Biocompatible oxygen-sensitive materials

Abstract

Provided herein are polysiloxane matrices and biocompatible membranes thereof for the detection of oxygen. In particular, oxygen-sensitive fluorophores embedded within the polysiloxane matrices are utilized to detect oxygen consumption rates in living cells.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 397,209, filed August 11, 2022, which is incorporated herein by reference.

[0002] Provided herein are polysiloxane matrices and biocompatible membranes thereof for the detection of oxygen. In particular, oxygen-sensitive fluorophores embedded within the polysiloxane matrices are utilized to detect oxygen consumption rates in living cells. [Background technology]

[0003] Cells consume oxygen during respiration, and the rate of oxygen consumption can be used to characterize the metabolic phenotype of cells. Currently, the most common methods for measuring oxygen consumption are using stand-alone devices and specialized equipment. Summary of the Invention

[0004] Provided herein are polysiloxane matrices and biocompatible membranes thereof for the detection of oxygen. In particular, oxygen-sensitive fluorophores embedded within the polysiloxane matrices are utilized to detect oxygen consumption rates in living cells.

[0005] In some embodiments, provided herein is a polysiloxane matrix, (a) a bis(trialkoxysilyl) monomer of formula (I): [ka] (In the formula, R 1 , R 1’ , R 1” , R 2 , R 2’ and R 2”is independently selected from CH3, CH2CH3, (CH2)2CH3, and L is C1-C6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 heteroalkenyl, C2-C6 alkynyl, C1-C6 heteroalkynyl, [ka] L' and L" are independently selected from a covalent bond, C1-C6 alkyl or heteroalkyl, C2-C6 alkenyl or heteroalkenyl, C2-C6 alkynyl or heteroalkynyl, and L 1 and L 2 is independently selected from an aryl or heteroaryl ring; L 1 and L 2 are linked by a covalent bond, and L, L', L'', and L 1 and L 2 is optionally branched or substituted) and (b) a dialkyldialkoxysilane monomer of formula (II): [ka] (In the formula, R 3 , R 3’ , R 4 and R 4’ is independently selected from CH, CHCH, and (CH)CH. In some embodiments, (i) R 1 , R 1’ , R 1” , R 2 , R 2’ and R 2” are the same; (ii) if present, L' and L" are the same; and (iii) if present, L 1 and L 2 are the same, and (iv) R 3 and R 3’ are the same, and / or (v) R 4 and R 4” and are the same. In some embodiments, R 1 , R 1’ , R 1” , R2 , R 2’ and R 2” is CH3 or CH2CH3. In some embodiments, L is (CH2) 1-6 , (CH2) 1-6 -NH-(CH2) 1-6 , (CH2) 1-6 -O-(CH2) 1-6 , (CH2) 0-6 -aryl-(CH2) 1-6 , and (CH2) 1-6 -aryl-aryl-(CH2) 1-6 In some embodiments, the bis(trialkoxysilyl) monomer is selected from 1,4-bis(trimethoxysilylethyl)benzene, 1,4-bis(trimethoxysilylmethyl)benzene, 1,4-bis(trimethoxysilyl)benzene, 4,4'-bis(triethoxysilyl)-1,1'-biphenyl, 1,2-bis(triethoxysilyl)ethane, and bis[3-(trimethoxysilyl)propyl]amine. In some embodiments, R 3 , R 3’ , R 4 , R 4’ is CH3 or CH2CH3. In some embodiments, the bis(trialkoxysilyl) monomer is 1,4-bis(trimethoxysilylethyl)benzene and the dialkyldialkoxysilane monomer is dimethyldimethoxysilane.

[0006] In some embodiments, a composition is provided herein comprising the polysiloxane matrix described herein and an oxygen-sensitive fluorophore. In some embodiments, the oxygen-sensitive fluorophore is selected from the group consisting of tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) chloride (Ru-dpp), platinum octaethylporphyrin, platinum(II) 2,3,7,8,12,13,17,18-octaethyl-21H,23H-porphyrin (PtOEP), palladium(II) octaethylporphine (PdOEP), platinum(II)-5,10,15,20-tetrakis-(2,3,4,5,6-pentafluorophenyl)-porphyrin (PtTfPP), palladium(II)-5,10,15,20-tetrakis-(2,3,4,5,6-pentafluorophenyl)-porphyrin (PtTfPP), and palladium(II)-5,10,15,20-tetrakis-(2,3,4,5,6-pentafluorophenyl)-porphyrin (PtTfPP). The compound is selected from the group consisting of platinum(II) octaethylporphyrin ketone (PtOEPK), palladium(II) octaethylporphyrin ketone (PdOEPK), platinum(II) tetraphenyltetrabenzoporphyrin (PtTPTBP), meso-tetraphenyl-tetrabenzoporphyrin palladium complex (PdTPTBP), platinum(II) tetraphenyltetranaphthoporphyrin (PtTPTNP), and palladium(II) tetraphenyltetranaphthoporphyrin (PdTPTNP).

[0007] In some embodiments, the composition further comprises an oxygen-insensitive fluorophore. In some embodiments, the oxygen-insensitive fluorophore is selected from Nile blue chloride, tris(8-hydroxyquinolinato)aluminum (AlQ3), TAMRA, and coumarin-6. In some embodiments, the oxygen-insensitive fluorophore is coumarin-6 and the oxygen-sensitive fluorophore is PtTfPP.

[0008] In some embodiments, provided herein are thin films comprising a polysiloxane matrix (eg, having embedded fluorophore(s)) described herein.

[0009] In some embodiments, provided herein is a device comprising a solid surface having deposited thereon a polysiloxane matrix thin film (with embedded fluorophore(s)) described herein. In some embodiments, the solid surface is a glass or plastic (e.g., polystyrene) surface. In some embodiments, the solid surface is a slide or the bottom of a microwell. In some embodiments, the device further comprises a layer of bioactive extracellular matrix proteins or biocompatible molecules on the thin film. In some embodiments, the layer of bioactive extracellular matrix proteins comprises one or more proteins selected from type I collagen, type IV collagen, fibronectin, and laminin. In some embodiments, the layer of biocompatible molecules comprises polydopamine. In some embodiments, the bioactive extracellular matrix proteins are passively coated on the thin film. In some embodiments, the bioactive extracellular matrix proteins are covalently or non-covalently attached to the thin film.

[0010] In some embodiments, a method is provided comprising: (a) contacting (i) an oxygen-sensitive fluorophore embedded in a polysiloxane matrix with (ii) a test solution; (b) exposing the oxygen-sensitive fluorophore to light within the excitation spectrum of the oxygen-sensitive fluorophore; (c) detecting light within the emission spectrum of the oxygen-sensitive fluorophore; and (d) determining the oxygen concentration in the test solution. In some embodiments, determining the oxygen concentration in the test solution comprises comparing the light within the emission spectrum of the oxygen-sensitive fluorophore to a reference value. In some embodiments, the reference value is the amount of light emitted when the test solution is saturated with oxygen and the amount of light emitted when the test solution is depleted of oxygen. In some embodiments, the reference value is the amount of light emitted at various oxygen concentrations. In some embodiments, the test solution comprises cells. In some embodiments, steps (b)-(d) are repeated to monitor oxygen consumption by the cells over time. In some embodiments, the polysiloxane matrix is ​​a film deposited on a transparent solid surface. In some embodiments, the solid surface is glass or plastic (e.g., polystyrene). In some embodiments, the solid surface is a slide or the bottom of a microwell. In some embodiments, the membrane further comprises a layer of bioactive extracellular matrix proteins or biocompatible molecules deposited thereon. In some embodiments, the layer of bioactive extracellular matrix proteins comprises one or more proteins selected from type I collagen, type IV collagen, fibronectin, and laminin. In some embodiments, the layer of biocompatible molecules comprises polydopamine.

[0011] In some methods, an oxygen-insensitive fluorophore is also embedded within the polysiloxane matrix. In some embodiments, the oxygen-insensitive fluorophore is selected from Nile blue chloride, tris(8-hydroxyquinolinato)aluminum (AlQ3), TAMRA, and coumarin-6. In some embodiments, the method further comprises exposing the oxygen-insensitive fluorophore to light within the excitation spectrum of the oxygen-insensitive fluorophore and detecting light within the emission spectrum of the oxygen-insensitive fluorophore. In some embodiments, determining the oxygen concentration in the test solution comprises comparing a reference value to the ratio of (i) light within the emission spectrum of the oxygen-sensitive fluorophore to (ii) light within the emission spectrum of the oxygen-insensitive fluorophore. In some embodiments, the reference value is the ratio of light emitted when the test solution is saturated with oxygen and the ratio of light emitted when the test solution is depleted of oxygen. In some embodiments, the reference value is the ratio of light emitted at various oxygen concentrations.

[0012] In some methods, the polysiloxane matrix comprises (a) a bis(trialkoxysilyl) monomer of formula (I): [ka] (In the formula, R 1 , R 1’ , R 1” , R 2 , R 2’ and R 2” is independently selected from CH3, CH2CH3, (CH2)2CH3, and L is C1-C6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 heteroalkenyl, C2-C6 alkynyl, C1-C6 heteroalkynyl, [ka] L' and L" are independently selected from a covalent bond, C1-C6 alkyl or heteroalkyl, C2-C6 alkenyl or heteroalkenyl, C2-C6 alkynyl or heteroalkynyl, and L1 and L 2 is independently selected from an aryl or heteroaryl ring; L 1 and L 2 are linked by a covalent bond, and L, L', L'', and L 1 and L 2 is optionally branched or substituted), and (b) a dialkyldialkoxysilane monomer of formula (II): [ka] (In the formula, R 3 , R 3’ , R 4 and R 4’ is independently selected from CH, CHCH, and (CH)CH. In some embodiments, (i) R 1 , R 1’ , R 1” , R 2 , R 2’ and R 2” are the same; (ii) if present, L' and L" are the same; and (iii) if present, L 1 and L 2 are the same, and (iv) R 3 and R 3’ are the same, and / or (v) R 4 and R 4” are the same). In some embodiments, R 1 , R 1’ , R 1” , R 2 , R 2’ and R 2” is CH3 or CH2CH3. In some embodiments, L is (CH2) 1-6 , (CH2) 1-6 -NH-(CH2) 1-6 , (CH2) 1-6 -O-(CH2) 1-6 , (CH2) 0-6 -aryl-(CH2) 1-6 , and (CH2) 1-6 -aryl-aryl-(CH2) 1-6In some embodiments, the bis(trialkoxysilyl) monomer is selected from 1,4-bis(trimethoxysilylethyl)benzene, 1,4-bis(trimethoxysilylmethyl)benzene, 1,4-bis(trimethoxysilyl)benzene, 4,4'-bis(triethoxysilyl)-1,1'-biphenyl, 1,2-bis(triethoxysilyl)ethane, and bis[3-(trimethoxysilyl)propyl]amine. In some embodiments, R 3 , R 3’ , R 4 , R 4’ is CH or CHCH. In some embodiments, the bis(trialkoxysilyl) monomer is 1,4-bis(trimethoxysilylethyl)benzene and the dialkyldialkoxysilane monomer is dimethyldimethoxysilane. In some embodiments, the oxygen-sensitive fluorophore is selected from Ru-dpp, PtOEP, PdOEP, PtTfPP, PdTFPP, PtOEPK, PdOEPK, PtTPTBP, PdTPTBP, PtTPTNP, and PdTPTNP. [Brief explanation of the drawings]

[0013] [Figure 1] Schematic of an exemplary embodiment. The oxygen-sensitive dye PtTFPP is immobilized on a cell substrate. This dye is highly fluorescent at low oxygen concentrations. As cells consume oxygen, the concentration of oxygen decreases, thereby increasing the fluorescence of the sensor dye. The fluorescence of the dye is monitored by excitation with a plate reader from the bottom of the substrate. [Figure 2] Components of an exemplary oxygen sensor matrix. [Figure 3] Image of an exemplary sensor matrix coating the bottom of a 96-well plate. [Figure 4] Change in oxygen concentration with 0.05u to 1u / well of glucose oxidase in wells of a 96-well plate coated with an exemplary oxygen-sensing membrane. [Figure 5]HCT-116 titration from 0 to 120,000 cells / well. Oxygen concentration decreased over time compared to cell controls in a cell density-dependent manner (n=16 replicates). [Figure 6] HCT-116 treatment with FCCP and rotenone. Mean oxygen concentration decreased over time compared to cell controls in a dose-dependent manner with FCCP (n=16 replicates). Changes in oxygen concentration over 30 minutes show trends in oxygen consumption rate with FCCP treatment (n=16). Changes in oxygen concentration over 30 minutes with rotenone (n=8). [Figure 7] HEP-G2 titration from 0 to 120,000 cells / well with or without rotenone (2 uM) (n=4 replicates). [Figure 8] PC-3 titration from 0 to 100,000 cells / well (n=4 replicates). Changes in oxygen concentration in PC-3 after FCCP and rotenone treatment. [Figure 9] Comparison of systems sealed with oxygen barriers (silver seal, aluminum foil) and systems open to the atmosphere. The oxygen barrier significantly increased the apparent oxygen consumption rate. DETAILED DESCRIPTION OF THE INVENTION

[0014] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and are not intended to be limiting.

[0015] Definitions of certain functional groups and chemical terms are explained in more detail below. For purposes of this disclosure, chemical elements are identified according to the Periodic Table of the Elements (CAS version, Handbook of Chemistry and Physics, 75th Ed, inside cover), and certain functional groups are generally defined as set forth therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivities, are described in: Sorrell, Organic Chemistry, 2nd edition, University Science Books, Sausalito, 2006; Smith, March's Advanced Organic Chemistry: Reactions, Mechanism, and Structure, 7th Edition, John Wiley & Sons, Inc., New York, 2013; Larock, Comprehensive Organic Transformations, 3rd Edition, John Wiley & Sons, Inc., New York, 2018; and Carruthers, Some Modern Methods of Organic Synthesis, 3rd Edition, Cambridge University Press, Cambridge, 1987 (the entire contents of each of which are incorporated herein by reference).

[0016] As used herein, the term "alkyl" refers to an alkyl group having 1 to 30 carbon atoms, e.g., 1 to 16 carbon atoms (C-C 16 alkyl), 1 to 14 carbon atoms (C1-C 14 alkyl), 1 to 12 carbon atoms (C1-C 12 alkyl), 1 to 10 carbon atoms (C1-C 10 alkyl), 1 to 8 carbon atoms (C1-C8 alkyl), 1 to 6 carbon atoms (C1-C6 alkyl), 1 to 4 carbon atoms (C1-C4 alkyl), 6 to 20 carbon atoms (C6-C 20 alkyl), or 8 to 14 carbon atoms (C8-C 14"Alkyl" refers to a straight or branched saturated hydrocarbon chain, including alkyl. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, and n-dodecyl.

[0017] As used herein, the term "alkylene" refers to a straight or branched chain hydrocarbon of 1 to 10 carbon atoms (C-C 10

[0033] "Alkylene" refers to a divalent group derived from a straight or branched chain hydrocarbon of 1 to 6 carbon atoms (C1-C6 alkylene), for example. Representative examples of alkylene include, but are not limited to, -CH2-, -CH2CH2-, -CH(CH3)-, -CH2CH2CH2-, -CH2CH(CH3)-, -CH2CH2CH2CH2-, -CH2CH2CH(CH3)-, -CH2CH2CH2CH2-, -CH2CH2CH2CH2-, -CH2CH2CH2CH2-, -CH2CH2CH2CH2-, -CH2CH2CH(CH3)CH2CH2-, -CH2CH2CH2CH2CH2-, -CH2CH2CH(CH3)CH2CH2-, -CH2CH2CH2CH2CH2-, -CH2CH2CH(CH3)CH2CH2-, -CH2CH2CH2CH2CH2-, and -CH(CH3)CH2CH2CH2CH2-.

[0018] As used herein, the term "alkenyl" refers to a straight or branched hydrocarbon chain containing 2 to 30 carbon atoms and containing at least one carbon-carbon double bond. Representative examples of alkenyl include, but are not limited to, ethenyl, 2-propenyl, 2-methyl-2-propenyl, 3-butenyl, 4-pentenyl, 5-hexenyl, 2-heptenyl, 2-methyl-1-heptenyl, and 3-decenyl.

[0019] As used herein, the term "alkynyl" refers to a straight or branched hydrocarbon chain containing 2 to 30 carbon atoms and containing at least one carbon-carbon triple bond. Representative examples of alkynyl include, but are not limited to, ethynyl, propynyl, and butynyl.

[0020] As used herein, the term "heteroalkyl" refers to an alkyl group, as defined herein, in which one or more carbon atoms (and any associated hydrogen atoms) are each independently replaced with a heteroatomic group, such as -NR-, -O-, -S-, -S(O)-, or -S(O)-, where R is H, alkyl, aryl, cycloalkyl, heteroalkyl, heteroaryl, or heterocyclyl, each of which may be optionally substituted. By way of example, one, two, or three carbon atoms may be independently replaced with the same or different heteroatomic groups. Examples of heteroalkyl groups include, but are not limited to, -OCH, -CHOCH, -SCH, -CHSCH, -NRCH, and -CHNRCH, where R is hydrogen, alkyl, aryl, arylalkyl, heteroalkyl, or heteroaryl, each of which may be optionally substituted. Heteroalkyl also includes groups in which an alkyl carbon atom is oxidized (i.e., -C(O)-).

[0021] As used herein, the term "heteroalkylene" refers to an alkylene group, as defined herein, in which one or more carbon atoms (and any associated hydrogen atoms) are each independently replaced with a heteroatom group, such as -NR-, -O-, -S-, -S(O)-, or -S(O)-, where R is H, alkyl, aryl, cycloalkyl, heteroalkyl, heteroaryl, or heterocyclyl, each of which may be optionally substituted. As an example, one, two, or three carbon atoms may be independently replaced with the same or different heteroatom groups. Heteroalkylene also includes groups in which an alkyl carbon atom is oxidized (i.e., -C(O)-). Examples of heteroalkylene groups include, but are not limited to, -CH-O-CH-, -CH-S-CH-, -CH-NR-CH-, -CH-NH-C(O)-CH, and the like, as well as polyethylene oxide chains, polypropylene oxide chains, and polyethyleneimine chains.

[0022] As used herein, the term "aryl" refers to an aromatic carbocyclic ring system having a single ring (monocyclic) or multiple rings (bicyclic or tricyclic), including fused ring systems, and zero heteroatoms. As used herein, aryl refers to an aromatic ring system having 6 to 20 carbon atoms (C-C 20 aryl), 6 to 14 ring carbon atoms (C6-C 14 aryl), 6 to 12 ring carbon atoms (C6-C 12 aryl), or 6 to 10 ring carbon atoms (C6-C 10 Representative examples of aryl groups include, but are not limited to, phenyl, naphthyl, anthracenyl, phenanthrenyl, and the like.

[0023] As used herein, the term "arylene" refers to a divalent aryl group. Representative examples of arylene groups include, but are not limited to, phenylene groups (e.g., 1,2-phenylene, 1,3-phenylene, and 1,4-phenylene).

[0024] As used herein, the term "heteroaryl" refers to an aromatic group having a single ring (monocyclic) or multiple rings (bicyclic or tricyclic) with one or more ring heteroatoms independently selected from O, N, and S. An aromatic monocyclic ring is a 5- or 6-membered ring containing at least one heteroatom independently selected from O, N, and S (e.g., 1, 2, 3, or 4 heteroatoms independently selected from O, N, and S). A 5-membered aromatic monocyclic ring has two double bonds, and a 6-membered aromatic monocyclic ring has three double bonds. Exemplary bicyclic heteroaryl groups include a monocyclic aryl group, as defined herein, or a monocyclic heteroaryl group, as defined herein, to which an additional monocyclic heteroaryl ring is fused. Exemplary tricyclic heteroaryl groups include a monocyclic heteroaryl ring fused to two rings independently selected from a monocyclic aryl group, as defined herein, and a monocyclic heteroaryl group, as defined herein. Representative examples of monocyclic heteroaryl include, but are not limited to, pyridinyl (e.g., pyridin-2-yl, pyridin-3-yl, pyridin-4-yl), pyrimidinyl, pyrazinyl, pyridazinyl, pyrrolyl, benzopyrazolyl, 1,2,3-triazolyl, 1,3,4-thiadiazolyl, 1,2,4-thiadiazolyl, 1,3,4-oxadiazolyl, 1,2,4-oxadiazolyl, imidazolyl, thiazolyl, isothiazolyl, thienyl, furanyl, oxazolyl, isoxazolyl, 1,2,4-triazinyl, and 1,3,5-triazinyl. Representative examples of bicyclic heteroaryl include, but are not limited to, benzimidazolyl, benzodioxolyl, benzofuranyl, benzoxadiazolyl, benzopyrazolyl, benzothiazolyl, benzothienyl, benzotriazolyl, benzoxadiazolyl, benzoxazolyl, chromenyl, imidazopyridine, imidazothiazolyl, indazolyl, indolyl, isobenzofuranyl, isoindolyl, isoquinolinyl, naphthyridinyl, purinyl, pyridoimidazolyl, quinazolinyl, quinolinyl, quinoxalinyl, thiazolopyridinyl, thiazolopyrimidinyl, thienopyrrolyl, and thienothienyl.Representative examples of tricyclic heteroaryls include, but are not limited to, dibenzofuranyl and dibenzothienyl. Monocyclic, bicyclic, and tricyclic heteroaryls are connected to the parent molecular moiety through any carbon atom or nitrogen atom contained within the ring.

[0025] As used herein, the term "substituent" refers to a group substituted on an atom of the indicated group.

[0026] Where a group or moiety can be substituted, the term "substituted" indicates that one or more (e.g., 1, 2, 3, 4, 5, or 6; in some embodiments, 1, 2, or 3; and in other embodiments, 1 or 2) hydrogens on the group designated in the phrase using "substituted" can be replaced with a selection of the designated groups listed or with suitable groups known to those of ordinary skill in the art (e.g., one or more of the groups listed below), provided that the normal valence of the designated atom is not exceeded. Substituents include, but are not limited to, alkyl, alkenyl, alkynyl, alkoxy, acyl, amino, amido, amidino, aryl, azido, carbamoyl, carboxyl, carboxyl ester, cyano, cycloalkyl, cycloalkenyl, guanidino, halo, haloalkyl, haloalkoxy, heteroalkyl, heteroaryl, heterocyclyl, hydroxy, hydrazino, imino, oxo, nitro, phosphate, phosphonate, sulfonic acid, thiol, thione, or combinations thereof.

[0027] For the compounds described herein, these groups and substituents may be selected according to the allowed valences of atoms and substituents, so that the selection and substitution results in stable compounds, e.g., compounds that do not spontaneously undergo transformation by rearrangement, cyclization, elimination, and the like.

[0028] Where substituents are specified by their conventional chemical formula written from left to right, this optionally includes the substituents resulting from writing the structure from right to left, e.g., -CHO- optionally also lists -OCH-, and -OC(O)NH- optionally also lists -NHC(O)O-.

[0029] When numerical ranges are recited herein, each intervening number with the same degree of precision is expressly contemplated. For example, in the range of 6 to 9, the numbers 7 and 8 are also contemplated in addition to 6 and 9, and in the range of 6.0 to 7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are also expressly contemplated.

[0030] Provided herein are polysiloxane matrices and biocompatible membranes thereof for the detection of oxygen. In particular, oxygen-sensitive fluorophores embedded within the polysiloxane matrices are utilized to detect oxygen consumption rates in living cells.

[0031] In some embodiments, provided herein are biocompatible sensor membranes that enable measurement of the oxygen content of a solution, particularly the rate of cellular oxygen consumption. In certain embodiments, the materials and systems herein enable monitoring of oxygen concentration and / or its rate of change in standard formats (e.g., 96-well plates, 384-well plates, etc.) using standard laboratory equipment (e.g., plate readers). Some embodiments herein provide functionalized surfaces that can (a) detect oxygen concentration over time in a solution (e.g., containing cells) and (b) support cell attachment.

[0032] In some embodiments, the oxygen-sensing component of the membrane consists of a porous siloxane matrix impregnated with a porphyrin dye, whose fluorescence intensity changes in response to oxygen concentration. An advantage of the oxygen-sensitive membranes provided herein is the membrane's dynamic range, specifically the ratio of fluorescence intensity under hypoxic conditions to that under air-saturated conditions (I / I). While the I / I ratios of the exemplary materials described herein are typically between 30 and 40, other materials utilizing the same oxygen-sensitive porphyrin dyes are characterized by I / I ratios between 4 and 5. An advantage of the embodiments herein is that they allow users to measure oxygen consumption on a standard plate reader.

[0033] In some embodiments, provided herein are polysiloxane matrices comprising bis(trialkoxysilyl) and dialkyldialkoxysilane monomers with oxygen-sensitive fluorophores embedded within the matrix. In some embodiments, a film of the matrix with embedded oxygen-sensitive fluorophores is deposited as a film on a solid substrate (e.g., a glass plate, the bottom of a well, etc.). In some embodiments, a biocompatible and / or bioactive protein (e.g., an extracellular matrix protein) or molecule (e.g., polydopamine) is a layer on top of the film to promote cell attachment. In some embodiments, an oxygen-insensitive fluorophore is also embedded within the matrix (e.g., to serve as a reference for the oxygen-sensitive fluorophore). In some embodiments, monitoring the fluorescence output of the oxygen-sensitive fluorophore (e.g., relative to the oxygen-insensitive fluorophore) allows for detection / quantification of the oxygen content in a solution in contact with the matrix film and / or monitoring cellular uptake of oxygen over time.

[0034] In some embodiments, the materials and devices herein comprise a polysiloxane matrix. In some embodiments, the polysiloxane matrix herein is sufficiently transparent (e.g., greater than 50%, 60%, 70%, 80%, 90%, or 95% transmittance) to allow wavelengths of light emitted from the fluorophores embedded therein to pass through the matrix. In some embodiments, the polysiloxane matrix is ​​capable of adhering to glass, plastic (e.g., polystyrene), or other materials commonly used in laboratory plates, tubes, and the like. In some embodiments, the polysiloxane matrix herein is biocompatible (e.g., non-toxic to cells). In some embodiments, the polysiloxane matrix herein is stable when in contact with water and is non-biodegradable.

[0035] In some embodiments, the polysiloxane matrix herein is a bis(trialkoxysilyl) monomer and a dialkyldialkoxysilane monomer. In some embodiments, the polysiloxane matrix is ​​prepared from the bis(trialkoxysilyl) and dialkyldialkoxysilane components by mixing them under acidic conditions (e.g., for 10 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, or more), followed by drying the material. In some embodiments, the bis(trialkoxysilyl) and dialkyldialkoxysilane monomers are mixed in a ratio of 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. In some embodiments, including one or more additional components (e.g., a fluorophore (e.g., an oxygen-sensitive fluorophore, an oxygen-insensitive fluorophore, a pH-sensitive fluorophore, a temperature-sensitive fluorophore, a fluorophore sensitive to the concentration of a particular chemical species (e.g., a chemical species involved in cellular metabolism) (e.g., reactive oxygen species (ROS), hydrogen peroxide, superoxide, nitric oxide, etc.))) within the mixture allows for embedding of the additional components within the matrix and the resulting membrane.

[0036] In some embodiments, the bis(trialkoxysilyl) monomer of the polysiloxane matrix has formula (I): [ka] (In the formula, R 1 , R 1’ , R 1” , R 2 , R 2’ and R 2” is independently selected from CH3, CH2CH3, (CH2)2CH3, and L is C1-C6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 heteroalkenyl, C2-C6 alkynyl, C1-C6 heteroalkynyl, [ka] L' and L" are independently selected from a covalent bond, C1-C6 alkyl or heteroalkyl, C2-C6 alkenyl or heteroalkenyl, C2-C6 alkynyl or heteroalkynyl, and L 1 and L 2 is independently selected from an aryl or heteroaryl ring; L 1 and L 2 are covalently linked. In some embodiments, R 1 , R 1’ and R 1” are the same functional group. 2 , R 2’ and R 2” are the same functional group. 1 , R 1’ , R 1” , R 2 , R 2’ and R 2” are the same functional group. 1 , R 1’ , R 1” , R 2 , R 2’ and R2” In some embodiments, one or more of R 1 , R 1’ , R 1” , R 2 , R 2’ and R 2” is CH3 or CH2CH3. In some embodiments, when present, L' and L" are the same functional group. In some embodiments, when present, L' and L" are different functional groups. In some embodiments, when present, L 1 and L 2 are the same functional group. In some embodiments, when present, L 1 and L 2 are different functional groups. In some embodiments, L is (CH) 1-6 , (CH2) 1-6 -NH-(CH2) 1-6 , (CH2) 1-6 -O-(CH2) 1-6 , (CH2) 0-6 -aryl-(CH2) 1-6 , and (CH2) 1-6 -aryl-aryl-(CH2) 1-6 is selected from.

[0037] In some embodiments, L, L', L", L 1 , L 2 , R 1 , R 1’ , R 1” , R 2 , R 2’ , and R 2” One or more of L, L', L", L are optionally branched or substituted. In some embodiments, L, L', L", L 1 , L 2 , R 1 , R 1’ , R 1” , R 2 , R 2’ , and / or R 2” In some embodiments, one or more CH groups in L, L', L", L 1 , L 2, R 1 , R 1’ , R 1” , R 2 , R 2’ , and / or R 2” In some embodiments, one or more CH groups of L, L', L", L 1 , L 2 , R 1 , R 1’ , R 1” , R 2 , R 2’ , and / or R 2” contains substituents selected from, for example, alkyl, alkenyl, alkynyl, alkoxy, acyl, amino, amido, amidino, aryl, azido, carbamoyl, carboxyl, carboxyl ester, cyano, cycloalkyl, cycloalkenyl, guanidino, halo, haloalkyl, haloalkoxy, heteroalkyl, heteroaryl, heterocyclyl, hydroxy, hydrazino, imino, oxo, nitro, phosphate, phosphonate, sulfonic acid, thiol, or thione groups, or combinations thereof.

[0038] In some embodiments, the bis(trialkoxysilyl) monomer can be, for example, 1,4-bis(trimethoxysilylethyl)benzene, 1,4-bis(trimethoxysilylmethyl)benzene, 1,4-bis(trimethoxysilyl)benzene, 1,4-bis(triethoxysilylethyl)benzene, 1,4-bis(triethoxysilyl)benzene, 1,2-bis(trimethoxysilylethyl)benzene, 1,2-bis(trimethoxysilylmethyl)benzene, 1,2-bis(trimethoxysilyl)benzene, 1,2-bis(triethoxysilylethyl)benzene, 1,2-bis(triethoxysilyl)benzene, 1,3-bis(trimethoxysilylethyl)benzene, 1,3-bis(trimethoxysilylmethyl)benzene, 1,3-bis(trimethoxysilyl)benzene, 1,3-bis(triethoxysilylethyl)benzene, bis(triethoxysilylethyl)benzene, 1,3-bis(triethoxysilyl)benzene, 4,4'-bis(triethoxysilyl)-1,1'-biphenyl, 4,4'-bis(trimethoxysilyl)-1,1'-biphenyl, 4,4'-bis(triethoxysilylmethyl)-1,1'-biphenyl, 4,4'-bis(trimethoxysilylmethyl)-1,1'-biphenyl, 4,4'-bis(triethoxysilylethyl)-1,1'-biphenyl, 4,4'-bis(trimethoxysilylethyl)-1,1'-biphenyl, 1,2-bis(triethoxysilyl)ethane, 1,2-bis(trimethoxysilyl)ethane, bis(triethoxysilyl)methane, bis(trimethoxysilyl)methane, bis[3-(trimethoxysilyl)propyl]amine, and bis[3-(triethoxysilyl)propyl]amine.

[0039] In some embodiments, the dialkyldialkoxysilane monomer of the polysiloxane matrix has formula (II): [ka] where R 3 , R 3’ , R 4 and R 4’ is independently selected from CH, CHCH, and (CH)CH. In some embodiments, R3 and R 3’ are identical. In some embodiments, R 3 and R 3’ In some embodiments, R 4 and R 4” are identical. In some embodiments, R 4 and R 4” In some embodiments, the dialkyldialkoxysilane monomer is selected from, for example, dimethyldimethoxysilane, diethyldimethoxysilane, and diethyldiethoxysilane.

[0040] In some embodiments, the polysiloxane matrix comprises, for example, 1,4-bis(trimethoxysilylethyl)benzene and dimethyldimethoxysilane.

[0041] In some embodiments, the dialkyldialkoxysilane and bis(trialkoxysilyl) monomer are mixed in the presence of an acid such as sulfuric acid, nitric acid, hydrochloric acid, citric acid, and acetic acid.

[0042] In some embodiments, the polysiloxane matrix is ​​formed from dialkyldialkoxysilane and bis(trialkoxysilyl) monomers in the presence of one or more fluorophores (e.g., oxygen-sensitive fluorophores, oxygen-insensitive fluorophores, etc.), thereby embedding the fluorophores within the polysiloxane matrix.

[0043] In some embodiments, the polysiloxane matrix herein comprises an oxygen-sensitive fluorophore. In some embodiments, provided herein is an oxygen-sensing material (e.g., a film) comprising an oxygen-sensitive fluorophore embedded within a polysiloxane matrix. In some embodiments, the oxygen-sensitive fluorophore is selected from one of the following, where M = Pt(II) or M = Pd(II). [ka]

[0044] Other oxygen-sensitive fluorophores that are understood by those of skill in the art are within the scope of the present invention and can be included in the polysiloxane matrices described herein.

[0045] In some embodiments, the polysiloxane matrix herein comprises an oxygen-insensitive fluorophore. In some embodiments, the oxygen-insensitive fluorophore is embedded (along with the oxygen-sensitive fluorophore) within the polysiloxane matrix. In some embodiments, provided herein are oxygen-sensing materials (e.g., membranes) comprising an oxygen-sensitive fluorophore and an oxygen-insensitive fluorophore embedded within a polysiloxane matrix, where the oxygen-sensitive fluorophore is used to monitor the oxygen concentration of an adjacent solution and the oxygen-insensitive fluorophore is used as a reference independent of oxygen concentration. Examples of oxygen-insensitive fluorophores that find use in embodiments herein include coumarin-6, Nile blue chloride, tris(8-hydroxyquinolinato)aluminum (AlQ3), TAMRA, and fluorescein derivatives.

[0046] In some embodiments, the polysiloxane matrix herein comprises coumarin-6 and PtTfPP.

[0047] In some embodiments, the polysiloxane matrix comprises a functionalized silane of formula (III): [ka] wherein R 5 is a functional handle such as a thiol, acrylate, methacrylate, maleimide, amine, COOH, azide, alkyne, or other click chemistry group; L is a covalent bond, C-C alkyl or heteroalkyl, C-C alkenyl or heteroalkenyl, C-C alkynyl or heteroalkynyl; and R 6 , R 6’ and R 6”is independently selected from CH, CHCH, and (CH)CH. In some embodiments, R 6 , R 6’ and R 6” are identical. In some embodiments, R 6 , R 6’ and R 6” An exemplary functionalized silane that may find use in embodiments herein is (3-mercaptopropyl)methyldimethoxysilane. [ka] Different L, R 5 , R 6 , R 6’ and R 6” Other functionalized silanes having groups are within the scope of this specification. In some embodiments, the functionalized silane is included along with the dialkyldialkoxysilane monomer and the bis(trialkoxysilyl) monomer in the formation of the polysiloxane matrix. In some embodiments, the functionalized silane is added after the initial formation of the polysiloxane matrix, for example, to add chemical functional groups on the surface of the polysiloxane matrix film. In some embodiments, the functional handle is used to covalently attach proteins or small molecules to the surface (e.g., environmentally sensitive fluorophores, biocompatible proteins, cells, etc.).

[0048] In some embodiments, the surface of the polysiloxane matrix is ​​functionalized with a heterobifunctional compound that can be used to further functionalize the surface. An exemplary heterobifunctional compound for use in surface functionalization of the polysiloxane matrix is ​​Sulfo-SANPAH (sulfosuccinimidyl 6-(4'-azido-2'-nitrophenylamino)hexanoate). [ka] In some embodiments, after formation of the polysiloxane matrix herein, the surface is functionalized with Sulfo-SANPAH. The surface is first functionalized with reactive groups, followed by covalent conjugation of biopolymers to the surface. Specifically, the surface is exposed to SANPAH and activated with UV light to form NHS moieties on the surface. Biomolecules and / or cells are then introduced, forming covalent amide bonds between the surface and the biomolecules and / or cells.

[0049] In some embodiments, provided herein are films (e.g., thin layers) of the polysiloxane matrices described herein (e.g., comprising oxygen-sensitive, or oxygen-sensitive and oxygen-insensitive fluorophores). In some embodiments, the films herein are less than 5 mm deep (e.g., 5 mm, 4 mm, 3 mm, 2 mm, 1 mm, 900 μm, 800 μm, 700 μm, 600 μm, 500 μm, or less, or ranges therebetween). In some embodiments, the films are formed by depositing a layer of a desired thickness of the polysiloxane matrices described herein (e.g., comprising oxygen-sensitive, or oxygen-sensitive and oxygen-insensitive fluorophores) on a solid surface (e.g., the bottom of a plate, a well, etc.).

[0050] In some embodiments, the cell-interacting properties of the polysiloxane matrix membranes herein are enhanced by the deposition of bioactive and / or biocompatible protein(s) and / or molecules on top of the membrane. In some embodiments, the proteins / molecules are passively deposited on the membrane. In other embodiments, the proteins / molecules are covalently or non-covalently conjugated to the polysiloxane matrix. In some embodiments, the proteins / molecules are conjugated to the surface of the polysiloxane matrix membrane by functionalized silanes or heterobifunctional compounds (e.g., SANPAH). In some embodiments, one or more bioactive extracellular matrix proteins are deposited / conjugated on the membrane. Examples of bioactive extracellular matrix proteins suitable for use with the membranes herein include type I collagen, type IV collagen, fibronectin, laminin, vitronectin, and Matrigel. In some embodiments, one or more biocompatible molecules are deposited / conjugated on the membrane. An example of a biocompatible molecule suitable for use with the membranes herein includes polydopamine.

[0051] In some embodiments, provided herein are methods for detecting oxygen concentration in a solution. In some embodiments, methods are provided for determining absolute oxygen concentration, and in other embodiments, methods are provided for determining oxygen concentration relative to a reference or control. In certain embodiments, the methods herein allow for monitoring oxygen concentration over time, thereby providing a way to monitor the rate of change of oxygen concentration in a sample.

[0052] In certain embodiments, methods are provided for monitoring the rate of oxygen consumption and / or production by a system. For example, cells consume oxygen when they respire. Thus, monitoring the rate of oxygen depletion in a system containing cells provides a method for monitoring cellular metabolism.

[0053] In some embodiments, the methods herein utilize oxygen-sensitive fluorophores embedded within a polysiloxane matrix, as described herein. When oxygen-sensitive fluorophores are excited by exposure to light of an appropriate excitation wavelength, they relax to a ground state by emitting light at an emission wavelength. The intensity of the emitted light depends on the oxygen concentration. Thus, the signal emitted from the fluorophore is a function of the oxygen concentration. By monitoring the fluorophore signal, it is possible to monitor the oxygen concentration of a sample in contact with the polysiloxane matrix material.

[0054] In certain embodiments described herein, the polysiloxane matrix contains both oxygen-sensitive and oxygen-insensitive fluorophores. The oxygen-sensitive fluorophores function as described in the previous paragraph, varying the intensity of the light they emit depending on the concentration of oxygen in the vicinity. However, the intensity of the light emitted by the oxygen-insensitive fluorophores remains constant regardless of the local oxygen concentration. The oxygen-insensitive fluorophores serve as a reference or control for accurately calculating the oxygen concentration.

[0055] As described below in the experimental section, oxygen concentration in a sample can be measured using the materials described herein by applying the Stern-Volmer equation, which relates the relative fluorescence intensities of oxygen-sensitive and oxygen-insensitive samples to oxygen concentration.

number

[0056] In some embodiments, the fluorophore materials embedded in the polysiloxane matrix described herein are provided as a film on a solid surface to measure the oxygen concentration of a sample disposed on the solid surface. In certain embodiments, the methods and materials described herein are used to measure oxygen consumption by cells in the sample. In such embodiments, it may be desirable for the cells to be located adjacent to the polysiloxane matrix film. In such embodiments, bioactive / biocompatible proteins / molecules are presented on the surface of the film facing the sample. Such proteins / molecules promote cell aggregation along the film surface. Monitoring oxygen concentration over time in such a system allows for monitoring the rate of cellular oxygen consumption.

[0057] In some embodiments, the membranes herein are placed in the bottom of wells of a 96-well or 384-well plate. In such a system, a standard laboratory plate reader can be used to determine the oxygen concentration of multiple samples (including, for example, cells) under various conditions. [Example]

[0058] experiment During development of embodiments herein, experiments were conducted to fabricate and test an exemplary device composed of a siloxane matrix containing two dyes: platinum(II) meso-(2,3,4,5,6-pentafluoro)phenylporphyrin (PtTFPP) and 3-(2-benzothiazolyl)-N,N-diethylumbelliferylamine (coumarin-6). The cell interface of the siloxane matrix was functionalized with a biocompatible layer containing collagen I. The dye-impregnated siloxane matrix was deposited on a glass substrate.

[0059] To fabricate the oxygen-sensitive matrix, bis(trimethoxysilylethyl)benzene (18.8 v / v%) was mixed with dimethyldiethoxysilane (37.6 v / v%), 0.1 N hydrochloric acid (5.6 v / v%), PtTFPP (1.5 mg mL in ethanol, 37.6 v / v%), and coumarin-6 (10 mg mL in THF, 0.2 v / v%). The mixture was stirred for 3 h, then deposited on a substrate, air-dried for 24 h, and oven-cured at 60 °C overnight. The substrate was not washed.

[0060] After fabrication of the silane membrane, the membrane surface was further modified with a biopolymer coating to enhance cell attachment. Biopolymers evaluated for cell attachment included type I collagen (rat tail, acid-extracted) and laminin. Passive coating of type I collagen was determined to be sufficient for cell attachment. Passive coating of the substrate was achieved by incubating a solution of biopolymer (e.g., 300 μg mL of type I collagen in 17 mM acetic acid) on the membrane for 1 hour at room temperature or overnight at 4°C. Excess biopolymer was removed by washing the substrate with phosphate-buffered saline (PBS).

[0061] The oxygen-sensitive component of the present invention is a PtTFPP compound. Molecular oxygen quenches the phosphorescence of PtTFPP, resulting in low phosphorescence in high-oxygen conditions and vice versa. The oxygen concentration in the film is calculated from the Stern-Volmer equation, which relates the relative phosphorescence intensity of PtTFPP to the concentration of oxygen. Here, I is the ratiometric fluorescence intensity (650 nm / 490 nm) of the film in the absence of oxygen. I sat is the fluorescence intensity of the membrane at the oxygen saturation ratio.

number

[0062] The chemical properties were first evaluated using a glucose oxidase / glucose biochemical assay. Glucose oxidase can consume glucose and O2 to generate different dissolved O2 concentrations in solution. Different concentrations of glucose oxidase (0.05–1 U / well) were added to wells of a plate with an oxygen-sensing membrane immobilized at the bottom of the wells. Glucose was then added, and the fluorescence intensity of the oxygen-sensing membrane was measured for 1–2 hours. The dissolved oxygen concentration in the solution was calculated based on the Stern-Volmer equation. Different oxygen consumption rates were observed and were found to correspond to the concentration of glucose oxidase added. Furthermore, as shown in Figure 4, the data were reproducible with small error bars.

[0063] Surfaces were covalently or passively coated with collagen I and laminin. Covalent conjugation was achieved using the sulfo-SANPAH conjugation method, and passive coating was achieved by incubating the siloxane surface in the presence of protein for 1 hour at room temperature. Protein was titrated from 100 μg mL to 3,000 μg mL. C2C12 cells were grown on the coated surface at 60,000 cells / cm. 2 Cells were seeded at 100 μg mL-1 and the cell coverage on the substrate was assessed the following day. It was found that coating the substrate with collagen I and laminin improved cell coverage on the substrate, but there was no difference between covalent and passive coating. The results also showed that 300 μg mL-1 collagen I and laminin were sufficient to enhance cell attachment to the surface.

[0064] Oxygen consumption experiments were successfully performed using the HCT-116 cell line. To determine the minimum cell number required to observe signal changes, HCT-116 cells were cultured at 15k–120k cells / well (95k–750k cells / cm). 2The next day, the cell culture medium was replaced with fresh DMEM (high glucose, phenol red-free), and the ratiometric fluorescence at 650 / 490 nm was recorded over time using a plate reader at a controlled temperature of 37°C. The results showed that as the cell density increased, the oxygen concentration decreased more rapidly (oxygen consumption rate, OCR) (Figure 5). Experiments showed that the oxygen concentration decreased more rapidly with increasing cell density (40,000 cells / well, 250 kJ / cm). 2 ) was determined to produce a sufficient increase in the ratiometric signal over a 2-hour period for measurement of oxygen consumption. Further experiments with drug treatments were performed at 250k cells / cm 2 It was carried out at.

[0065] HCT-116 cells were plated at 40,000 cells / well (250,000 cells / cm) in half-area 96-well plates onto an oxygen-sensing surface functionalized with 300 μg mL collagen I. 2 ) were seeded. The next day, the culture medium was replaced with medium containing 2-[[4-(trifluoromethoxy)phenyl]hydrazinylidene]propanedinitrile (FCCP), an uncoupler of mitochondrial oxidative phosphorylation that increases oxygen consumption. The results showed that oxygen consumption increased as the FCCP concentration increased up to 4 μM (Figure 6, upper panel). HCT-116 cells were further treated with 2 μM rotenone, an inhibitor of oxidative phosphorylation. The results showed that rotenone treatment suppressed oxygen consumption in these cells (Figure 6, lower panel).

[0066] Similar studies were performed with additional cell lines, HEP-G2 (Figure 7) and PC-3 (Figure 8). Results with these cells also showed that oxygen consumption increased with increasing cell density. Similarly, in HEP-G2 and PC-3, FCCP treatment increased oxygen consumption, while rotenone treatment decreased it.

[0067] Experiments conducted during the development of embodiments herein demonstrated that the use of an oxygen barrier in the form of aluminum foil over the wells containing assay reagents further enhanced the oxygen rate changes observed with the present invention (Figure 9).

Claims

1. A polysiloxane matrix, (a) a bis(trialkoxysilyl) monomer of formula (I): 【Chemical 1】 (In the formula, R 1 , R 1’ , R 1” , R 2 , R 2’ and R 2” are independently CH 3 , C.H. 2 CH 3 , (CH 2 ) 2 CH 3 and L is selected from C 1 -C 6 Alkyl, C 1 -C 6 Heteroalkyl, C 2 -C 6 Alkenyl, C 2 -C 6 Heteroalkenyl, C 2 -C 6 Alkynyl, C 1 -C 6 heteroalkynyl, 【Chemistry 2】 L′ and L″ are independently selected from a covalent bond, C 1 -C 6 alkyl or heteroalkyl, C 2 -C 6 alkenyl or heteroalkenyl, C 2 -C 6 alkynyl or heteroalkynyl; 1 and L 2 is independently selected from an aryl or heteroaryl ring; L 1 and L 2 are linked by a covalent bond, and L, L', L", and L 1 and L 2 is optionally branched or substituted), and (b) Dialkyldialkoxysilane monomers of formula (II): 【Chemistry 3】 (In the formula, R 3 , R 3’ , R 4 and R 4’ are independently CH 3 , C.H. 2 CH 3 , and (CH 2 ) 2 CH 3 and

2. (i) R 1 , R 1’ , R 1” , R 2 , R 2’ and R 2” are the same, (ii) if present, L′ and L″ are the same; (iii) if present, L 1 and L 2 are the same, (iv) R 3 and R 3’ are the same, and / or (v) R 4 and R 4” The polysiloxane matrix of claim 1 , wherein

3. R 1 , R 1’ , R 1” , R 2 , R 2’ and R 2” is CH 3 or CH 2 CH 3 2. The polysiloxane matrix of claim 1, wherein:

4. L is (CH 2 ) 1-6 , (CH 2 ) 1-6 -NH-(CH 2 ) 1-6 , (CH 2 ) 1-6 -O-(CH 2 ) 1-6 , (CH 2 ) 0-6 -aryl-(CH 2 ) 1-6 , and (CH 2 ) 1-6 -aryl-aryl-(CH 2 ) 1-6 2. The polysiloxane matrix of claim 1, wherein the polysiloxane matrix is ​​selected from:

5. 2. The polysiloxane matrix of claim 1, wherein the bis(trialkoxysilyl) monomer is selected from 1,4-bis(trimethoxysilylethyl)benzene, 1,4-bis(trimethoxysilylmethyl)benzene, 1,4-bis(trimethoxysilyl)benzene, 4,4'-bis(triethoxysilyl)-1,1'-biphenyl, 1,2-bis(triethoxysilyl)ethane, and bis[3-(trimethoxysilyl)propyl]amine.

6. R 3 , R 3’ , R 4 , R 4’ is CH 3 or CH 2 CH 3 2. The polysiloxane matrix of claim 1, wherein:

7. 2. The polysiloxane matrix of claim 1, wherein the bis(trialkoxysilyl) monomer is 1,4-bis(trimethoxysilylethyl)benzene and the dialkyldialkoxysilane monomer is dimethyldimethoxysilane.

8. A composition comprising the polysiloxane matrix of any one of claims 1 to 7 and an oxygen-sensitive fluorophore.

9. The oxygen-sensitive fluorophores include tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) chloride (Ru-dpp), platinum octaethylporphyrin, platinum(II) 2,3,7,8,12,13,17,18-octaethyl-21H,23H-porphyrin (PtOEP), palladium(II) octaethylporphine (PdOEP), platinum(II)-5,10,15,20-tetrakis-(2,3,4,5,6-pentafluorophenyl)-porphyrin (PtTfPP), palladium(II)-5,10,15,20-tetrakis-(2,3,4,5, 9. The composition of claim 8, wherein the palladium(II) octaethylporphyrin ketone (PtOEPK), platinum(II) octaethylporphyrin ketone (PdOEPK), platinum(II) tetraphenyltetrabenzoporphyrin (PtTPTBP), meso-tetraphenyl-tetrabenzoporphyrin palladium complex (PdTPTBP), platinum(II) tetraphenyltetranaphthoporphyrin (PtTPTNP), and palladium(II) tetraphenyltetranaphthoporphyrin (PdTPTNP).

10. The composition of claim 8 , further comprising an oxygen-insensitive fluorophore.

11. 11. The composition of claim 10, wherein the oxygen-insensitive fluorophore is selected from Nile blue chloride, tris(8-hydroxyquinolinato)aluminum (AlQ3), TAMRA, and coumarin-6.

12. The composition of claim 10, wherein the oxygen-insensitive fluorophore is coumarin-6 and the oxygen-sensitive fluorophore is PtTfPP.

13. A thin film comprising the composition of any one of claims 8 to 12.

14. A device comprising a solid surface having the thin film of claim 13 deposited thereon.

15. The device of claim 14 , wherein the solid surface is a glass or polystyrene surface.

16. 15. The device of claim 14, wherein the solid surface is a slide or the bottom of a microwell.

17. The device of claim 14 , further comprising a layer of bioactive extracellular matrix proteins or biocompatible molecules over the thin film.

18. 18. The device of claim 17, wherein the layer of bioactive extracellular matrix proteins comprises one or more proteins selected from type I collagen, type IV collagen, fibronectin, and laminin.

19. The device of claim 17 , wherein the layer of biocompatible molecules comprises polydopamine.

20. 18. The device of claim 17, wherein the bioactive extracellular matrix protein is passively coated onto the thin film.

21. 18. The device of claim 17, wherein the bioactive extracellular matrix protein is covalently or non-covalently attached to the thin film.

22. 1. A method comprising: (a) (i) contacting an oxygen-sensitive fluorophore embedded within a polysiloxane matrix with (ii) a test solution; (b) exposing the oxygen-sensitive fluorophore to light within the excitation spectrum of the oxygen-sensitive fluorophore; (c) detecting light within the emission spectrum of the oxygen-sensitive fluorophore; and (d) determining the oxygen concentration in the test solution.

23. 23. The method of claim 22, wherein determining the oxygen concentration in the test solution comprises comparing light within the emission spectrum of the oxygen-sensitive fluorophore to a reference value.

24. 24. The method of claim 23, wherein the reference values ​​are the amount of light emitted when the test solution is saturated with oxygen and the amount of light emitted when the test solution is depleted of oxygen.

25. 24. The method of claim 23, wherein the reference values ​​are the amount of light emitted at different oxygen concentrations.

26. 23. The method of claim 22, wherein the test solution comprises cells.

27. 27. The method of claim 26, wherein steps (b) through (d) are repeated to monitor oxygen consumption by the cells over time.

28. 23. The method of claim 22, wherein the polysiloxane matrix is ​​a film deposited on a transparent solid surface.

29. 30. The method of claim 28, wherein the solid surface is glass or polystyrene.

30. 29. The method of claim 28, wherein the solid surface is a slide or the bottom of a microwell.

31. 30. The method of claim 28, further comprising a layer of a bioactive extracellular matrix protein or biocompatible molecule on the membrane.

32. 32. The method of claim 31, wherein the layer of bioactive extracellular matrix proteins comprises one or more proteins selected from type I collagen, type IV collagen, fibronectin, and laminin.

33. 32. The method of claim 31 , wherein the layer of biocompatible molecules comprises polydopamine.

34. 23. The method of claim 22, wherein the polysiloxane matrix also has embedded therein an oxygen-insensitive fluorophore.

35. 35. The method of claim 34, wherein the oxygen-insensitive fluorophore is selected from Nile blue chloride, tris(8-hydroxyquinolinato)aluminum (AlQ3), TAMRA, and coumarin-6.

36. 35. The method of claim 34, further comprising exposing the oxygen-insensitive fluorophore to light within the excitation spectrum of the oxygen-insensitive fluorophore and detecting light within the emission spectrum of the oxygen-insensitive fluorophore.

37. 37. The method of claim 36, wherein determining the oxygen concentration in the test solution comprises comparing a reference value to the ratio of (i) light within the emission spectrum of the oxygen-sensitive fluorophore to (ii) light within the emission spectrum of the oxygen-insensitive fluorophore.

38. 38. The method of claim 37, wherein the reference values ​​are the ratio of light emitted when the test solution is saturated with oxygen and the ratio of light emitted when the test solution is depleted of oxygen.

39. 38. The method of claim 37, wherein the reference value is a ratio of light emitted at different oxygen concentrations.

40. The polysiloxane matrix is (a) a bis(trialkoxysilyl) monomer of formula (I): 【Chemistry 4】 (In the formula, R 1 , R 1’ , R 1” , R 2 , R 2’ and R 2” are independently CH 3 , C.H. 2 CH 3 , (CH 2 ) 2 CH 3 and L is selected from C 1 -C 6 Alkyl, C 1 -C 6 Heteroalkyl, C 2 -C 6 Alkenyl, C 2 -C 6 Heteroalkenyl, C 2 -C 6 Alkynyl, C 1 -C 6 heteroalkynyl, 【Chemistry 5】 L′ and L″ are independently selected from a covalent bond, C 1 -C 6 alkyl or heteroalkyl, C 2 -C 6 alkenyl or heteroalkenyl, C 2 -C 6 alkynyl or heteroalkynyl; 1 and L 2 is independently selected from an aryl or heteroaryl ring; L 1 and L 2 are linked by a covalent bond, and L, L', L", and L 1 and L 2 is optionally branched or substituted), and (b) Dialkyldialkoxysilane monomers of formula (II): 【Chemistry 6】 (In the formula, R 3 , R 3’ , R 4 and R 4’ are independently CH 3 , C.H. 2 CH 3 , and (CH 2 ) 2 CH 3 and

41. (i) R 1 , R 1’ , R 1” , R 2 , R 2’ and R 2” are the same, (ii) if present, L′ and L″ are the same; (iii) if present, L 1 and L 2 are the same, (iv) R 3 and R 3’ are the same, and / or (v) R 4 and R 4” 41. The method of claim 40, wherein

42. R 1 , R 1’ , R 1” , R 2 , R 2’ and R 2” is CH 3 or CH 2 CH 3 41. The method of claim 40, wherein:

43. L is (CH 2 ) 1-6 , (CH 2 ) 1-6 -NH-(CH 2 ) 1-6 , (CH 2 ) 1-6 -O-(CH 2 ) 1-6 , (CH 2 ) 0-6 -aryl-(CH 2 ) 1-6 , and (CH 2 ) 1-6 -aryl-aryl-(CH 2 ) 1-6 41. The method of claim 40, wherein the

44. 41. The method of claim 40, wherein the bis(trialkoxysilyl) monomer is selected from 1,4-bis(trimethoxysilylethyl)benzene, 1,4-bis(trimethoxysilylmethyl)benzene, 1,4-bis(trimethoxysilyl)benzene, 4,4'-bis(triethoxysilyl)-1,1'-biphenyl, 1,2-bis(triethoxysilyl)ethane, and bis[3-(trimethoxysilyl)propyl]amine.

45. R 3 , R 3’ , R 4 , R 4’ is CH 3 or CH 2 CH 3 41. The method of claim 40, wherein:

46. 41. The method of claim 40, wherein the bis(trialkoxysilyl) monomer is 1,4-bis(trimethoxysilylethyl)benzene and the dialkyldialkoxysilane monomer is dimethyldimethoxysilane.

47. 41. The method of claim 40, wherein the oxygen-sensitive fluorophore is selected from Ru-dpp, PtOEP, PdOEP, PtTfPP, PdTFPP, PtOEPK, PdOEPK, PtTPTBP, PdTPTBP, PtTPTNP, and PdTPTNP.

Images