions for 24 hours:

[0698] • Buffer solution (negative control)

[0699] • Synthetic saliva (negative control containing interferon)

[0700] • 0.005ppm IL6 buffer (positive control)

[0701] • Synthetic saliva containing 0.005 ppm IL6 (positive control containing interferon)

[0702] The hydrogel was washed in PBS overnight and then exposed to 365 nm light for a total of 60 min. The supernatant was incubated in the wells of an anti-IL6 coated microtiter plate for 30 min, followed by washing with PBS to remove any unbound material, and the fluorescence of each well was measured. Figure 42 The fluorescence signal of four solutions as a function of exposure time is shown. The fluorescence intensity of wells treated with the supernatant obtained after exposing the hydrogel to 365 nm light was compared, where the hydrogel was pre-incubated in buffer and 0.005 ppm IL6 (i.e., Figure 42 The black and blue lines (represented by the black and blue lines respectively) clearly demonstrate that our hydrogel is well-suited for determining IL-6 levels of at least 0.005 ppm. The fluorescence intensity of wells treated with the supernatant of the hydrogel, pre-incubated in synthetic saliva and exposed to 365 nm, was comparable to that of the buffer (i.e., Figure 42 The black lines and red marks in the image indicate that the synthetic saliva had no effect on our hydrogel. Finally, the supernatant of the hydrogels exposed to 365 nm after pre-incubation in IL6 buffer and synthetic saliva was comparable. Figure 42The blue and dark yellow lines in the image indicate that our PEG hydrogel is well-suited for determining IL6 in synthetic saliva.

[0703] References:

[0704] (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.

[0705] (2) M, A.; A., F. Enzyme linked immunosorbent assay; StatPearlsPublishing, 2023.

[0706] (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.

[0707] (4) Chen, P.;huang, N.-T.; Chung, M.-T.; Cornell, TT; Kurabayashi,K. Label-free cytokine micro-and nano-biosensing towards personalized medicine of systemic inflammatory disorders. Advanced Drug Delivery Reviews2015, 95, 90.

[0708] (5) Goddard, N. J.; Gupta, R. A novel manifestation at optical leakywaveguide modes for sensing applications. Sensors and Actuators B 2020, 309,127776. DOI: 10.1016 / j.snb.2020.127776.

[0709] (6) Gupta, R.; Goddard, N. J. Leaky waveguides (LWs) for chemical andbiological sensing − a review and future perspective. Sensors and ActuatorsB-Chemical 2020, 322, 128628. DOI:

[0710] 10.1016 / j.snb.2020.128628.

[0711] (7) Chen, C.; Wand, J. Optical biosensors: an exhaustive andcomprehensive review. Analyst 2020, 145, 1605.

[0712] (8) Bahadir, E. B.; Sezgintürk, M. F. A review on impedimetricbiosensors. Artificial Cells,

[0713] Nanomedicine, and Biotechnology 2016, 44, 248.

[0714] (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 impedancebiosensors with microfluidic chips for single-cell analysis. Biosensors 2021,11, 412.

[0715] (10) Alanazi, N.; Almutairi, M.; Alodhayb, A. N. A review of quartzcrystal microbalance for chemical and biological sensing applications.Sensing and Imaging 2023, 24, 10.

[0716] (11) Lim,h. J.; Saha, T.; Tey, B. T.; Tan, W. S.; OOi, C. W. Quartzcrystal microbalance-based

[0717] biosensors as rapid diagnostic devices for infectious diseases.Biosensors and Bioelectronics 2020, 168, 112513.

[0718] (12) Abbate, G.; Bernini, U.; Ragozzino, E.; Somma, F. Thetemperature dependence of the refractive index of water. Journal of PhysicsD: Applied Physics 1978, 11, 1167.

[0719] (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 forultrasensitive microfluidic immunoassays of cancer biomarkers. Biosensors andBioelectronics 2014, 53, 268.

[0720] (14) Osbourn, D. M.; Weiss, D. J.; Lunte, C. E. On-linepreconcentration methods for capillary

[0721] electrophoresis. Electrophoresis 2000, 21, 2768.

[0722] (15) Goddard, N. J.; Gupta, R. Speed and sensitivity - integration ofelectrokinetic preconcentration with a leaky waveguide biosensor. Sensors andActuators B-Chemical 2019, 301, 127063. DOI: 10.1016 / j.snb.2019.127063.

[0723] (16) Gupta, R.; Baldock, S. J.; Fielden, P. R.; Prest, J. E.; Grieve,B. D. Isotachophoresis-based sample preparation of cellulases in sugarcanejuice using bovine serum albumin as a model protein. Journal ofChromatography A 2010, 1217, 8026.

[0724] (17) Twining, S. S. Fluorescein isothiocyanate-labeled casein assayfor proteolytic enzymes. Analytical biochemistry 1984, 143 (1), 30-34.

[0725] (18)hungerford, G.; Benesch, J.; Mano, J. F.; Reis, R. L. Effect ofthe labelling ratio on the photophysics of fluorescein isothiocyanate (FITC)conjugated to bovine serum albumin. Photochemical & Photobiological Sciences2007, 6 (2), 152-158.

[0726] (19) Xing, Y.; Zeng, B.; Yang, W. Light responsivehydrogels forcontrolled drug delivery. Frontiers in Bioengineering and Biotechnology 2022,10.

[0727] (20) Kim, M. S.; Diamond, S. L. Photocleavage of o-nitrobenzyl etherderivatives for rapid biomedical release applications. Bioorganic & medicinalchemistry letters 2006, 16 (15), 4007-4010.

[0728] (21) Zhao,h.; Sterner, E. S.; Coughlin, E. B.; Theato, P. o-Nitrobenzyl alcohol derivatives: Opportunities in polymer and materialsscience. Macromolecules 2012, 45 (4), 1723-1736.

[0729] (22) Kloxin, A. M.; Kasko, A. M.; Salinas, C. N.; Anseth, K. S.Photodegradablehydrogels for dynamic tuning of physical and chemicalproperties. Science 2009, 324 (5923), 59-63.

[0730] (23) Thomas III, S. W. New applications of photolabile nitrobenzylgroups in polymers. Macromolecular Chemistry and Physics 2012, 213 (23),2443-2449.

[0731] (24) Rosales, A. M.; Vega, S. L.; DelRio, F. W.; Burdick, J. A.;Anseth, K. S.hydrogels with reversible mechanics to probe dynamic cellmicroenvironments. Angewandte Chemie International Edition 2017, 56 (40),12132-12136.

[0732] (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- induceddegradation. Macromolecules 2011, 44 (24), 9758-9772.

[0733] (26) Pal, A. K.; Labella, E.; Goddard, N. J.; Gupta, R.Photofunctionalizablehydrogel for fabricating volume optical diffractivesensors. Macromolecular Chemistry and Physics 2019, 220 (16), 1900228.

[0734] (27) Gupta, R.; El Sayed, S.; Goddard, N. J.hydrogel gratings withpatterned analyte responsive dyes for spectroscopic sensing. RSC Advances2021, 11, 40197.

[0735] (28) holmes, C. P. Model studies for new o-nitrobenzyl photolabilelinkers: Substituent effects on the rates of photochemical cleavage. TheJournal of organic chemistry 1997, 62 (8), 2370-2380.

[0736] (29) Mathew, T.; Ajayaghosh, A.; Das, S.; Kamat, P. V.; George, M. Anew photodegradable polyamide containing o-nitrobenzyl chromophore. Steadystate and laser flash photolysis studies. Journal of Photochemistry andPhotobiology A: Chemistry 1993, 71 (2), 181-189.

[0737] (30) Sternson, S. M.; Schreiber, S. L. An acid-and base-stable o-nitrobenzyl photolabile linker for solid phase organic synthesis. Tetrahedronletters 1998, 39 (41), 7451-7454.

[0738] (31) Buchmeiser, M. R. Polymeric monolithic materials: Syntheses,properties, functionalization and applications. Polymer 2007, 48 (8), 2187-2198.

[0739] (32) Davidson, R. S.;hilchenbach, M. M. The use of fluorescent probesin immunochemistry. Photochemistry and photobiology 1990, 52 (2), 431-438.

Claims

1. 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 linked to the active monomer via a cleavable bond.

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

3. The hydrogel according to claim 1, wherein the plurality of active monomers are selected from polyethylene glycol acrylamide, polyethylene glycol N-isopropylacrylamide, polyethylene glycol methacrylamide, polyethylene glycol methacrylate, allylamide, and polyethylene glycol azide.

4. The hydrogel according to any one of the preceding claims, wherein the at least one fluorophore is selected from fluorescein, fluorescein isothiocyanate (FITC), eosin Y, eosin B, tetrachlorofluorescein, carbofluorescein, naphthalenefluorescein and (semi)naphthalenefluorescein having suitable protein reactive groups, preferably wherein the at least one fluorophore is fluorescein isothiocyanate (FITC).

5. The hydrogel according to any one of the preceding claims, wherein the molar ratio of the inactive monomer to the active monomer is 100:1 to 5:1, preferably wherein the ratio of the inactive monomer to the active monomer is 40:

1.

6. The hydrogel according to any one of the preceding claims, wherein the plurality of inactive monomers are acrylamide or polyethylene glycol bisazide.

7. The hydrogel according to any one of the preceding claims, wherein the plurality of active monomers are polyethylene glycol methacrylamide, preferably FITC-NVOC-PEG. 3400 1-Methylacrylamide.

8. The hydrogel according to any one of claims 1 to 6, wherein the plurality of active monomers are polyethylene glycol azide, preferably FITC-NVOC-PEG. 1000 -Azides.

9. The hydrogel according to any one of the preceding claims, wherein the cleavable bond is based on nitrobenzyl or carbonyl, preferably wherein the cleavable bond is selected from ortho-nitrobenzyl, meta-nitrobenzyl and para-nitrobenzyl.

10. The hydrogel according to claims 1 to 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 contains at least one fluorescein isothiocyanate capable of covalently binding to a protein, wherein the molar ratio of the inactive monomer to the active monomer is 100:1 to 5:1, and wherein each fluorescein isothiocyanate is linked to the polyethylene glycol methacrylamide monomer via a cleavable bond, wherein the cleavable bond is optionally o-nitrobenzyl.

11. The hydrogel according to any one of claims 1 to 6, 8 or 9, wherein the plurality of inactive monomers are polyethylene glycol bisazides, wherein the plurality of active monomers are polyethylene glycol azides, wherein each polyethylene glycol azide monomer contains at least one fluorescein isothiocyanate capable of covalently binding to a protein, wherein the molar ratio of the inactive monomer to the active monomer is 100:1 to 5:1, and wherein each fluorescein isothiocyanate is linked to the polyethylene glycol azide monomer via a cleavable bond, wherein the cleavable bond is optionally an o-nitrobenzyl group.

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

13. An oral formulation comprising the hydrogel according to any one of 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, lozenge, lollipop, sampling material, or round tablet.

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

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

17. A container including an inner surface operable to contact a reaction mixture, wherein the inner surface is coated with a base layer, and a reaction layer is coated on the base layer, the reaction layer comprising a mixture of binding molecules and a blocking agent, wherein the blocking agent comprises an amine-free compound.

18. A multi-component reagent kit 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. Instruction manual.

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

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

21. Use of 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 for concentrating and labeling proteins in a sample.

22. Use of 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 in a method for diagnosing a disease or condition.

23. A method for concentrating and labeling proteins in a sample, the method comprising: a. Contacting a sample with a hydrogel according to any one of claims 1 to 11, a formulation according to claim 12, an oral formulation according to claim 13 or 14, a sampling device according to claim 15, or an 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 labeled proteins bound to the hydrogel.

24. A method for detecting proteins in a sample, the method comprising: a. Contacting a sample with a hydrogel according to any one of claims 1 to 11, a formulation according to claim 12, an oral formulation according to claim 13 or 14, a sampling device according to claim 15, or an 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 labeled proteins bound to the hydrogel; b. Exposing the hydrogel from step (a) to a cleavage inducer under suitable conditions to cleave cleavable bonds, thereby releasing the fluorescently labeled protein from the hydrogel; as well as c. Determine the presence of the protein in the sample, wherein the presence of fluorescence indicates the presence of the protein in the sample.

25. A method for determining 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 of claim 12, the oral formulation of claim 13 or 14, the sampling device of claim 15, or the inert substrate of claim 16 under suitable conditions to allow proteins in the sample to bind to the hydrogel via the fluorophore, thereby obtaining fluorescently labeled proteins bound to the hydrogel; b. Exposing the hydrogel from step (a) to a cleavage inducer under suitable conditions to cleave the cleavable bonds, thereby releasing the fluorescently labeled protein from the hydrogel; c. Separate the fluorescently labeled protein; d. Contact the fluorescently labeled protein with a binding molecule capable of specifically binding to the protein of interest; e. Remove any unbound fluorescently labeled proteins; and f. Measure the fluorescence level, wherein the fluorescence level indicates the amount of the protein of interest in the sample.

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

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

28. A method for determining whether an object suffers from a disease or symptom, the method comprising: a. Contacting a sample from the object with a hydrogel according to any one of claims 1 to 11, a formulation according to claim 12, a formulation according to claim 13 or 14, a sampling device according to claim 15, or an 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 labeled proteins bound to the hydrogel; b. Exposing the hydrogel from step (a) to a cleavage inducer under suitable conditions to cleave the cleavable bonds, thereby releasing the fluorescently labeled protein from the hydrogel; c. Separate the fluorescently labeled protein; d. Contact the fluorescently labeled protein with a binding molecule that can specifically bind to a protein biomarker of a disease or symptom; e. Remove any unbound fluorescently labeled proteins; f. Detecting the presence of fluorescence or determining the level of said fluorescence, wherein the presence of fluorescence indicates the presence of said protein biomarker in the sample, or wherein the level of fluorescence indicates the level of said protein biomarker in the sample. g. Based on f., determine that the object suffers from a disease or condition, wherein the presence of fluorescence or the level of fluorescence indicates the disease or condition.

29. The method of claim 28, wherein the disease or condition is cancer or cardiovascular disease.

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

31. The method of claim 30, wherein the ultraviolet light comprises a wavelength range of approximately 200 nm to 400 nm, preferably wherein the ultraviolet light comprises a wavelength of approximately 365 nm.

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

Images