Compound for glucose sensing and uses thereof

Abstract

The present invention is directed to intracellular and extracellular glucose indicators and use thereof in a method for determining glucose concentration within a cell and its surrounding. Furthermore, there is provided a method for intracellular delivery of glucose using the intracellular indicator of the invention.

Description

COMPOUND FOR GLUCOSE SENSING AND USES THEREOFCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 63 / 530,493, filed 03 Aug. 2023 and U.S. Provisional Patent Application No. 63 / 636,787, filed 21 April 2024. The contents of the above applications are all incorporated by reference as if fully set forth herein in their entirety.FIELD OF INVENTION

[0002] The present invention relates to the field of glucose sensing and glucose quantification.BACKGROUND OF THE INVENTION

[0003] There is a need for a non-invasive and highly specific method for a real-time measurement of intracellular glucose concertation. Currently, glucose concentration in the cell culture medium can be detected using water-soluble molecular fluorescent indicators. However, due to their hydrophilic nature, these water-soluble indicators usually have an almost neglecting cell penetration ability. Thus, there is a long-felt but unsolved need for new fluorescent molecular indicators characterized by efficient cell penetration and cell accumulation, for selective real-time intracellular detection of glucose.SUMMARY OF THE INVENTION

[0004] In one aspect of the invention, provided herein a compound, including any salt, any hydrate, or any solvate thereof, wherein the compound is represented by Formula 1 :, wherein X is -O- or -N-; each a and b is an integer being independently between 1 and 10; if X is O than R is H, and if X is N than each R independently is selected from hydrogen, optionally substituted C1-C10 alkyl, optionally substituted C3-C10 cycloalkyl, optionally substituted C3-C10 heterocyclyl, optionally substituted heteroaryl, optionally substituted aryl, or a combination thereof; each R2 independently is selected from (i) electron withdrawing group and (ii) halo, haloalkyl, a cyano group, carboxy, amide, carbonyl, anhydride, carbonate ester, carbamate, a sulfonyl group, a sulfonate group, a sulfinyl group, a sulfonamide group, an azo group, a guanidine group, and ammonium group, or any combination thereof; and A represents any of cycloalkyl, aryl, and heteroaryl, a fused aryl, or a fused cycloalkyl or any combination thereof, each option of the above may be substituted or non-substituted.

[0005] In another aspect of the invention, provided herein a compound represented by Formula 1A including any salt, any hydrate, or any solvate thereof:wherein each a and b is an integer being independently between1 and 10; each R independently is selected from H, an optionally substituted Cl -CIO alkyl, -OR’, -NR’R’, optionally substituted C3-C10 cycloalkyl, optionally substituted C3- C10 heterocyclyl, optionally substituted heteroaryl, optionally substituted aryl, or a combination thereof, or both R are interconnected to form a cyclic structure; each R’ independently is selected from H, optionally substituted Cl -CIO alkyl, optionally substituted C3-C10 cycloalkyl, optionally substituted C3-C10 heterocyclyl, optionally substituted heteroaryl, optionally substituted aryl, or a combination thereof; each R2 independently is selected from halo, haloalkyl, a cyano group, carboxy, amide, carbonyl, anhydride, carbonate ester, carbamate, a sulfonyl group, a sulfonate group, a sulfinyl group, a sulfonamide group, an azo group, a guanidine group, and ammonium group, or any combination thereof; and A represents any of cycloalkyl, aryl, and heteroaryl, a fused aryl, or a fused cycloalkyl or any combination thereof, each option of the above may be substituted or non-substituted.

[0006] In one embodiment, the compound is represented by Formula:

[0007] In one embodiment, the haloalkyl is a fluoroalkyl.

[0008] In one embodiment, R2 is in para position to B(OH)2 moiety; and wherein the a is between 1 and 5.

[0009] In one embodiment, the compound is:

[0010] In one embodiment, the compound is a glucose indicator configured to emit fluorescence upon complexing glucose.

[0011] In one embodiment, the glucose indicator is configured to selectively detect intracellular glucose at a pH in a range between about 4 and 10.

[0012] In another aspect, there is provided a composition, comprising the compound of the invention and a liquid carrier.

[0013] In one embodiment, the liquid carrier is an aqueous carrier.

[0014] In one embodiment, the composition is a solution and wherein a concentration of the compound within the is between 10 uM and 0.1 M.

[0015] In one embodiment, the compound is represented by Formula 6:wherein each R2 is an electron-withdrawing group; each a is an integer being independently between 1 and 10; and each A represents any of cycloalkyl, aryl, and heteroaryl, a fused aryl, a fused cycloalkyl, substituted or non-substitute or any combination thereof.

[0016] In one embodiment, the electron- withdrawing group is a moiety selected from: nitro, cyano, ammonium, haloalkyl, halo, sulfonyl and carbonyl.

[0017] In one embodiment, A is aryl.

[0018] In one embodiment, the compound is represented by Formula 7:

[0019] In one embodiment, R2 is selected from halo and haloalkyl, and wherein R2 is in para position to the B(OH)2 moiety.

[0020] In one embodiment, the haloalkyl is a fluoroalkyl.

[0021] In one embodiment, a is 1.

[0022] In one embodiment, the compound comprises:including any salt thereof.

[0023] In one embodiment, the compound is a glucose indicator is configured to selectively detect intracellular glucose at a pH in a range between about 4 and 10.

[0024] In another aspect, there is provided a method for determining a concentration of glucose within a cell, the method comprising: (i) contacting the cell with the compound of the invention, or with the composition of the invention, to obtain a complex; (ii) irradiating the cell at a first wavelength suitable for excitation of the complex; (iii) measuring fluorescence emitted from the cell to obtain a fluorescence value, and (iv) determining the concentration based on the fluorescence value.

[0025] In one embodiment, the method is for determining glucose concentration in- vitro.

[0026] In one embodiment, measuring is performed by a fluorescence detector at a second wavelength, wherein the second wavelength is within an emission spectrum of the glucose complex.

[0027] In one embodiment, the first wavelength is in a range between 300 and 400 nm; and wherein the second wavelength is in a range between 400 and 500 nm.

[0028] In one embodiment, the cell is an aggregate of cells.

[0029] In one embodiment, the aggregate of cells comprises a spheroid, and wherein the contacting is for a time sufficient for internalization of the compound into the spheroid.

[0030] In one embodiment, the concentration is in a range between 0.0001 and 10 mM; and wherein the cell further comprises a pharmaceutically active ingredient.

[0031] In another aspect, there is provided an immobilized extracellular glucose indicator including any salt, any hydrate, or any solvate thereof, wherein the extracellular glucose indicator is represented by Formula 4:wherein each wavy bond represents an attachment point to a polymeric material or H, and at least one wavy bond is the attachment point; each a is an integer being independently between 3 and 20; each R2 independently is selected from halo, haloalkyl, a cyano group, carboxy, amide, carbonyl, anhydride, carbonate ester, carbamate, a sulfonyl group, a sulfonate group, a sulfinyl group, a sulfonamide group, an azo group, a guanidine group, and ammonium group, or any combination thereof; X represents ethyl, CNR’2, NH, O, S, -CONH-, -CONR’-, -C(=NH)NR’-, -C(=S)NR’-, - NC(=O)-, -NC(=O)O-, -NC(=O)N-, -NC(=S)O-, -NC(=S)N-, -C(=O)-, -C(=O)O-, -OC(=O)O-, -OC(=O)N-, -OC(=S)O-, -OC(=S)N-,, or phosphate; wherein each R is selected from H and Cl -CIO alkyl; and A represents any of cycloalkyl, aryl, and heteroaryl, a fused aryl, or a fused cycloalkyl or any combination thereof.

[0032] In one embodiment, R2is haloalkyl, wherein X is -CONH-; and wherein A is phenyl.

[0033] In one embodiment, the extracellular glucose indicator is represented byFormula 5:wherein the a is between 5 and 10.

[0034] In one embodiment, each a is 6, and wherein each wavy bond represents the attachment point.

[0035] In another aspect, there is provided a method for determining a concentration of glucose both in a cell and in a cell medium, the method comprising: (i) contacting the cell attached to a cell support with the compound of the invention, to obtain a first complex comprising the compound bound to glucose within the cell; wherein the cell support comprises the immobilized extracellular glucose indicator of the invention; and wherein the immobilized extracellular glucose indicator is configured for binding glucose within the cell medium to obtain a second complex; (ii) irradiating the cell at a first wavelength suitable for excitation of the first complex, measuring a first fluorescence to obtain a first fluorescence value, and determining a concentration of glucose within the cell based on the first fluorescence value; and (iii) irradiating the cell support at a second wavelength suitable for excitation of the second complex, measuring a second fluorescence to obtain a second fluorescence value, and determining a concentration of glucose within the cell medium based on the second fluorescence value.

[0036] In another aspect, there is provided a method for determining glucose concentration within an aqueous liquid, the method comprising: (i) contacting the aqueous liquid with the compound of the invention, to obtain a obtain a complex comprising the compound bound to glucose; (ii) irradiating the aqueous liquid at a wavelength suitable for excitation of the complex; (iii) measuring fluorescence emitted by complex to obtain a fluorescence value, and (iv) determining glucose concentration within the aqueous liquid based on the fluorescence value.

[0037] In one embodiment, the aqueous liquid is selected from an aqueous solution, a biological fluid, a cell culture medium or a cell culture medium in contact with a cell.

[0038] In one embodiment, the compound of the invention is for use in the delivery of glucose into a cell.

[0039] In one embodiment, delivery comprises in-vivo, ex-vivo and in-vitro; and wherein the cell is a human cell.

[0040] In one embodiment, the cell is an aggregate of cells.

[0041] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[0042] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0043] The subject matter of the invention is particularly pointed out and distinctly claimed in the concluding section of the specification. The invention, however, both as to organization and method of operation, together with objects, features, and advantages thereof, may be best understood by reference to the following detailed description when read with the accompanying drawings in which:

[0044] Figs. 1A1-1B6 present chemical structure representation of the boronic acid based glucose sensors (1A1) BA, (1A2) BA_2, (1A3) BA_3, (1A4) BA_4, (1A5) BA_5 and (1A6) Mc-CDB A depicting fluorescence emission / excitation spectra for each glucosesensor, respectively (Figs. 1A1-1A6). Fluorescent microscopy images of the boronic acid based glucose sensors (same as in Figs. 1A1-1A6, respectively) in 3D InSight™ human liver microtissues incubated with 20 pM glucose probes treated for 2h (0.1% DMSO), in (-) glucose 3D InSight™ TOX liver medium and imaged 48h later (Figs. 1B1-1B6).

[0045] Figs. 2A-2F. Photophysical properties of extracellular glucose probe (BA), structure depicted in Fig. 1A1. (2A) Schematic representation of the sensing mechanism of BA for glucose. (2B) UV-visible absorption spectrum of 25 pM BA in TLM + / - glucose. (2C) Fluorescence excitation and emission spectra of BA before and after glucose addition (Xex= 378 nm, Z.em = 430 nm). (2D) Fluorescence response of BA (100 pM) in response to glucose, xylose, mannose, galactose, and sucrose (0-10 mM). (2E) Linear relationship of the fluorescence intensity of BA (800 pM) versus different glucose concentrations, in TLM (pH 7.4). (2F) pH-dependent (pH from 2 to 12) fluorescence emission intensity of BA (100 pM) in TLM + / - glucose. Schematic representation of the proposed mechanism of the fluorescence sensing of BA for the turn-on detection of glucose. Data are presented as the mean+SD (n=4).

[0046] Figs. 3A-3F. (3A) Schematic illustration of the sensing mechanism of BA 5 before and after glucose addition (Xex = 378 nm, Z.em = 430 nm). (3B) UV-visible absorption spectrum of BA_5 (100 pM) in (-) and (+) glucose 3D InSight™ TOX Liver Medium. (3C) Fluorescence excitation and emission spectra of BA_5 before and after glucose addition (Xex = 378 nm, Z.em = 430 nm) (3D) Fluorescence intensity of BA 5 (100 pM) upon the addition of glucose concentrations (0-200 mM) in PBS. (3E) pH- dependent (pH from 2 to 12) fluorescence emission intensity of BA 5 (100 pM) in TLM + / - glucose. Schematic representation of the proposed fluorescence BA 5 sensing mechanism vs Mc-CDBA for the turn-on detection of glucose. (3F) Microscopy imaging and intensity quantification of the level of cellular permeability of BA 5 vs BA and Mc- CDBA. 3D human liver spheroids incubated with BA, BA 5 and Mc-CDBA (all in 20 pM) for 2 h (0.1% DMSO), in glucose-free TLM and imaged 24 h later. Data are presented as the mean+SD (n=4).

[0047] Figs. 4A-4E. Application and photophysical properties of hydrogel-based glucose probe (BA_21). (4A) Schematic representation of the sensing mechanism of BA_21. (4B) Calculated differences in spectra obtained by subtracting the UV-visibleabsorption spectrum of BA_21 hydrogel from the hydrogel only (+) glucose 3D InSight™ TOX Liver Medium. (4C) Glucose dose dependent fluorescence emission spectra of hydrogel embedded BA_21 in 3D InSight™ TOX Liver Medium (Lex = 370 nm, Xem = 390-580 nm, step 2 nm, gain 50). BA_21 emission spectra at pH 2 and pH 7.4 (4D) pH- dependent (pH from 2 to 12) fluorescence emission intensity of BA-21, 5 pL hydrogel in (-) vs (+) glucose 3D InSight™ TOX Liver Medium. (4E) BA_21 hydrogel fluorescent behavior in a glucose dose dependent manner . Data are presented as the means ± SD (n = 4).

[0048] Figs. 5A-5C. (5A) Representative images of 3D InSight™ Human Liver Microtissues stained with BA_5 (5) Glucose probe at 20 pM (Blue) for 2h. Once stained the microtissues were treated for 24 hours with Cyclospurine at 5, 2.5, 1.25 and 0.625 pM respectively and imaged 48h later. (5B) Fluorescence image intensity quantification upon the addition of increased concentrations of Cyclospurine. (5C) Media based glucose uptake quantification in response to Cyclospurine treatment at 5, 2.5, 1.25 and 0.625 pM respectively after 72h.

[0049] Figs. 6A-6F present non-limiting configurations of the exemplary article of the invention compatible with the 96-well cell culture plate. Cross-section view of the article (6A), zoom-in (6B), and side view of the article (6C). Cross-section view of the article inserted into 96-well cell culture plate (6D) and zoom-in (6E). Figure 6F is an illustration of a cross-section view of a single insert with the cell support immersed into a well containing cell culture medium.

[0050] Fig. 7 is a schematic illustration depicting a non-limiting exemplary configuration of the disclosed sensing device.

[0051] Figs. 8A-8B are graphs demonstrating the fluorescence intensity emitted by the compound of the invention BA (1) upon incubation with glucose (8A) and lactate (8B).

[0052] Figs. 9A-9B are bar graphs demonstrating the fluorescence intensity of the exemplary compound of the invention BA (1) (termed herein as “indicator 1”) in response to varying glucose concentration in the liver spheroid culture cell medium over time in the presence of Tolcapone (9A) and Entacapone (9B).

[0053] Figs. 10A-10B Glucose probe derivatives activity (10A) Fluorescence intensity of BA at 0, 25, 50, 100 pM in 0.1% DMSO at 3D InSight™ TOX Liver Medium. (10B)Fluorescence intensity of BA_2 at 0, 25, 50, 100 pM in 0.1% DMSO at 3D InSight™ TOX Liver Medium. (10C) Fluorescence intensity of BA_3 at 0, 25, 50, 100 pM in 0.1% DMSO at 3D InSight™ TOX Liver Medium. (10D) pH-dependent (pH from 2 to 12) fluorescence emission intensity of BA (100 pM) in (-) vs (+) glucose 3D InSight™ TOX Liver Medium. Schematic representation of the proposed fluores-cence sensing mechanism of B A_2 vs BA_3 for the tum-on detection of glucose. Data are presented as the mean+SD (n=4). Statistical analysis was performed using Mann Whitney test and normality test. ***p<0.001.

[0054] Figs. 11A-11B include bar graphs demonstrating a real-time detection of glucose in low and high glucose concentration cell culture medium of brain spheroids, before and after addition of a drug(s).

[0055] Fig. 12 includes a vertical bar showing a real time detection of glucose in low and high glucose concentration cell culture medium of brain spheroids.DETAILED DESCRIPTION OF THE PRESENT INVENTION

[0056] In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the invention. However, it will be understood by those skilled in the art that the present invention may be practiced without these specific details. In other instances, well-known methods, procedures, and components have not been described in detail so as not to obscure the present invention.

[0057] In one aspect of the invention, there is provided a compound represented by Formula 1 including any salt, any hydrate, or any solvate thereof:, wherein X is -O- or -N-; each a and b is an integer being independently between 1 and 10; if X is O than R is H, and if X is N than each R independently is selected from hydrogen, optionally substituted C1-C10 alkyl, optionally substituted C3-C10 cycloalkyl, optionally substituted C3-C10 heterocyclyl, optionally substituted heteroaryl, optionally substituted aryl, or a combination thereof; each R2 independently is selected from (i) electron withdrawing group and (ii) halo, haloalkyl, a cyano group, carboxy, amide, carbonyl, anhydride, carbonate ester, carbamate, a sulfonyl group, a sulfonate group, a sulfinyl group, a sulfonamide group, an azo group, a guanidine group, and ammonium group, or any combination thereof; and A represents any of cycloalkyl, aryl, and heteroaryl, a fused aryl, or a fused cycloalkyl or any combination thereof, each option of the above may be substituted or non-substituted.

[0058] In some embodiments, the compound of the invention is water soluble. In some embodiments, the compound of the invention is a glucose indicator configured to emit fluorescence upon complexing glucose in a liquid. In some embodiments, the compound of the invention is capable of selectively detecting glucose in the liquid, wherein the liquid is a solution (e.g., an aqueous solution, an organic solution, or a combination of an aqueous and organic solvent).

[0059] The term “detecting”, as used herein, encompasses generation of a fluorescent signal upon binding to glucose. A skilled artisan will appreciate that the fluorescent signal can be detected by conventional means, such as a fluorimeter.

[0060] In some embodiments, the compound of the invention comprises an intracellular glucose indicator (e.g. a compound of Formula 1A), as described hereinbelow. In some embodiments, the compound of the invention comprises an extracellular glucose indicator (e.g. a compound of Formula 6), as described hereinbelow.Intracellular Glucose Indicator

[0061] In some embodiments, the compound is represented by Formula 1, wherein X is N. In some embodiments, the compound is represented by Formula 1A:each a is an integer being independently between 1 and 10; and wherein each b is independently between 0 and 10; each R independently is H or is selected from an optionally substituted C1-C10 alkyl, -OR’, -NR’R’, optionally substituted C3-C10 cycloalkyl, optionally substituted C3-C10 heterocyclyl, optionally substituted heteroaryl, optionally substituted aryl, or a combination thereof, or both R are interconnected to form a cyclic structure; each R’ independently is selected from H, optionally substituted Cl- C10 alkyl, optionally substituted C3-C10 cycloalkyl, optionally substituted C3-C10 heterocyclyl, optionally substituted heteroaryl, optionally substituted aryl, or a combination thereof; each R2 independently is selected from halo, haloalkyl, a cyano group, carboxy, amide, carbonyl, anhydride, carbonate ester, carbamate, a sulfonyl group,a sulfonate group, a sulfinyl group, a sulfonamide group, an azo group, a guanidine group, and ammonium group, or any combination thereof; and A represents any of cycloalkyl, aryl, and heteroaryl, a fused aryl, or a fused cycloalkyl or any combination thereof, each option of the above may be independently substituted or non-substituted.

[0062] In some embodiments, a is between 1 and 10, including any range in between. In some embodiments, a is between 1 and 8, including any range in between. In some embodiments, a is between 1 and 5, including any range in between. In some embodiments, a is between 1 and 3, including any range in between. In some embodiments, a is between 1 and 3 and b is 1 or 2. In some embodiments, a is between 1 and 3 and b is 1.

[0063] In some embodiments, A is an aromatic ring.

[0064] In some embodiments, R2 represents 1, 2, 3 or 4 substituents, each independently selected from halo, haloalkyl, a cyano group, carboxy, amide, carbonyl, anhydride, carbonate ester, carbamate, a sulfonyl group, a sulfonate group, a sulfinyl group, a sulfonamide group, an azo group, a guanidine group, and ammonium group, or any combination thereof. In some embodiments, R2 represents 1, 2, 3 or 4 haloalkyl substituents (e.g., CF3, CI3, CC13 or CBr3). In some embodiments, at least one R2 is halo or haloalkyl.

[0065] In some embodiments, the compound is represented by Formula 1, wherein a is between 1 and 3; b is 1 or 2 and R2 is selected from halo and haloalkyl. In some embodiments, the compound is represented by Formula 1, wherein a is between 1 and 3; b is 1 or 2 and R2 is a fluoroalkyl. In some embodiments, R2 represent CF3.

[0066] The term “haloalkyl” refers to C1-C10 alkyl as described herein, substituted by at least one (e.g. 1, 2, 3, 4 or more) halide atoms, wherein halide is selected from F, Br, Cl, and I, or a combination thereof.

[0067] In some embodiments, R2 is in an ortho-, meta-, and / or para-position to the B(OH)2 moiety. In some embodiments, R2 is in a para position to the B(OH)2 moiety.

[0068] In some embodiments, the compound is represented by Formula 1, wherein a is between 1 and 3; b is 1 or 2; wherein R2 is selected from halo and haloalkyl; and wherein A is an aromatic / heteroaromatic ring. In some embodiments, the compound is representedby Formula 1, wherein a is between 1 and 3; b is 1; wherein R2 is selected from halo and haloalkyl; and wherein A is an aromatic / heteroaromatic ring; and wherein each R independently is H, or C1-C10 alkyl.

[0069] In some embodiments, the compound is represented by Formula 2:, 2 are as disclosed herein above; and wherein each R is independently H or C1-C10 alkyl. In some embodiments, the compound is represented by Formula 2 or 2 A, wherein R2 is selected from fluoro and fluoroalkyl. In some embodiments, R2 is CF3. In some embodiments, a is between 2 and 5, or is selected from 1, 2, 3, 4, 5 and 6.

[0070] In some embodiments, the compound is represented by Formula 2 or 2 A, wherein a is between 1 and 3, between 1 and 4 or between 1 and 5; and wherein R2 represent a single substituent in para position to B(OH)2 moiety.

[0071] In some embodiments, the compound is represented by Formula 3:, g y hereof, wherein R and R2 are as described above; and wherein R” represents one or more substituents each independently selected from H, OH, oxo, carbonyl, halogen, OR’, -NO2, -CN, -CONH2, -CONR’2, -CNNR’2, - CSNR’2, -CONH-OH, -CONH-NR’2, -NHCOR’, -NHCSR’, -NHCNR, -NC(=O)OR’, -NC(=O)NR’, -NC(=S)OR’, -NC(=S)NR’, -SO2R’, -SOR’, -SR’, -SO2OR’, -SO2N(R’)2, -NHNR’2, -NNR’, C1-C6 haloalkyl, optionally substituted C1-C6 alkyl, - NR’2, -NH(C1-C6 alkyl), -N(C1-C6 alkyl)2, C1-C6 alkoxy, C1-C6 haloalkoxy,hydroxy(Cl-C6 alkyl), hydroxy(Cl-C6 alkoxy), alkoxy(Cl-C6 alkyl), alkoxy(Cl-C6 alkoxy), C1-C6 alkyl-NR’2, C1-C6 alkyl-SR’, -CONH(C1-C6 alkyl), -CON(C1-C6 alkyl)2, -CO2H, -CO2R’, -OCOR, -OCOR’, -OC(=O)OR’, -OC(=O)NR’, -OC(=S)OR’, -OC(=S)NR’, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, optionally substituted heterocyclic alkyl, or a combination thereof; wherein each R’ is independently hydrogen or is selected from the group comprising: optionally substituted Cl-C 10 alkyl, optionally substituted C3-C10 cycloalkyl, optionally substituted C3-C10 heterocyclyl, optionally substituted heteroaryl, optionally substituted aryl, or a combination thereof.

[0072] In some embodiments, the compound is represented by Formula 3, 3” or 3A, wherein each R is independently H or Cl -CIO alkyl, and wherein R2 is selected from halo (e.g. fluoro) and haloalkyl (e.g. fluoroalkyl). In some embodiments, the compound is represented by Formula 3 or 3 A, wherein each R is independently H or Cl -CIO alkyl, a is between 1 and 5, or between 1 and 3; and wherein R2 is selected from halo (e.g. fluoro) and haloalkyl (e.g. fluoroalkyl). In some embodiments, the compound is represented by Formula 3, 3” or 3A, wherein R2 is selected from fluoro and fluoroalkyl; and wherein a is between 1 and 5, or between 1 and 3. In some embodiments, R2 is haloalkyl; and wherein b is 1 or 2.

[0073] In some embodiments, the compound is:, including any salt, any hydrate, and / or any solvate thereof.

[0074] In some embodiments, the compound of the invention (intracellular glucose indicator) has a cell membrane penetration ability. In some embodiments, the cell comprises an aggregate of cells. In some embodiments, the aggregate of cells comprises a spheroid.

[0075] In some embodiments, the compound of the invention (i.e. the intracellular glucose indicator) is configured of complexing glucose in a liquid. In some embodiments, the intracellular glucose indicator is configured of selectively complexing glucose in a liquid, wherein the liquid further comprises additional natural compounds (e.g. additional sugars other than glucose, such as fructose, proteins, nucleotides or any other natural compounds).

[0076] In some embodiments, the intracellular glucose indicator is a glucose indicator. In some embodiments, the intracellular glucose indicator is a fluorescent glucose indicator. In some embodiments, the intracellular glucose indicator has an enhanced binding affinity (and / or selectivity) to glucose, as compared to additional sugars, such as lactose, fructose, etc. In some embodiments, the intracellular glucose indicator is a selective glucose indicator.

[0077] In some embodiments, enhanced binding affinity (or selectivity) comprises at least 5, at least 10, at least 100, at least 1000, at least 10,000 times greater binding affinity to glucose, as compared to structural similar molecules (e.g., mono-, or di-sugars such as lactose, lactate, fructose, isomers of glucose, etc.). In some embodiments, the intracellular glucose indicator is capable of emitting a fluorescent signal upon binding to glucose in a liquid (e.g. cytoplasm), wherein the intensity of the fluorescent signal is in direct correlation with the glucose concentration within the liquid, wherein the liquid is as described herein. In some embodiments, the liquid is a biological fluid. In some embodiments, the liquid is a cytoplasm.

[0078] In some embodiments, the intracellular glucose indicator is capable of emitting a concentration dependent fluorescent signal in response to glucose concentration within the liquid being up to 10 mM, up to 1 mM, up to 0.5 mM, between 0.0001 and 10 mM, between 0.001 and 10 mM, between 0.01 and 10 mM, including any range between. In some embodiments, the intracellular glucose indicator is capable of selectively detecting intracellular glucose. In some embodiments, intracellular glucose indicator is capable of emitting a concentration dependent fluorescent signal in response to glucose concentration within a cell being up to 1 mM, up to 0.5 mM, between 0.0001 and 10 mM, between 0.001 and 10 mM, between 0.01 and 10 mM, including any range between.

[0079] In some embodiments, the compound of the invention (i.e. the intracellular glucose indicator) is a glucose indicator configured to emit fluorescence upon complexing glucose in a liquid. In some embodiments, the compound of the invention is capable of selectively detecting glucose in the liquid, wherein the liquid is a solution (e.g., an aqueous solution, an organic solution, or a combination of an aqueous and organic solvent). Non-limiting structure of a complex between the intracellular glucose indicator and glucose is represented in Figure 3A.

[0080] In some embodiments, the compound of the invention (i.e. the intracellular glucose indicator) is a glucose indicator. In some embodiments, the compound of the invention is a fluorescent glucose indicator. In some embodiments, the compound of the invention has an enhanced binding affinity (and / or selectivity) to glucose, as compared to additional sugars, such as lactose, fructose, etc. In some embodiments, the compound of the invention is a selective glucose indicator. In some embodiments, enhanced binding affinity (or selectivity) comprises at least 5, at least 10, at least 100, at least 1000, at least 10,000 times greater binding affinity to glucose, as compared to structural similar molecules (e.g., mono-, or di-sugars such as lactose, lactate, fructose, isomers of glucose, etc.). In some embodiments, the compound of the invention is capable of emitting a fluorescent signal upon binding to glucose in a liquid (e.g. cytoplasm), wherein the intensity of the fluorescent signal is in direct correlation with the glucose concentration within the liquid.

[0081] In some embodiments, the intracellular glucose indicator is a selective glucose indicator within the liquid having a pH value below 10, below 8, below 9, or between 2 and 10, between 3 and 10, between 4 and 10, between 4.3 and 10, between 4.5 and 9.5, between 4.5 and 9, between 4 and 9, including any range between.

[0082] In some embodiments, the intracellular glucose indicator has a cell membrane penetration ability. In some embodiments, the cell comprises an aggregate of cells. In some embodiments, the aggregate of cells comprises a spheroid. In some embodiments, the intracellular glucose indicator is devoid of cell toxicity up to a concertation of 1 mM within a cell medium.

[0083] In some embodiments, the intracellular glucose indicator is water soluble. In some embodiments, the intracellular glucose indicator is characterized by a watersolubility of at least 0.075 g / L, at least 0.08 g / L, at least 0.09 g / L, at least 0.1 g / L, and between 0.075 and 0.75 g / L, between 0.075 and 0.75 g / L, between 0.075 and 0.75 g / L, between 0.075 and 0.75 g / L, between 0.075 and 0.09 g / L, between 0.075 and 0.1 g / L, between 0.075 and 0.15 g / L, between 0.075 and 0.2 g / L, between 0.075 and 0.3 g / L, between 0.075 and 0.4 g / L, between 0.075 and 0. 5 g / L, between 0.075 and 0.6 g / L, between 0.075 and 0.75 g / L, between 0.1 and 0.6 g / L, between 0.3 and 0.5 g / L including any range in between

[0084] In some embodiments, the compound of the invention (intracellular-, and extracellular glucose indicator) is devoid of cell toxicity up to a concertation of 1 mM within a cell medium.Extracellular Glucose Indicator

[0085] In another aspect of the invention, there is provided a compound (also used herein as "immobilized extracellular glucose indicator”) represented by Formula 4 including any salt, any hydrate, or any solvate thereof:, wherein each wavy bond represents an attachment point to a polymeric material or H, and at least one wavy bond is the attachment point; each a is an integer being independently between 3 and 20; each R2 independently is selected from halo, haloalkyl, a cyano group, carboxy, amide, carbonyl, anhydride, carbonate ester, carbamate, a sulfonyl group, a sulfonate group, a sulfinyl group, a sulfonamide group, an azo group, a guanidine group, and ammonium group, or any combination thereof; X represents ethyl, CNR’2, NH, O, S, -CONH-, -CONR’-, -C(=NH)NR’-, -C(=S)NR’-, - NC(=O)-, -NC(=O)O-, -NC(=O)N-, -NC(=S)O-, -NC(=S)N-, -C(=O)-, -C(=O)O-, -0C(=0)0-, -OC(=O)N-, -OC(=S)O-, -OC(=S)N-,, or phosphate; wherein each R is selected from H and Cl -CIO alkyl; and A represents any of cycloalkyl, aryl, anthracene, and heteroaryl, a fused aryl, or a fused cycloalkyl or any combination thereof, each option of the above may be substituted or non-substituted or any combination thereof. In some embodiments, the compound of Formula 4 (immobilized extracellular glucose indicator) is covalently attached (or bound) to the polymeric material. In some embodiments, the covalent bond to the polymeric material is stable in a aqueous solution. In some embodiments, immobilized extracellular glucose indicator is water insoluble.

[0086] In some embodiments, the compound is represented by Formula 4, wherein a is between 3 and 20, between 3 and 15, between 5 and 20, between 5 and 15, between 4 and 10, between 5 and 10, between 5 and 7, between 5 and 8, between 4 and 8, including any range in between. In some embodiments, a is between 1 and 5, including any range in between. In some embodiments, the compound is represented by Formula 4, wherein R2 is haloalkyl (e.g. CF3), and wherein A is phenyl. In some embodiments, the compound is represented by Formula 4, wherein R is haloalkyl (e.g. CF3), wherein X is -CONH-; and wherein A is phenyl.

[0087] In some embodiments, the immobilized extracellular glucose indicator is represented by Formula 5:, wherein the wavy bond and a are as described above. In some embodiments, the immobilized extracellular glucose indicator is represented by Formula5 wherein a is between 5 and 10, between 5 and 9, between 5 and 8, including any range between.

[0088] In some embodiments, the immobilized extracellular glucose indicator is

[0089] In some embodiments, the immobilized extracellular glucose indicator is configured of complexing glucose in a liquid. Non-limiting structure of a complex between the immobilized extracellular glucose indicator and glucose is represented in Figures 4A.

[0090] In some embodiments, the immobilized extracellular glucose indicator is configured of selectively complexing glucose in a liquid, wherein the liquid further comprises additional natural compounds (e.g. additional sugars other than glucose, such as fructose, proteins, nucleotides or any other natural compounds). In some embodiments, the liquid is a biological fluid. In some embodiments, the liquid is a cytoplasm. In some embodiments, the liquid is an aqueous solution or water.

[0091] In some embodiments, the immobilized extracellular glucose indicator is configured to detect glucose concentration in the extracellular liquid (e.g. extracellular aqueous solution). In some embodiments, the immobilized extracellular glucose indicator is a fluorescent glucose indicator. In some embodiments, the e immobilized extracellular glucose indicator has an enhanced binding affinity (and / or selectivity) to glucose, as compared to additional sugars, such as lactose, fructose, galactose, mannose, etc. In some embodiments, the extracellular glucose indicator is a selective glucose indicator.

[0092] In some embodiments, the immobilized extracellular glucose indicator is a selective glucose indicator within the liquid at a pH value below 8.5, below 8, or between2 and 8.5, between 3 and 8.5, between 4 and 8.5, between 4 and 8, between 5 and 7, including range between.

[0093] In some embodiments, enhanced binding affinity (or selectivity) comprises at least 5, at least 10, at least 100, at least 1000, at least 10,000 times greater binding affinity to glucose, as compared to structural similar molecules (e.g., mono-, or di-sugars such as lactose, lactate, fructose, isomers of glucose, etc.). In some embodiments, the immobilized extracellular glucose indicator is capable of emitting a fluorescent signal upon binding to glucose in a liquid (e.g. the extracellular liquid) in contact with the polymeric material to which the immobilized extracellular glucose indicator is attached.

[0094] In some embodiments, the immobilized extracellular glucose indicator is capable of emitting a concentration dependent fluorescent signal in response to glucose concentration within the liquid being up to 500 mM, up to 300 mM, up to 200 mM, up to 100 mM, up to 70 mM, up to 50 mM, up to 30 mM, up to 20 mM, up to 15 mM, up to 10 mM, between 0.01 and 500 mM, between 0.01 and 100 mM, between 0.01 and 50 mM, between 0.01 and 30 mM, between 0.01 and 20 mM, between 0.1 and 30 mM, between 0.1 and 20 mM, between 0.1 and 15 mM, between 0.1 and 10 mM, between 1 and 10 mM, including any range between. In some embodiments, the immobilized extracellular glucose indicator is capable of selectively detecting extracellular glucose concentration.

[0095] In another aspect, there is provided a compound represented by Formula 6 including any salt, any hydrate, or any solvate thereof:(also used herein as "water-soluble extracellular glucose indicator” or “extracellular glucose indicator”), wherein: each “a” is an integer being independently between 1 and 10; each R2 independently represents one or more electron-withdrawing group(s); and each A represents any of cycloalkyl, aryl, and heteroaryl, a fused aryl, a fused cycloalkyl, substituted or non-substituted. In some embodiments, the compound of Formula 6 is water soluble.

[0096] In some embodiments, a is between 1 and 10, including any range in between. In some embodiments, a is between 1 and 8, including any range in between. In some embodiments, a is between 1 and 5, including any range in between. In some embodiments, a is between 1 and 3, including any range in between.

[0097] In some embodiments, A is an aromatic ring.

[0098] In some embodiments, the electron-withdrawing group is selected from: nitro, cyano, guanidine, haloalkyl, halo, phosphate, sulfate, sulfonate, sulfonyl, sulfoxide, carbonyl, imine, carboxy, azo, azide, and thiocarbonyl.

[0099] In some embodiments, R2 represents 1, 2, 3 or 4 substituents, each independently being an electron-withdrawing group. The term "electron-withdrawing group" is well known to those skilled in the art as a functional group that draws electrons to itself more than a hydrogen atom would if it occupied the same position in the molecule, as described in J. March, Advanced Organic Chemistry, third edition, Pub: John Wiley & Sons, Inc (1985).

[0100] Exemplary electron-withdrawing group include, but are not limited to, nitro group, fluoro, haloalkyl, halocycloalkyl, haloaryl, halo heteroaryl, a cyano group, an alkyloxy carboxylic ester bond, a sulfonyl group, a sulfonate group, a sulfinyl group, a sulfonamide group, an azo group, a guanidine group, and a carboxylic acid derivative, or any combination thereof. The term "carboxylic acid derivative" as used herein encompasses carboxy, amide, carbonyl, anhydride, carbonate ester, and carbamate.

[0101] In some embodiments, R2 represents 1, 2, 3 or 4 haloalkyl substituents (e.g., CF3, CI3, CC13 or CBr3). In some embodiments, at least one R2 is haloalkyl. In some embodiments, R2 represent CF3. In some embodiments, R2 represents 1, 2, 3 or 4 halo substituents (e.g. F).

[0102] The term “haloalkyl” refers to C1-C10 alkyl as described herein substituted by 1, 2, 3 halide atoms, wherein halide is selected from F, Br, Cl, and I, or a combination thereof.

[0103] In some embodiments, R2 is in an ortho-, meta-, and / or para-position to the B(OH)2 moiety. In some embodiments, R2 is in a para position to the B(OH)2 moiety.

[0104] In some embodiments, the electron-withdrawing group is selected from: nitro, cyano, ammonium, haloalkyl, halo, sulfonyl and carbonyl. In some embodiments, A is an aromatic ring.

[0105] In some embodiments, the compound (water-soluble extracellular glucose indicator) is represented by Formula 7:, R2 are as disclosed herein above. In some embodiments, the compound is represented by Formula 2, wherein R2 is an electron withdrawing group. In some embodiments, Formula 1 or Formula 2 comprises a fluoroalkyl.

[0106] In some embodiments, R2 is CF3.In some embodiments, R2 is a fluoroalkyl. In some embodiments, the fluoroalkyl is in para position to the B(OH)2 moiety, and wherein a is 1.

[0107] In some embodiments, the compound (water-soluble extracellular glucose indicator is or comprises:, including any salt, any hydrate, or any solvate thereof.

[0108] In some embodiments, the water-soluble extracellular indicator is capable of emitting a concentration dependent fluorescent signal in response to glucose concentration within the liquid being up to 10 mM, up to 1 mM, up to 0.5 mM, between 0.0001 and 10 mM, between 0.001 and 10 mM, between 0.01 and 10 mM, including any range between. In some embodiments, the water-soluble extracellular is capable of selectively detecting extracellular glucose. In some embodiments, the water-soluble extracellular glucose indicator is capable of emitting a concentration dependent fluorescent signal in response to glucose concentration within a cell being up to 1 mM, up to 0.5 mM, between 0.0001 and 10 mM, between 0.001 and 10 mM, between 0.01 and 10 mM, including any range between.

[0109] In some embodiments, the water-soluble extracellular glucose indicator is a selective glucose indicator within the liquid having a pH value below 10, below 8, below 9, or between 2 and 10, between 3 and 10, between 4 and 10, between 4.3 and 10, between 4.5 and 9.5, between 4.5 and 9, between 4 and 9, including any range between. In some embodiments, the water-soluble extracellular glucose indicator is capable of selectively detecting glucose in a liquid (e.g., an aqueous liquid). In some embodiments, the water- soluble extracellular glucose indicator is configured to selectively detect intracellular glucose at a pH in a range between about 5 and about 11.5. In some embodiments, the water-soluble extracellular glucose indicator has an enhanced binding affinity (and / or selectivity) to glucose, as compared to additional sugars, such as lactose, fructose, sucrose, mannose, galactose, etc.

[0110] In some embodiments, the water-soluble extracellular glucose indicator has an enhanced binding affinity (and / or selectivity) to glucose, as compared to additional sugars at a concentration range of up to 500mM, up to 300mM, up to 200mM, up to lOOmM, up to 50mM, up to 20mM, up to 15mM, up to 10 mM, or between O.luM and 500mM, between O.luM and 200mM, between O.luM and lOOmM, between O.luM and 50mM, between O.luM and 20mM, between luM and 15mM, between 1 uM and lOmM, including any range between. In some embodiments, enhanced binding affinity comprises at least 5, at least 10, at least 100, at least 1000, at least 10,000 times greater binding affinity to glucose, as compared to additional sugar(s).

[0111] In some embodiments, the liquid is an aqueous liquid (or aqueous fluid). In some embodiments, the aqueous liquid is an aqueous buffer. In some embodiments, the aqueous liquid is substantially devoid of organic solvents (i.e., the concentration of organic solvent within the aqueous liquid is below 5%, below 1%, below 0.5% by volume of the aqueous liquid, including any range between). In some embodiments, the liquid is devoid of an organic solvent.

[0112] The terms “aqueous liquid” encompasses any one of an aqueous solution, a solution in which the solvent is water, or water that contains one or more dissolved substance(s) which are not organic solvent. The terms “liquid”, “aqueous liquid” are used herein interchangeably.

[0113] In some embodiments, the liquid is a cell-culture medium. In some embodiments, the liquid is a biological sample derived from a subject. In some embodiments, the liquid is a biological fluid derived from a subject. In some embodiments, the biological fluid encompasses any extracellular bodily fluid such as blood, plasma, saliva, urine, lymph, aqueous humor, cerebrospinal fluid, buccal swab, tears, peritoneal fluid, semen, sebum, sputum, gastrointestinal fluid, etc.

[0114] In some embodiments, the compound of the invention is configured to detect glucose in an extra-cellular biological fluid. In some embodiments, the extra-cellular biological fluid is a cell culture medium.

[0115] In some embodiments, the compound of the invention (i.e. water-soluble extracellular indicator and immobilized extracellular indicator) is substantially devoid of cell membrane penetration ability.

[0116] In some embodiments, the compound of the invention (i.e. water-soluble extracellular indicator and intracellular indicator) is water soluble. In some embodiments, the water soluble compound of the invention is characterized by a water solubility of at least 0.075 g / L, at least 0.08 g / L, at least 0.09 g / L, at least 0.1 g / L, and between 0.075 and 0.75 g / L, between 0.075 and 0.75 g / L, between 0.075 and 0.75 g / L, between 0.075 and 0.75 g / L, between 0.075 and 0.09 g / L, between 0.075 and 0.1 g / L, between 0.075 and 0.15 g / L, between 0.075 and 0.2 g / L, between 0.075 and 0.3 g / L, between 0.075 and 0.4 g / L, between 0.075 and 0. 5 g / L, between 0.075 and 0.6 g / L, between 0.075 and 0.75 g / L, between 0.1 and 0.6 g / L, between 0.3 and 0.5 g / L including any range in between. The term “water soluble” including any grammatical form thereof refers to the ability of the compound to dissolve in water (or in an aqueous solution), so as to form an aqueous solution, substantially devoid of a particulate matter (i.e., undissolved aggregates or solid particles of the compound). The presence of particles / aggregates in the aqueous solution can be determined by various methods, such as DLS. In some embodiments, the compound of the invention is characterized by water solubility as disclosed herein, wherein water solubility encompasses the ability of the compound to undergo complete dissolution in water or in an aqueous solution and wherein the aqueous solution is devoid of an organic solvent.

[0117] In another aspect, there is provided a composition comprising the compound of the invention and a liquid carrier. In some embodiments, the compound of the invention is a water soluble compound. In some embodiments, the compound of the invention is dissolved in the liquid carrier.

[0118] In some embodiments, the liquid carrier is capable of dissolving the compound of the invention to obtain an aqueous solution, wherein a concentration of the compound of the invention within the aqueous solution is at least 0.075 g / L, at least 0.08 g / L, at least 0.09 g / L, at least 0.1 g / L, and between 0.075 and 0.75 g / L, between 0.075 and 0.75 g / L, between 0.075 and 0.75 g / L, between 0.075 and 0.75 g / L, between 0.075 and 0.09 g / L, between 0.075 and 0.1 g / L, between 0.075 and 0.15 g / L, between 0.075 and 0.2 g / L, between 0.075 and 0.3 g / L, between 0.075 and 0.4 g / L, between 0.075 and 0. 5 g / L, between 0.075 and 0.6 g / L, between 0.075 and 0.75 g / L, between 0.1 and 0.6 g / L, between 0.3 and 0.5 g / L including any range in between. In some embodiments, the compositionis a liquid glucose indicator. In some embodiments, the composition is intended for use in a method / kit and / or an apparatus of the invention.

[0119] In some embodiments, the liquid carrier is an aqueous solution, such as an aqueous buffer solution, a cell medium, etc. In some embodiments, the liquid carrier is substantially devoid of ethanol (contains not more than 10%, not more than 1%, or not more than 0.1% ethanol by volume of the liquid carrier).

[0120] In some embodiments, the liquid carrier is an aqueous buffer. In some embodiments, the carrier is characterized by a pH value of at least 6, at least 6.3, at least 6.5 and between 6 and 9, between 6 and 8.5, between 6.5 and 8.5, between 7 and 8.5, between 7 and 8, between 7 and 9, including any range in between.

[0121] In some embodiments, the liquid carrier constitutes between about 0.1% to about 99.99%, between 0.1% to about 30%, between 0.1% to about 50%, between 0.1% to about 70%, between 50 to about 99.99% by weight of the composition present herein.

[0122] In some embodiments, the composition is an aqueous solution. In some embodiments, the concentration of the compound of the invention within the aqueous solution is up to 0.1M, up to 0.01M, up to 5mM, up to ImM, or between 1 uM and 10 mM, between 10 uM and 10 mM, between 10 uM and 5 mM, between 1 uM and 100 uM, between 100 uM and 10 mM, between 100 uM and 300 uM, between 100 uM and 500 uM, between 100 uM and 700 uM, between 300 uM and 1000 uM, between 300 uM and 700 uM, between 400 uM and 600 uM, between 500 uM and 1000 uM, between 700 uM and 900 uM, between 100 uM and 2 mM, between 500 uM and 10 mM, between 100 uM and 5 mM, including any range in between.

[0123] In some embodiments, the composition comprises an indicator effective amount of the compound of the invention. In some embodiments, the indicator effective amount of the compound of the invention comprises a concentration of the compound of the invention sufficient for detecting the presence and / or concentration of glucose with a liquid. In some embodiments, the indicator effective amount is at least lOuM, at least 25uM, at least 50uM, at least lOOuM, including any range in between.

[0124] In some embodiments, the composition consists essentially of the liquid carrier and the compound of the invention. In some embodiments, the composition comprises between 80 and 99.99%, between 85 and 99.99%, between 90 and 99.99%, between 95and 99.99%, between 90 and 95%, including any range in between of the liquid carrier and the compound of the invention by the total weight of the composition.

[0125] In some embodiments, the compound of the invention is devoid of cell toxicity up to a concertation of 1 mM within a cell medium.Sensing device

[0126] In another aspect, there is provided a sensing device comprising (i) a chamber comprising the composition of the invention; (ii) a fluorescence detector and (iii) a light source in an operable communication with the chamber and with the fluorescence detector; wherein the chamber is configured to receive a liquid sample; and wherein the sensing device is configured for detecting the concentration of glucose within the liquid sample.

[0127] Reference is now made to Figure 7, which shows a perspective view of the disclosed sensing device in a non-limiting embodiment thereof. In some embodiments, the sensing device comprises a sensing unit (1) comprising a holder 2, configured to accommodate a chamber 3 comprising the composition of the invention. The chamber 3 is further configured to contain the liquid sample. The chamber 3 may alternatively comprise a reference solution, as disclosed hereinbelow.

[0128] In some embodiments, the chamber is light transparent. In some embodiments, the chamber is configured for light penetration, wherein light is at a wavelength between 300 and 500 nm, or between 300 and 400 nm. In some embodiments, the chamber is made of a light transparent material, such as plastic, glass, quartz, etc. In some embodiments, the chamber is a sample cuvette. In some embodiments, the chamber is a sample plate (e.g., 96 well-plate).

[0129] In some embodiments, the chamber volume is between 0.1 ul and 10 ml, between 0.1 and 2000 ul, between 1 and 1000 ul, between 100 and 1000 ul, including any range between. In some embodiments, the volume of the liquid sample is between 50 nL and 200 ul, between 50 nL and 10 ul, between 0.1 ul and 200 ul, between 0.1 and 10 ul, between 0.1 and 100 ul, including any range between.

[0130] The sensing unit 1 further comprises a fluorescence detector 4 and a light source 5. The fluorescence detector 4, the light source 5, and the chamber 3 are positioned onthe same plane. The light source 5 and the chamber 3 are positioned on the same optical axis 6. The fluorescence detector 4 is positioned perpendicular to the optical axis 6.

[0131] The light source 5 may comprise a plurality of light sources.

[0132] The holder 2 is further configured to accommodate an additional chamber containing, receiving or maintaining the reference solution. The holder 2 may be configured to contain at least two chambers: chamber 3 configured for holding a liquid sample comprising the compound / composition of the invention and an additional chamber configured for holding the reference solution, wherein chamber 3 is in an operable communication with a first light source (i.e., chamber 3 and the first light source are positioned along the same optical axis) and the additional chamber is in an operable communication with a second light source. In some embodiments, chamber 3 and the additional chamber are each in an operable communication with one or more fluorescence detector(s).

[0133] The sensing unit 1 has a housing which fully or partially encloses the elements of the sensing unitl. In some embodiments, the housing is selected from a rigid durable material such as aluminum, stainless steel, a hard polymer and / or any of the like. In some embodiments, the housing prevents unwanted foreign elements from entering the sensing unit 1 (e.g., dust, light etc.).

[0134] In some embodiments, the light source is configured to emit light at a wavelength between 300 and 400 nm, and the light sensor is configured to detect light emitted (by the compound of the invention) at a wavelength between 400 and 500 nm.

[0135] In some embodiments, light source 5 is selected from light emitting diodes (LEDs), a laser diode, a mercury or xenon arc-lamp and halogen lamp.

[0136] The fluorescence detector 4 may be any suitable fluorescence detector known in the art. Fluorescence may also be detected using a standard fluorescence microscope fitted with a camera and software.

[0137] In some embodiments, the sensing device further comprises a power source, and a control unit in an operable communication with the sensing unit. In some embodiments, the control unit comprises an electronic circuitry unit. In some embodiments, the control unit comprises at least one hardware processor. In some embodiments, the hardwareprocessor may analyze the fluorescence values obtained from the fluorescence detector 4, and to calculate the glucose concentration in the liquid sample based on the reference value and optionally based on a calibration curve. The calibration curve may be obtained by contacting the composition of the invention with increasing concentrations of glucose, receiving fluorescence values, and plotting the received fluorescence values for each glucose concentration on a graph, wherein the fluorescence values are calculated from the fluorescent signal intensity detected by the fluorescence detector 4, and wherein the fluorescent signal is emitted by the complex (resulting from glucose complexation by the compound of the invention).

[0138] In some embodiments, the control unit comprises at least one hardware processor; and a non-transitory computer-readable storage medium having stored thereon program instructions, the program instructions executable by the at least one hardware processor to: receive (i) a fluorescence value obtained from the liquid sample, and (ii) a fluorescence value obtained from the reference solution; and to calculate the glucose concentration in the liquid sample based on the received fluorescence values and based on the calibration curve.

[0139] The computer-readable storage medium may have a program code embodied therewith. The computer readable storage medium can be a tangible device that can retain and store instructions for use by an instruction execution device. The computer readable storage medium may be, for example, but is not limited to, an electronic storage device, a magnetic storage device, an optical storage device, an electromagnetic storage device, a semiconductor storage device, or any suitable combination of the foregoing. A non- exhaustive list of more specific examples of the computer readable storage medium includes the following: a portable computer diskette, a hard disk, a random access memory (RAM), a read-only memory (ROM), an erasable programmable read-only memory (EPROM or Flash memory), a static random access memory (SRAM), a portable compact disc read-only memory (CD-ROM), a digital versatile disk (DVD), a memory stick, a floppy disk, a mechanically encoded device such as punch-cards or raised structures in a groove having instructions recorded thereon, and any suitable combination of the foregoing. A computer readable storage medium, as used herein, is not to be construed as being transitory signals per se, such as radio waves or other freely propagating electromagnetic waves, electromagnetic waves propagating through awaveguide or other transmission medium (e.g., light pulses passing through a fiber-optic cable), or electrical signals transmitted through a wire.

[0140] Computer readable program instructions described herein can be downloaded to respective computing / processing devices from a computer readable storage medium or to an external computer or external storage device via a network, for example, the Internet, a local area network, a wide area network and / or a wireless network. The network may comprise copper transmission cables, optical transmission fibers, wireless transmission, routers, firewalls, switches, gateway computers and / or edge servers. A network adapter card or network interface in each computing / processing device receives computer readable program instructions from the network and forwards the computer readable program instructions for storage in a computer readable storage medium within the respective computing / processing device.

[0141] Computer readable program instructions for carrying out operations of the present invention may be assembler instructions, instruction-set-architecture (ISA) instructions, machine instructions, machine dependent instructions, microcode, firmware instructions, state-setting data, or either source code or object code written in any combination of one or more programming languages, including an object oriented programming language such as Java, Smalltalk, C++ or the like, and conventional procedural programming languages, such as the "C" programming language or similar programming languages. The computer readable program instructions may execute entirely on the user's computer, partly on the user's computer, as a stand-alone software package, partly on the user's computer and partly on a remote computer or entirely on the remote computer or server. In the latter scenario, the remote computer may be connected to the user's computer through any type of network, including a local area network (LAN) or a wide area network (WAN), or the connection may be made to an external computer (for example, through the Internet using an Internet Service Provider). In some embodiments, electronic circuitry including, for example, programmable logic circuitry, field-programmable gate arrays (FPGA), or programmable logic arrays (PLA) may execute the computer readable program instructions by utilizing state information of the computer readable program instructions to personalize the electronic circuitry, in order to perform aspects of the present invention.

[0142] These computer readable program instructions may also be stored in a computer readable storage medium that can direct a computer, a programmable data processing apparatus, and / or other devices to function in a particular manner, such that the computer readable storage medium having instructions stored therein comprises an article of manufacture including instructions which implement aspects of the function / act specified in the drawings.

[0143] In some embodiments, the program code is executable by a hardware processor. In some embodiments, the hardware processor is a part of the control unit.

[0144] In some embodiments, the sensing device further comprises an optical unit in an operable communication with the fluorescence detector. In some embodiments, the optical unit comprises an emission filter, excitation filter, a dichroic mirror, an ocular and an objective.

[0145] In some embodiments, the sensing device is configured for detection of the concentration of glucose within the liquid sample (e.g., an aqueous solution, a cell medium, or a sample derived from a subject). In some embodiments, the sensing device is configured for detection of the concentration of glucose (i) within the liquid sample comprising a cell medium in contact with at least one cell (i.e., extracellular glucose concentration), (ii) within a cell (i.e., intracellular glucose concentration), or both (i) and (ii), wherein (i) and (ii) are detected simultaneously or subsequently. In some embodiments, the sensing device is configured for detection of the concentration of glucose according to any of the methods disclosed herein.

[0146] In some embodiments, the method for determining glucose concentration within a liquid sample comprises contacting the liquid sample with the sensing device of the invention (e.g., by introducing a predetermined amount of the aqueous liquid into chamber 3 of the sensing device), irradiating the aqueous liquid via the light source, measuring fluorescence signal detected by the fluorescence detector to obtain a fluorescence value, and determining the glucose concentration within the liquid sample based on the fluorescence value.

[0147] In some embodiments, the upper detection limit of the sensing device (i.e. a maximum glucose concentration detectable by the device) is about 100 mM, about 80 mM, about 50 mM, about 30 mM, about 10 mM, including any range between. A skilledartisan will appreciate that the major challenge in the glucose sensing is to detect elevated glucose concentrations. Thus, it should be apparent that the lowest detection limit of the present sensing device is mainly dependent on the concentration of the compound within the composition of the invention.

[0148] In some embodiments, the sensing device of the invention is configured to detect a concentration of glucose within the liquid sample in the range between 0.001 and 100 mM, between 0.001 and 50 mM, between 0.01 and 100 mM, between 0.1 and 100 mM, including any range between.Article

[0149] In another aspect, there is provided an article comprising a cell support, wherein the cell support is a hydrogel comprising the immobilized extracellular glucose indicator. In some embodiments, the cell support is a solid (i.e. non-flowable) hydrogel. In some embodiments, the cell support is attached or mounted to an insert. In some embodiments, the insert has a proximal end attached to a base and a distal end. In some embodiments, the cell support is embedded inside the distal end of the insert. In some embodiments, the insert is vertically emerging from the base.

[0150] In some embodiments, the cell support attached to the insert is configured for insertion into a container. In some embodiments, the cross-section and thickness dimensions of the cell support are compatible for insertion into the container. In some embodiments, the container is configured for holding a liquid volume. In some embodiments, the container is configured for holding a liquid volume. In some embodiments, the cell support is configured for immersion into the liquid volume. In some embodiments, the liquid volume is or comprises a cell culture medium.

[0151] In some embodiments, the length of the insert is sufficient for immersing the cell support into the liquid volume. In some embodiments, the cell support remains attached to the insert upon immersion into the liquid volume. In some embodiments, the cell support is configured to support growth of a cell within the liquid volume. In some embodiments, the cell support is configured to support growth of a cell attached thereto within the liquid volume, wherein the cell is at least partially immersed within the liquidvolume. In some embodiments, the length of the insert is sufficient for at least partially immersing the cell attached to the cell support into the liquid volume.

[0152] In some embodiments, the container is or comprises at least one well of a cell culture plate (e.g. a 96-well plate). In some embodiments, the cross-section of the cell support is compatible with a cross-section of the well. In some embodiments, the crosssection of the cell support is at least 2%, at least 5%, at least 10%, at least 20% lower than the inner cross-section of the well. In some embodiments, the cross-section of the cell support is between about 2 and about 4mm, between about 2 and about 3 mm, or about 2.8 mm. In some embodiments, the thickness of the cell support is between 0.1 and 3, between 0.1 and 2, between 0.5 and 1mm. In some embodiments, the cross-section of the cell support is about 2.8 mm and the thickness of the cell support is about 0.8 mm.

[0153] In some embodiments, the article comprises a plurality of inserts attached to the base. In some embodiments, the plurality of inserts are vertically attached to the base. In some embodiments, the plurality of inserts are parallel to each other. In some embodiments, the plurality of inserts are arranged in a pattern on a surface of the base. In some embodiments, the pattern is compatible with the locations of the wells within the cell culture plate. In some embodiments, the article is compatible with within the cell culture plate (i.e. the location of each of the plurality of inserts is compatible with the location of each well within the cell culture plate). In some embodiments, the article is compatible for use with the cell culture plate, so as to allow insertion each of the cell supports into a well of the cell culture plate.

[0154] In some embodiments, the length of the insert is sufficient for at least partially immersing the cell attached to the cell support into the liquid volume. In some embodiments, the length of the insert is at least 10%, at least 20%, at least 30, at least 50% less than the depth of the container (e.g. the depth of the well of a cell culture plate).

[0155] In some embodiments, the length of the insert is between about 7 and about 8mm. In some embodiments, the length of the insert is between 7.3 and 7.8mm, or about 7.6mm. In some embodiments, the cross-section of the insert is between 2 and 4, between 3 and 4, or about 3.8 mm.

[0156] In some embodiments, the article is adapted for being in operable communication with a microscope. In some embodiments, the article is suitable fordetection of a fluorescent signal emitted by the cell support (i.e. by the immobilized extracellular glucose indicator on the surface of the cell support), or by the cell bound thereto (i.e. by the intracellular glucose indicator located within the cell), wherein detection is performed by a confocal microscope.

[0157] Reference is now made to Fig. 6B, which is a nonlimiting example of an article according to some embodiments of the invention. The article 100 may have a base 10 and at least one insert 20 attached thereto. The article 100 may have a cell support 22 attached to or mounted into the insert 20.

[0158] The insert 20 may vertically emerge from the base 10 (the longitudinal axis of the insert 20 is perpendicular to the plane of the base 10). The insert 20 may have a proximal end 25 in contact with the base 10, and a distal end 26 in contact with the cell support 22. The distal end 26 may have a flat shape. The distal end 26 may have a rim defining a lumen for holding the cell support 22.

[0159] Non-limiting article of the invention having a pattern of inserts compatible with a 96-well cell culturing plate is depicted in Figure 6C. Cross-section view of an exemplary article inserted into the wells of the 96-well cell culturing plate is depicted in Figures 6D and 6F.MethodsMeasuring intracellular glucose levels

[0160] According to another aspect, there is provided a method for determining glucose concentration within a cell, the method comprises (i) contacting the cell with the composition or with the compound of the invention, thereby obtaining a complex; (ii) irradiating the cell at a first wavelength suitable for excitation of the complex; (iii) measuring fluorescence emitted from the cell to obtain a fluorescence value, and (iv) determining glucose concentration within the cell based on the fluorescence value; and wherein the composition or the compound is or comprises the intracellular glucose indicator disclosed hereinabove.

[0161] In some embodiments, the method comprises contacting the cell, a composition comprising thereof, or a cell culture medium in contacting with the cell, with the compound or with the composition of the invention, for a time period sufficient for thecompound to penetrate cell membrane. In some embodiments, contacting comprises adding the compound or the composition to the cell culture medium, there by obtaining a concentration of the compound within the cell culture medium ranging between luMand 1 mM, between 1 uM and lOOuM, between 100 uM and 1 mM, between 10 and 1000 uM, between 5 and 1000 uM, between 100 and 1000 uM, between 100 and 300 uM, between 100 and 500 uM, between 100 and 700 uM, between 300 and 1000 uM, between 300 and 700 uM, between 400 and 600 uM, between 500 and 1000 uM, between 700 and 900 uM, including any range in between.

[0162] In some embodiments, contacting is performed at a temperature of the cell culture medium ranging between 10 and 40 °C. In some embodiments, contacting further comprises incubating the liquid mixture prior to performing step (ii) for a time period ranging between 1 second and 10 h, between 1 minute and lOh, including any range between.

[0163] In some embodiments, the step (i) is performed while the composition comprising the cell, or the cell culture medium is devoid or substantially devoid (i.e. below 0.0001, below O.OlmM glucose) of glucose.

[0164] In some embodiments, irradiation is performed at a wavelength suitable for excitation of the complex. The term “complex” refers to a complex between the compound of the invention and glucose. Glucose hydroxy groups are complexed to boron atoms of the compound of the invention.

[0165] In some embodiments, the first wavelength is between 300 and 400 nm, between 300 and 380 nm, between 330 and 400 nm, between 330 and 380 nm, between 350 and 380 nm, between 350 and 380 nm, between 360 and 380nm, between 365 and 400 nm, including any range in between. In some embodiments, the steps (ii)-(iv) are performed simultaneously. In some embodiments, the steps (ii) and (iii) are performed simultaneously.

[0166] In some embodiments, step (iii) comprises measuring the fluorescence emitted from the cell, wherein the fluorescence is emitted by the complex located inside the cell (e.g. in the cytosol). In some embodiments, step (iii) is performed by a fluorescence detection device (various detection devices are well known in the art, such as a fluorimeter, ELISA, or any suitable fluorescence detector). In some embodiments,measuring comprises detecting the fluorescent radiation emitted from the liquid mixture and determining the intensity of the fluorescent radiation. In some embodiments, the numerical value of the intensity of the fluorescent radiation is the fluorescence value.

[0167] In some embodiments, measuring is performed at a second wavelength of between 400 and 500 nm, between 400 and 450 nm, between 410 and 500 nm, between 410 and 450 nm, between 410 and 480 nm, between 410 and 440 nm, between 420 and 500 nm, between 420 and 450 nm, between 450 and 500 nm, including any range in between. In some embodiments, the first wavelength and the second wavelength also encompasses a wavelength range (i.e. excitation and measuring is performed at a single wavelength or at a plurality of wavelengths).

[0168] In some embodiments, the method is for ex-vivo / in-vitro measurement of glucose concentration within a cell. In some embodiments, the method is for ex-vivo / in- vitro measurement of glucose uptake / reabsorption by the cell. In some embodiments, the cell is a tissue sample derived from a subject. In some embodiments, the cell comprises a plurality of cells. In some embodiments, the cell comprises an aggregate / agglomerate of cells. In some embodiments, the aggregate of cells is a 3-dimensional cell aggregate.

[0169] In some embodiments, the cell is or comprises a cell spheroid. In some embodiments, the cell spheroid comprises a liver spheroid or a kidney spheroid. In some embodiments, the method is for ex-vivo / in-vitro measurement of glucose retake (or reabsorption) by the kidney spheroid.

[0170] In some embodiments, the method is for determining glucose concentration within the cell ranging between 0.0001 and 10 mM, between 0.0001 and 1 mM, between 0.0005 and 10 mM, between 0.001 and 10 mM, between 0.1 and 10 mM, between 0.01 and 10 mM, including any range between.

[0171] In some embodiments, determining the glucose concentration is based on a calibration curve, and optionally comprises subtracting a value of a reference from the fluorescence value.

[0172] In some embodiments, the reference comprises the cell or a composition comprising thereof, devoid of the compound of the invention.

[0173] In some embodiments, determining the glucose concentration in the cell is performed by subtracting the reference fluorescence value from the sample fluorescence value to obtain a normalized fluorescence value, and comparing the normalized fluorescence value to a calibration curve.

[0174] In some embodiments, the steps (i)-(iii) of the method are performed at a temperature between 5 and 50 °C, including any range between.

[0175] According to another aspect, there is provided a method for determining a concentration of glucose in a cell, in a liquid (i.e. a liquid which is not the cytoplasm), or both in the cell and in the liquid, the method comprising:(i) contacting the cell and / or the liquid in contact with the cell with the intracellular glucose indicator, to obtain a first complex comprising the intracellular glucose indicator bound to a glucose molecule inside the cell;(ii) contacting the liquid with the extracellular glucose indicator to obtain a second complex comprising the extracellular glucose indicator bound to a glucose molecule in the liquid;(iii) irradiating the liquid, the cell and / or the cell support at a first wavelength suitable for excitation of the first complex, measuring a first fluorescence emitted by the first complex to obtain a first fluorescence value, and determining a concentration of glucose within the cell based on the first fluorescence value; and(iv) irradiating the liquid, the cell and / or the cell support at a second wavelength suitable for excitation of the second complex, measuring a second fluorescence to obtain a second fluorescence value, and determining a concentration of glucose within the cell medium based on the second fluorescence value.

[0176] In some embodiments, the step (i) and the step (ii) of the method are performed subsequently or simultaneously In some embodiments, the step (ii) / (i) and the step (iii) of the method are performed subsequently or simultaneously. In some embodiments, the step (i) is performed while the liquid is devoid or substantially devoid (i.e. below 0.0001, below O.OlmM glucose) of glucose.

[0177] In some embodiments, irradiating and measuring in each of the steps (iii) and (iv) is performed simultaneously. In some embodiments, the step (iii) is performed beforeor after the step (iv). In some embodiments, the step (ii) is performed after the step (i). In some embodiments, the steps (iii) -(iv) are performed after the step (i) and / or after the step (ii).

[0178] In some embodiments, the step (ii) is performed while the cell is not immersed in the liquid. In some embodiments, the step (iii) is performed while the cell support is immersed in the liquid.

[0179] In some embodiments, the step (iii) and / or (iv) is / are performed using a confocal microscope.

[0180] In some embodiments, the step (iii) is performed while the cell and / or the cell support is immersed in the liquid. In some embodiments, the step (iv) is performed while the cell and / or the cell support is immersed in the liquid.

[0181] According to another aspect, there is provided a method for determining a concentration of glucose in a cell, in a liquid (i.e. a liquid which is not the cytoplasm), or both in the cell and in the liquid, the method comprising:(i) contacting the cell with the intracellular glucose indicator, to obtain a first complex comprising the intracellular glucose indicator bound to a glucose molecule inside the cell; wherein the cell is attached to or in contact with a cell support comprising the immobilized extracellular glucose indicator; and wherein the immobilized extracellular glucose indicator is configured for binding glucose within the liquid to obtain a second complex;(ii) irradiating the liquid, the cell and / or the cell support at a first wavelength suitable for excitation of the first complex, measuring a first fluorescence emitted by the first complex to obtain a first fluorescence value, and determining a concentration of glucose within the cell based on the first fluorescence value; and(iii) irradiating the liquid, the cell and / or the cell support at a second wavelength suitable for excitation of the second complex, measuring a second fluorescence to obtain a second fluorescence value, and determining a concentration of glucose within the cell medium based on the second fluorescence value.

[0182] In some embodiments, the step (ii) and the step (iii) of the method are performed subsequently. In some embodiments, the method further comprises a preliminary step performed prior to step (i), the preliminary step comprises providing a cell attached to acell support comprising the immobilized extracellular glucose indicator, and contacting the cell support with the liquid, thereby forming the second complex comprises a glucose molecule within the liquid bound to the immobilized extracellular glucose indicator.

[0183] In some embodiments, irradiating and measuring in each of the steps (ii) and (iii) is performed simultaneously. In some embodiments, the step (ii) is performed before or after the step (iii). In some embodiments, the step (ii) is performed after the step (i). In some embodiments, the step (iii) is performed before the step (i). In some embodiments, the step (iii) is performed after the step (i) and before the step (ii).

[0184] In some embodiments, the step (ii) is performed while the cell is not immersed in the liquid. In some embodiments, the step (iii) is performed while the cell support is immersed in the liquid.

[0185] In some embodiments, the step (ii) is performed using a confocal microscope.

[0186] In some embodiments, the first fluorescence is measured at a wavelength of between about 410 and about 450nm, or about 430 nm, including any range or value in between. In some embodiments, the second fluorescence is measured at a wavelength of between about 460 and about 510nm, or about 490nm, including any range or value in between.

[0187] In some embodiments, the liquid is a cell culture medium.

[0188] In some embodiments, the method is for determining intracellular glucose concentration in a range between 0.0001 and 10 mM and extracellular glucose concentration (i.e. concentration in the cell culture medium) in a range between 0.01 and 500 mM. In some embodiments, the cell, and / or cell medium comprises a pharmaceutically active agent (e.g. cyclosporine). In some embodiments, the method of the invention is performed while the cell and / or the liquid (e.g. cell medium) is / are in contact with a pharmaceutically active agent. In some embodiments, a concentration of pharmaceutically active agent within the liquid (e.g. cell medium) is between O.OluM and ImM, between O.luM and ImM, between O.luM and lOOuM, between O.luM and lOuM, including any range between.Measuring extracellular glucose levels

[0189] According to another aspect, there is provided a method for determining a concentration of glucose in a liquid (e.g. extracellular medium), the method comprises (i) contacting the extracellular glucose indicator with the liquid, thereby obtaining a complex; (ii) irradiating the liquid at a first wavelength suitable for excitation of the complex; (iii) measuring fluorescence to obtain a fluorescence value, and (iv) determining glucose concentration within the liquid based on the fluorescence value. In some embodiments, the extracellular glucose indicator is the immobilized extracellular glucose indicator (e.g. compound of Formulae 4-5). In some embodiments, the extracellular glucose indicator is the water-soluble extracellular glucose indicator.

[0190] In some embodiments, the step of irradiating, measuring, and / or determining of the method are performed using the sensing device disclosed herein.

[0191] In some embodiments, the method od the invention is for ex- vivo measurement of glucose concentration within a biological sample. In some embodiments, the method is for measuring or determining glucose concentration within a sample derived from a subject, wherein the sample comprises a biological fluid and / or a cell. In one embodiment, “liquid” includes aqueous solution.

[0192] In some embodiments, contacting comprises adding or mixing the compound or the composition of the invention with the liquid (sample) to obtain a concentration the compound of the invention within the liquid in a range between 10 um and 0. IM, between 10 um and 50mM, between 100 um and 50mM, between 100 um and 50mM, between 100 um and lOmM, between 100 um and 5mM, between 100 and 1000 uM, between 100 and 300 uM, between 100 and 500 uM, between 100 and 700 uM, between 300 and 1000 uM, between 300 and 700 uM, between 400 and 600 uM, between 500 and 1000 uM, between 700 and 900 uM, including any range in between.

[0193] In some embodiments, contacting is so as to obtain a solution of the compound. In some embodiments, the solution is a homogeneous aqueous solution of the compound, wherein the compound is substantially dissolved in the solution.

[0194] In some embodiments, contacting further comprises incubating the solution prior to performing step (ii) for a time period ranging between 1 second and 10 h, between 1 minute and lOh, including any range between,

[0195] In some embodiments, irradiation is performed at a wavelength suitable for excitation of the compound of the invention. In some embodiments, the wavelength suitable for excitation is between 300 and 400 nm, between 300 and 380 nm, between 330 and 400 nm, between 330 and 380 nm, between 350 and 380 nm, between 350 and 380 nm, between 360 and 380nm, between 365 and 400 nm, including any range in between. In some embodiments, the steps (ii)-(iv) are performed simultaneously. In some embodiments, the steps of irradiating and measuring of the method are performed simultaneously.

[0196] In some embodiments, measuring is performed by a fluorescence detection device (various detection devices are well known in the art, such as a fluorimeter, ELISA, etc.). In some embodiments, measuring comprises detecting the fluorescent radiation emitted from the liquid mixture and determining the intensity of the fluorescent radiation. In some embodiments, the numerical value of the intensity of the fluorescent radiation is the fluorescence value. In some embodiments, detecting the fluorescent radiation is at a wavelength of between 400 and 500 nm, between 400 and 450 nm, between 410 and 500 nm, between 410 and 450 nm, between 410 and 480 nm, between 410 and 440 nm, between 420 and 500 nm, between 420 and 450 nm, between 450 and 500 nm, including any range in between.

[0197] In some embodiments, the method is for determining glucose concentration within the aqueous liquid ranging between 0.001 and 100 mM, between 0.01 and 100 mM, between 0.1 and 100 mM, between 0.001 and 50 mM, including any range between.

[0198] In some embodiments, determining the glucose concentration is based on a calibration curve, and optionally comprises subtracting a value of a reference solution from the fluorescence value.

[0199] In some embodiments, the reference solution comprises the compound of the invention at a predetermined concentration, wherein the predetermined concentration is identical with the concentration of the compound of the invention in the liquid mixture. In some embodiment, the reference comprises the composition of the invention (i.e., the compound and the liquid carrier), wherein the concentration of the compound is the same as the concentration within the liquid mixture. In some embodiments, the reference solution further comprises a pharmaceutically active agent. If the sample comprises apharmaceutically active agent (or drug), the drug may be added to the reference solution to normalize possible indicator-drug interactions which may affect the intensity of the fluorescent signal emitted by the indicator, and, consequently, may affect the glucose detection accuracy of the herein disclosed method.

[0200] In some embodiments, determining the glucose concentration in the sample is performed by subtracting the reference fluorescence value from the sample fluorescence value to obtain a normalized fluorescence value, and comparing the normalized fluorescence value to a calibration curve.

[0201] In some embodiments, any one of the steps (i)-(iv) of the method are performed at a temperature between 5 and 50 °C, including any range between.Kit

[0202] In another aspect there is provided a kit comprising the compound of the invention, and the liquid carrier disclosed hereinabove, and wherein the liquid carrier and the compound are stored in separate containers.

[0203] In some embodiments, the kit further comprises instructions for mixing the compound and an appropriate amount of the liquid carrier, under suitable conditions so as to obtain the composition of the invention.

[0204] In some embodiments, the appropriate amount is to obtain a concentration of the compound of the invention within the composition in a range between luMand 1 mM, between 1 um and lOOuM, between 100 um and ImM, between 10 and 1000 uM, between 5 and 1000 uM, between 100 and 1000 uM, between 100 and 300 uM, between 100 and 500 uM, between 100 and 700 uM, between 300 and 1000 uM, between 300 and 700 uM, between 400 and 600 uM, between 500 and 1000 uM, between 700 and 900 uM, including any range in between.

[0205] In some embodiments, suitable conditions comprise a temperature between 5°C and 50°C), and further comprise mixing the compound and the liquid carrier to obtain a solution.

[0206] In some embodiments, the kit further comprises instructions for contacting the composition with a cell culture medium comprising the cell to be tested for the presence of glucose. In some embodiments, contacting is so as to obtain a predeterminedconcentration of the compound within cell culture medium sufficient for detection of glucose within the cell. In some embodiments, the predetermined concentration is between luMand 1 mM, between 1 uM and 100 uM, between 100 uM and 1 mM, between 10 and 1000 uM, between 5 and 1000 uM, between 100 and 1000 uM, between 100 and 300 uM, between 100 and 500 uM, between 100 and 700 uM, between 300 and 1000 uM, between 300 and 700 uM, between 400 and 600 uM, between 500 and 1000 uM, between 700 and 900 uM, including any range in between.

[0207] In some embodiments, measuring the glucose concentration within a cell. In some embodiments, the kit is for use in the method of the invention.Definitions

[0208] The term "alkyl", as used herein, also encompasses saturated or unsaturated hydrocarbon, hence this term further encompasses alkenyl and alkynyl.

[0209] As used herein, the term “Cl -CIO alkyl” encompasses any linear, cyclic or branched alkyl chain comprising between 1 and 10, between 1 and 3, between 1 and 5, between 1 and 8, between 1 and 6, between 1 and 2, between 2 and 3, between 3 and 4, between 4 and 5, between 5 and 6, carbon atoms, including any range therebetween. In some embodiments, Cl -CIO alkyl comprises any of methyl, ethyl, propyl, butyl, pentyl, iso-pentyl, hexyl, and tert-butyl or any combination thereof. In some embodiments, Cl- C 10 alkyl as described herein further comprises one or more unsaturated bond(s), wherein the unsaturated bond is located at any position (e.g. 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8, 9th, or 10th) position of the C1-C10 alkyl. In some embodiments, C1-C10 alkyl is C1-C6 alkyl.

[0210] As used herein, the terms “halogen”, "halo" and "halide", which are referred to herein interchangeably, describe an atom of a halogen, that is fluorine, chlorine, bromine, or iodine, also referred to herein as fluoride, chloride, bromide, and iodide.

[0211] As used herein, the term “substituted” encompasses one or more substituent(s) (e.g. 1, 2, 3, 4, 5 or more) selected from: -OH, oxo, carbonyl, halogen, OR’, -NO2, -CN, -CONH2, -CONR’2, -CNNR’2, -CSNR’2, -CONH-OH, -CONH-NR’2, -NHCOR’, - NHCSR’, -NHCNR, -NC(=O)OR’, -NC(=O)NR’, -NC(=S)OR’, -NC(=S)NR’, -SO2R’, -SOR’, -SR’, -SO2OR’, -SO2N(R’)2, -NHNR’2, -NNR’, C1-C6 haloalkyl, optionally substituted C1-C6 alkyl, -NR’2, -NH(C1-C6 alkyl), -N(C1-C6 alkyl)2, C1-C6 alkoxy, C1-C6 haloalkoxy, hydroxy(Cl-C6 alkyl), hydroxy(Cl-C6 alkoxy), alkoxy(Cl-C6alkyl), alkoxy(Cl-C6 alkoxy), C1-C6 alkyl-NR’2, C1-C6 alkyl-SR’, -CONH(C1-C6 alkyl), -CON(C1-C6 alkyl)2, -C02H, -C02R’, -OCOR, -OCOR’, -OC(=O)OR’, - OC(=O)NR’, -OC(=S)OR’, -OC(=S)NR’, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, optionally substituted heterocyclic alkyl, or a combination thereof; wherein each R’ is independently hydrogen or is selected from the group comprising: optionally substituted C1-C10 alkyl, optionally substituted C3-C10 cycloalkyl, optionally substituted C3-C10 heterocyclyl, optionally substituted heteroaryl, optionally substituted aryl, or a combination thereof.

[0212] The term “(C3-C10) cycloalkyl” is referred to an optionally substituted C3, C4, C5, C6, C7, C8, C9 or CIO ring. In some embodiments, (C3-C10) ring comprises optionally substituted cyclopropane, cyclobutene, cyclopentane, cyclohexane, or cycloheptane.

[0213] In some embodiments, the term “hydroxy(Cl-C6 alkyl)” and the term “C1-C6 alkoxy” are used herein interchangeably and refer to C1-C6 alkyl as described herein substituted by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 hydroxy group(s), wherein the hydroxy group(s) is located at 1st, 2nd, 3rd, 4th, 5th, or 6th position of the C1-C6 alkyl, including any combination thereof.

[0214] The term "aryl" describes an all-carbon monocyclic or fused-ring polycyclic (i.e., rings which share adjacent pairs of carbon atoms) groups having a completely conjugated pi-electron system. The aryl group may be substituted or unsubstituted, as indicated herein.

[0215] The term "alkoxy" describes both an O-alkyl and an -O-cycloalkyl group, as defined herein.

[0216] The term "aryloxy" describes an -O-aryl, as defined herein.

[0217] Each of the alkyl, cycloalkyl and aryl groups in the general formulas herein may be substituted by one or more substituents, whereby each substituent group can independently be, for example, halide, alkyl, alkoxy, cycloalkyl, nitro, amino, hydroxyl, thiol, thioalkoxy, carboxy, amide, aryl and aryloxy, depending on the substituted group and its position in the molecule. Additional substituents are also contemplated.

[0218] The term “hydroxyl” or "hydroxy" describes a -OH group.

[0219] The term "mercapto" or “thiol” describes a -SH group.

[0220] The term "thioalkoxy" describes both an -S-alkyl group, and a -S-cycloalkyl group, as defined herein.

[0221] The term "thioaryloxy" describes both an -S-aryl and a -S-heteroaryl group, as defined herein.

[0222] The term “amino” describes a -NR’R” group, with R’ and R” as described herein.

[0223] The term “C3-C10 heterocyclyl” is referred to an optionally substituted C3, C4, C5, C6, C7, C8, C9 or CIO heterocyclic aromatic and / or aliphatic, or unsaturated ring, and describes a monocyclic or fused ring group having in the ring(s) one or more atoms such as nitrogen, oxygen, and sulfur. The rings may also have one or more double bonds. However, the rings do not have a completely conjugated pi-electron system. Representative examples are piperidine, piperazine, tetrahydrofuran, tetrahydropyran, morpholino and the like.

[0224] The term "carboxy" or "carboxylate" describes a -C(O)OR' group, where R' is hydrogen, alkyl, cycloalkyl, alkenyl, aryl, heteroaryl (bonded through a ring carbon) or heterocyclyl (bonded through a ring carbon) as defined herein.

[0225] The term “carbonyl” describes a -C(O)R' group, where R' is as defined hereinabove.

[0226] The above-terms also encompass thio-derivatives thereof (thiocarboxy and thiocarbonyl).

[0227] The term “thiocarbonyl” describes a -C(S)R' group, where R' is as defined hereinabove.

[0228] A "thiocarboxy" group describes a -C(S)OR' group, where R' is as defined herein.

[0229] A "sulfinyl" group describes an -S(O)R' group, where R' is as defined herein.

[0230] A "sulfonyl" or “sulfonate” group describes an -S(O)2R' group, where R' is as defined herein.

[0231] A "carbamyl" or “carbamate” group describes an -OC(O)NR'R" group, where R' is as defined herein and R" is as defined for R'.

[0232] A "nitro" group refers to a -NO2 group.

[0233] The term "amide" as used herein encompasses C-amide and N-amide.

[0234] The term "C-amide" describes a -C(O)NR'R" end group or a -C(O)NR'-linking group, as these phrases are defined hereinabove, where R' and R" are as defined herein.

[0235] The term "N-amide" describes a -NR"C(O)R' end group or a -NR'C(O)- linking group, as these phrases are defined hereinabove, where R' and R" are as defined herein.

[0236] The term "carboxylic acid derivative" as used herein encompasses carboxy, amide, carbonyl, anhydride, carbonate ester, and carbamate.

[0237] A "cyano" or "nitrile" group refers to a -CN group.

[0238] The term "azo" or "diazo" describes an -N=NR' end group or an -N=N- linking group, as these phrases are defined hereinabove, with R' as defined hereinabove.

[0239] The term "guanidine" describes a -R'NC(N)NR"R"' end group or a -R'NC(N) NR"- linking group, as these phrases are defined hereinabove, where R', R" and R'" are as defined herein.

[0240] As used herein, the term “azide” refers to a -N3 group.

[0241] The term “sulfonamide” refers to a -S(O)2NR'R" group, with R' and R" as defined herein.

[0242] The term “phosphonyl” or “phosphonate” describes an -OP(O)-(OR')2 group, with R' as defined hereinabove.

[0243] The term “phosphinyl” describes a -PR'R" group, with R' and R" as defined hereinabove.

[0244] The term “alkylaryl” describes an alkyl, as defined herein, which substituted by an aryl, as described herein. An exemplary alkylaryl is benzyl.

[0245] The term "heteroaryl" describes a monocyclic (e.g. C5-C6 heteroaryl ring) or fused ring (i.e. rings which share an adjacent pair of atoms) group having in the ring(s) one or more atoms, such as, for example, nitrogen, oxygen, and sulfur and, in addition,having a completely conjugated pi-electron system. In some embodiments, the terms “heteroaryl” and “C5-C6 heteroaryl” are used herein interchangeably. Examples, without limitation, of heteroaryl groups include pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline and purine. The heteroaryl group may be substituted or unsubstituted by one or more substituents, as described hereinabove. Representative examples are thiadiazol, pyridine, pyrrole, oxazole, indole, purine, and the like.

[0246] In the discussion, unless otherwise stated, adjectives such as “substantially” and “about” modifying a condition or relationship characteristic of a feature or features of an embodiment of the invention, are understood to mean that the condition or characteristic is defined to within tolerances that are acceptable for operation of the embodiment for an application for which it is intended. Unless otherwise indicated, the word “or” in the specification and claims is considered to be the inclusive “or” rather than the exclusive or, and indicates at least one of, or any combination of items it conjoins.

[0247] It should be understood that the terms “a” and “an” as used above and elsewhere herein refer to “one or more” of the enumerated components. It will be clear to one of an ordinary skill in the art that the use of the singular includes the plural unless specifically stated otherwise. Therefore, the terms “a”, “an” and “at least one” are used interchangeably in this application.

[0248] For purposes of better understanding the present teachings and in no way limiting the scope of the teachings, unless otherwise indicated, all numbers expressing quantities, percentages or proportions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0249] In the description and claims of the present application, each of the verbs, “comprise”, “include”, and “have” and conjugates thereof, are used to indicate that theobject or objects of the verb are not necessarily a complete listing of components, elements or parts of the subject or subjects of the verb.

[0250] Other terms as used herein are meant to be defined by their well-known meanings in the art.

[0251] Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive.

[0252] Throughout this specification and claims, the word “comprise” or variations such as “comprises” or “comprising” indicate the inclusion of any recited integer or group of integers but not the exclusion of any other integer or group of integers.

[0253] As used herein, the term “consists essentially of’ or variations such as “consist essentially of’ or “consisting essentially of’ as used throughout the specification and claims, indicate the inclusion of any recited integer or group of integers, and the optional inclusion of any recited integer or group of integers that do not materially change the basic or novel properties of the specified method, structure, or composition.

[0254] As used herein, the terms "comprises", "comprising", "containing", "having" and the like can mean "includes", "including", and the like; "consisting essentially of or "consists essentially" likewise has the meaning ascribed in U.S. patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments. In one embodiment, the terms "comprises" "comprising", and "having" are / is interchangeable with "consisting".

[0255] Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

[0256] All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent, or patent application was specifically andindividually indicated to be incorporated herein by reference. In addition, citation, or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.EXAMPLES

[0257] Generally, the nomenclature used herein, and the laboratory procedures utilized in the present invention include molecular, biochemical, and microbiological techniques. Such techniques are thoroughly explained in the literature.EXAMPLE 1Synthesis

[0258] The inventors successfully synthesized an intracellular boronic acid-based indicatordepicted in Scheme 1.

[0259] Scheme 1. Synthesis route of the proposed diboronic acid probes BA (1), BA_5 (5) and BA_21: (i) R1 = tert-Butyl 3-aminopropanoate DIPEA, CHC13, 23 °C, 72 hrs. R2 = beta-Alanine amide, DIPEA, DMF, 50 °C, 12 hrs. R3 = tert-butyl N-(7- aminoheptyl)carbamate , DIPEA, DMSO, 20 °C, 2 hrs. (ii) R1 = Comp 3, DIPEA, CHC13, 23 °C, 72 hrs. R2 = Comp 3, DIPEA, DMF, 50 °C, 12 hrs. R3 = Comp 3, DIPEA, DMF, 20 °C, 2 hrs. (iii) R1 = TFA, DCM, 23 °C, 12 hrs. R2 = TFA, DCM, 25 °C, 1 h. R3 = TFA, DCM, 0 °C, 3 hrs. (iv) prop-2-enoyl chloride, DIPEA, DMF , 20 °C, 0.25 h. (v) PEGDA, PETA, DPPO, DMSO, 23 °C, 1 min.

[0260] A methyl ester analog has been synthesized as follows:

[0261] Scheme 2. Synthesis route of the proposed diboronic acid probe BA_3 (3): (i) Methyl p-alaninate HC1, DIPEA, CHC13, 23 °C, 12 hrs. (ii) Comp 3, DIPEA, CHC13, 23 °C, 72 hrs. (iii) TFA, DCM, 20 °C, 2 hrs.

[0262] The extracellular indicator precursor (BA_21) was synthesized as depicted in Scheme 1 above. The extracellular indicator immobilized on a crosslinked PEGDA gel was synthesized as follows.

[0263] The BA_21 polyethylene glycol diacrylate (PEGDA) hydrogel film was synthesized by the free-radical polymerization utilizing Diphenyl(2,4,6- trimethylbenzoyl)phosphine oxide (DPPO) as the photoinitiator and Pentaerythritol tetraacrylate (PETA) as the cross-linker. To remove the monomethyl ether hydroquinone inhibitor from PETA, inhibitor remover column was prepared by adding cotton to a 5 ml syringe as a filter and 3 ml of the inhibitor remover beads. Then the monomer solution was prepared from mixing PEGDA and PETA at 20: 1 ratio and added to the column, the prepared mix was washed trough the column twice. To a 45 uL monomer solution 20 uL BA_21 at 10 mM in DMSO was added. Then 5 uL photoinitiator DPPO at 30 mM in EtOH was added and the suspended monomer solution was stir-mixed for 10 sec at 24 °C. Prepolymer solution (5 pL) was drop-casted directly onto a hydrophobic slide. A photopolymerization process was initiated with a UV lamp for 1 min. The resulting replica hydrogel was formed within the master stamp, washed with deionized water, and kept in the dry condition prior to further experiments.EXAMPLE 2Glucose sensing

[0264] The immobilized extracellular indicator (BA_21) has been successfully used for selective detection of glucose concertation in the cell medium, as presented in Figures 4D-E. Figure 4E shows a pH dependent specific glucose detection by the immobilized extracellular indicator.

[0265] Glucose dose dependance emission spectra of BA_21 were obtained at 14, 7, 1.75 and 0.44 mM glucose concentrations, demonstrating its sensitivity in high and low glucose environments with three measure peaks for emission (kern = 410, 430 and 456 nm) (Figure 4B).

[0266] Further, the inventors have tested the intracellular indicator of the invention, as well as additional structurally similar compounds for their ability to detect glucose (the chemical structures are presented in Figures 1A1-1A6).

[0267] Glucose detection spectroscopy: Stock solutions were prepared by dissolving 10 mM glucose probes (l)-(6) in DMSO and stored at -20 °C until use. Prior to spectroscopic tests, the probe solutions were freshly prepared by diluting the DMSO stock solution in InSight™ TOX Liver Medium at 7.4 pH (Cat no. CS-07-001a, InSphero AG) to a final concentration of 25, 50 and 100 pM probe solutions. To a 96-well, half area black plate with clear flat bottom (Cat no. 3880, Coming Incorporated Life Sciences) 100 pL / well of solution was added. Fluorescence excitation / emission spectra were recorded using Cytation5 plate reader with Xenon Flash light source. Fixed excitation at kex: 370 nm, kern: 390-580 nm and fixed emission at kem:430 nm, kex: 300-550 nm.

[0268] Glucose escalation assay: Solutions of InSight™ TOX Liver medium at 0-200 mM glucose concentration was added to a 96-well, half area black plate with clear flat bottom at 100 pL / well in quadruplets. 10 pL / well of 10 mM stock solution of BA (l-(6) probes were added and mixed three times. After 30 min incubation in room temperature fluorescence intensity was recorded using Cytation5 plate reader (Excitation: 370 nm, Emission: 430 nm).

[0269] pH dependance glucose recognition assay: InSight™ TOX Liver medium with and without glucose at pH from 2 to 12 were prepared by adding 37% HC1 and NaOH IN solution. To a 96-well, half area black plate with clear flat bottom 100 pL / well of the prepared medium solutions were added in quadruplets for each pH condition. 10 pL / well of 10 mM stock solution of 1- (6) probe was added and mixed three times. After 30 min incubation in room temperature fluorescence intensities were recorded using Cytation5 plate reader as previously mentioned.

[0270] Glucose selectivity assay: InSight™ TOX Liver medium without glucose was used to prepare a 0-10 mM Glucose, Mannose, Galactose and Sucrose media solutions. To a 96-well, half area black plate with clear flat bottom 100 pL / well of the prepared medium solutions were added in quadruplets for each condition. Then 10 pL / well of 10 mM stock solution of (l)-(6) probe was added and mixed three times. After 30 minincubation in room temperature fluorescence intensity was recorded using Cytation5 plate reader as previously mentioned.

[0271] The results of this experiment are presented in Figures 1-4. As presented in Figure 1A5, the exemplary intracellular indicator of the invention (# 5) showed glucosedependent fluorescence, thus confirming glucose-indicator capability thereof.

[0272] Further the inventors have tested the above compounds for detection of glucose concentration in liver spheroid cell culture. In brief, initial stocks of boronic acid based probes (l)-(6) at 100 mM stock solution were prepared. Then, the stock solutions were diluted with free-glucose media to a final concentration of 100 uM. Human liver spheroids (3D InSight™ Human Liver Microtissue) were washed three times with glucose-free media and incubated with (l)-(6) probes at final 100 uM concentration for two hours at 37°C and 5% CO2. For imaging, the medium was exchanged and replaced with glucose-included maintenance media, and fluorescent activity of the probes were detected at Ex / Em= 370, 423 nm using Thorlabs fluorescence microscope.

[0273] Surprisingly, it has been observed that the exemplary intracellular indicator of the invention efficiently penetrates into the spheroids, as demonstrated in Figure 1B5 and 3F.

[0274] Liver spheroid culture: 3D InSight™ Human Liver Microtissues containing PHH and human KCs (PHH to KC ratio at 10:2) were provided by InSphero (InSphero AG, Schlieren, Switzerland). The human microtissues were incubated in Akura™ 96 spheroid microplate with 70 pL 3D InSight™ Human Liver Maintenance Medium - AF per well at 37 °C and 5% CO2.

[0275] 3D InSight™ Human Liver Microtissues in good growth state were passaged into Akura™ 384 Spheroid Microplate, keeping one spheroid in each well and cultured overnight. After 24 h, spheroids were cultured in glucose-free medium and treated with 20 uM of BA (1), BA_5 (5), Mc-CDBA (6) for 2 h at 37°C and 5% CO2. Then, spheroids were washed three times with PBS, and probes were imaged after 48h at Ex / Em= 370, 423 nm using Thorlabs fluorescence microscope As shown in Figure 3F.

[0276] After treatment, BA_5 (5) emitted bright fluorescence signal. In contrast, a carboxy analog BA (1) solely exhibited extracellular fluorescence. Surprisingly, the inventors have found that a methyl ester analog (BA_3) as well as BA_2 (2) showed anunspecific signal mainly due to solubility issue. Additional structurally similar control BA (4) had no intracellular signal and Mc-CDBA (6) only exhibited a weak intracellular fluorescent signal. Accordingly, the compound of the invention can be utilized for intracellular glucose detection.

[0277] Real time monitoring of the intracellular glucose was successfully performed by the inventors. In brief, human liver spheroids (3D InSight™ Human Liver Microtissue) were washed three times with glucose-free media and incubated with the intracellular indicator BA_5 (5) at final 20 uM concentration for two hours at 37°C and 5% CO2. At the same day spheroids were treated with Cyclosporine at 5, 2.5, 1.25 and 0.625 pM concentration for 24h. Cells were washed three times with glucose-included maintenance media and daily fluorescence imaging was used to detect intracellular glucose with (Ex / Em= 370, 423 nm) using Thorlabs fluorescence microscope. Additionally, daily 2 uL media samples were collected for 5 days and glucose levels quantification was conducted as earlier reported. The results of this experiment are present in Figure 5.EXAMPLE 3

[0278] A combination of extracellular media-based detection of glucose and cell-based glucose monitoring was conducted. The inventors have tested application of both intracellular indicator BA-5 (5) and extracellular indicator BA (1) in drug screening effects in the 3D cell models, human liver spheroids.

[0279] In brief, Spheroids were incubated in a free glucose media for 2 hours with 20 pM concentration of BA-5 (5). Spheroids were washed with media and immediately treated with cyclosporine at increasing doses (5, 2.5, 1.25 and 0.625 pM) for 24 hours. The changes of BA_5 (5) fluorescence intensity (which are indicative of intracellular glucose concentration) after treatment with different doses of cyclosporine are represented at (Figure 5A, B) compared to non-treated control. A dose-dependent effect was observed after 72h.

[0280] The extracellular medium glucose concertation was measured by collecting the cell medium and adding of the extracellular glucose probe BA (1) (to obtain a final concentration of 10 mM). As presented in Figure 5C a dose-dependent response of BA (1) was observed, as the increasing drug dose decreased extracellular glucose concentration (most significant reduction of glucose concentration was observed aftertreatment with 5 uM cyclosporine). Moreover, the glucose levels were lower in all concentrations compared to non-treated control.

[0281] Alternatively, it is possible to detect extracellular glucose concertation in cell medium by culturing the spheroids on top of the substrate with the immobilized extracellular indicator (BA_21). The extracellular glucose concertation can be easily determined in a real time by measuring the emission of the immobilized extracellular indicator (e.g. via a plate reader) (Figure 4E).EXAMPLE 4 - SAMPLE PREPARATION

[0282] For the purpose of simplicity, the terms “compound of the invention” and “indicator” are used interchangeably throughout the application.Detection of glucose

[0283] 2 uL / well of the indicator stock solution (concentration of 10 mM) was added to 2 uL / well of the cell culture medium comprising 12 mM of glucose, and further diluted with 21 uL / well of water at a pH of 7.4, to obtain a final indicator concentration of 800 uM / well. The samples were incubated at room temperature for 30 min on a shaker, and the fluorescence was monitored. Exemplary compound of the invention (e.g. Indicator 1, the structure is presented in Example 6) has been implemented for glucose detection.

[0284] Several parameters were optimized to improve the detection accuracy of the current method. Exemplary parameters are inter alia: sample volume, the indicator concentration, dilution of the liquid sample, pH of water added, reaction and incubation time.

[0285] Samples and reference solution were prepared based on the following table:

[0286] The inventors successfully performed real time monitoring of varying glucose concentrations in the cell culture medium or in an aqueous solution (e.g., between 10 and 25 mM) with high accuracy.EXAMPLE 5Indicator selectivity

[0287] Indicator 1 to glucose versus lactate was tested by comparing the florescence intensities upon incubation of Indicator 1 with lactate and glucose at the same concentrations, respectively. The results of this experiment are presented in Figs. 2D and 8B. Fig. 8B demonstrates an increased fluorescence intensity with increasing glucose concentration, whereas upon incubation of the Indicator BA (l)with lactate, the fluorescence intensity remains unchanged (Fig. 8B). Selectivity of BA for glucose vs other saccharides at a constant concentration (100 pM) across a broad range of saccharides concentrations (0-10 mM in PBS) (Fig. 2D).Indicator sensitivity

[0288] Indicator 1 was dissolved in DMSO to generate a stock solution of 10 mM, 2 ul / well of the stock solution was dissolved in 21 uL / well H2O at 7.4 pH reaching a final indicator concentration of 800 uM. Cell culture medium samples comprised of different known glucose concentrations at 2 uL / well were added to the stock solution and the ability of glucose detection via fluorescent emission was tested. As shown in Fig. 2E, the indicator’s fluorescence intensity of increases with the increasing glucose concentration. A linear graph is obtained with a correlation of 98%, Z’=0.93 and s / b=3.88 ratio demonstrating high sensitivity of the indicator to varying glucose concentrations even at a low indicator concentration. Furthermore, the indicator’s sensitivity was tested at two different pH values - 7.4 and 8.4. No major difference in the fluorescent signal was observed, indicating that the indicator has a pH tolerance at least in the range between 7 and 9.Real time glucose sensing - liver vs brain spheroid system

[0289] The inventors implemented the method of the invention to monitor glucose concentration after administration of varying concentrations of a drug to liver spheroids.

[0290] The ability of the indicator to detect the amount of glucose over time was tested in brain spheroids as well as in liver spheroids. 2 uL / well of the indicator stock solution (at a concentration of 10 mM) was diluted with 21 uL / well H2O (pH 7.4) to obtain a solution with a final indicator concentration of 800 uM. The indicator solution (2 uL / well) was added to a cell culture medium to evaluate the ability of the indicator to detect glucose in the presence and absence of varying concentrations of the drug of interest (such as Tolcapone or Entacapone). Specifically, 2 cell spheroid types have been tested: a) 3D InSight™ Liver Microtissues, produced by using primary hepatocytes and non- parenchymal liver cells, and b) iPSC-derived neurons from a healthy patient. A two to seven days study was conducted to evaluate glucose consumption over time by each cell type.

[0291] The inventors successfully performed real time detection of extracellular glucose concentration in a cell culture medium, comprising living liver / brain spheroids treated by various drugs. Exemplary results are summarized in Figure 9A-9B, demonstrating the effect of Tolcapone (Figure 9A) and Entacapone (Figure 9B) on the glucose consumption by liver spheroids. While Tolcapone already at a low concentration (30 pm) induced a decrease of the fluorescent signal by 20%, Entacapone started to affect glucose consumption only at a drug concentration higher than 100 pm.

[0292] Similarly, a real time detection of glucose in low and high glucose concentration cell culture medium of brain spheroids has also been successfully demonstrated, as presented in Fig. 12.EXAMPLE 6Glucose monitoring

[0293] The inventors successfully synthesized a water-soluble boronic acid-based indicator with sufficient water solubility and specificity to glucose such as for ex-vivo glucose detection in an aqueous solution, comprising inter alia a cell-culture medium.

[0294] The inventors successfully implemented exemplary compounds of the invention (indicator 1 and indicator 2) for specific detection of glucose concentration in a liquid.Using the real time glucose sensing, the selectivity of Indicator 1 was compared to an ester derivative thereof:

[0295] The ability of the ester derivative indicator to detect glucose was negligible, as demonstrated in Fig. 10C showing the same fluorescence signal intensity with and without glucose. At the same time, the compound of the invention exhibited a concentration dependent fluorescence signal (Figure 10A).

[0296] In addition, the inventors examined glucose sensitivity of two exemplary compounds of the invention having different length of the aliphatic chain spacing between the carboxy group and an amino group. The following two indicators were compared:Indicator 1 Indicator 2

[0297] As presented in Figs. 11A and 11B, Indicator 1 and Indicator 2 have substantially the same glucose affinity / sensitivity, demonstrating a solid concentrationdependent fluorescent signal in the presence of between about lOmM and 25 mM of glucose. Accordingly, the inventors postulated that the length of the aliphatic chain has no effect on the glucose detection capability of the compound of the invention.Intracellular glucose transport

[0298] The inventors have surprisingly observed that the exemplary intracellular indicator of the invention (see Example 1) is capable for delivery of glucose form the cell medium into the cell.

[0299] In brief, the inventors added the intracellular indicator to a medium containing high glucose levels (5-25mM). The intracellular indicator effectively recognized and complexed glucose present in the cell medium. Remarkably, we were able to detect (according to the procedure disclosed in Example 2) the glucose-sensor complex intracellularly, indicating that the sensor not only identified extracellular glucose but also facilitated its transport across the cellular membrane to significantly increase intracellular glucose concentration.

[0300] This discovery underscores the sensor's dual capability of glucose detection and translocation, revealing its potential for advanced applications in monitoring and managing cellular glucose levels.

Claims

CLAIMSWhat is claimed is:

1. A compound, including any salt, any hydrate, or any solvate thereof, wherein said compound is represented by Formula 1:wherein:X is -O- or -N-; each a and b is an integer being independently between 1 and 10; if X is O than R is H, and if X is N than each R independently is selected from hydrogen, optionally substituted Ci-Cio alkyl, optionally substituted C3-C10 cycloalkyl, optionally substituted C3-C10 heterocyclyl, optionally substituted heteroaryl, optionally substituted aryl, or a combination thereof; each R2 independently is selected from (i) electron withdrawing group and (ii) halo, haloalkyl, a cyano group, carboxy, amide, carbonyl, anhydride, carbonate ester, carbamate, a sulfonyl group, a sulfonate group, a sulfinyl group, a sulfonamide group, an azo group, a guanidine group, and ammonium group, or any combination thereof; andA represents any of cycloalkyl, aryl, and heteroaryl, a fused aryl, or a fused cycloalkyl or any combination thereof, each option of the above may be substituted or nonsubstituted.

2. The compound of claim 1, wherein R2 is haloalkyl; and wherein b is 1 or 2.

3. The compound of claim 2, wherein said haloalkyl is a fluoroalkyl.

4. The compound of any one of claims 1 to 3, represented by Formula 1A:

5. The compound of claim 4, wherein A is an aromatic ring; and wherein R is H.

6. The compound of claim 4 or 5, represented by Formula 2:

7. The compound of any one of claims 1 to 6, wherein R2 is located in para position to B(OH)2 moiety; and wherein a is between 1 and 5.

8. The compound of any of claims 1 to 7, wherein said a is between 1 and 3.

9. The compound of claim 1 to 8, wherein said compound comprises:each R2 is an electron-withdrawing group; each a is an integer being independently between 1 and 10; and each A represents any of cycloalkyl, aryl, and heteroaryl, a fused aryl, a fused cycloalkyl, substituted or non- substitute or any combination thereof.

11. The compound of claim 10, wherein said electron- withdrawing group is a moiety selected from: nitro, cyano, ammonium, haloalkyl, halo, sulfonyl and carbonyl.

12. The compound of claim 10 or 11, wherein A is aryl.

13. The compound of any of claims 10 to 12, wherein represented by Formula 7:

14. The compound of claim 13, wherein R2 is selected from halo and haloalkyl, and wherein R2 is in para position to the B(OH)2 moiety.

15. The compound of claim 14, wherein said haloalkyl is a fluoroalkyl.

16. The compound of any of claims 13 to 15, wherein a is 1.

17. The compound of any of claims 13 to 16, wherein said compound comprises:including any salt thereof.

18. The compound of any of claims 1 to 17, wherein said compound is a glucose indicator configured to emit fluorescence upon complexing glucose.

19. The compound of claim 18, wherein said glucose indicator is configured to selectively detect intracellular glucose at a pH in a range between about 4 and 10.

20. A composition, comprising the compound of any one of claims 1 to 19 and a liquid carrier.

21. The composition of claim 20, wherein said liquid carrier is an aqueous carrier.

22. The composition of claim 20 or 21, wherein said composition is a solution and wherein a concentration of said compound within the composition is between 10 pM and 0.1 M.

23. A method for determining a concentration of glucose within a cell, the method comprising: (i) contacting said cell or a cell medium in contact with the cell, with the compound of any one of claims 4 to 9, to obtain a complex; (ii) irradiating said cell at a first wavelength suitable for excitation of said complex; (iii) measuring fluorescence emitted from said cell to obtain a fluorescence value, and (iv) determining said concentration based on said fluorescence value.

24. The method of claim 23, wherein said method is for determining glucose concentration in-vitro.

25. The method of claim 23 or 24, wherein said measuring is performed by a fluorescence detector at a second wavelength, wherein the second wavelength is within an emission spectrum of said glucose complex.

26. The method of claim 25, wherein said first wavelength is in a range between 300 and 400 nm; and wherein said second wavelength is in a range between 400 and 500 nm.

27. The method of any one of claims 23 to 26, wherein said cell is an aggregate of cells.

28. The method of claim 27, wherein said aggregate of cells comprises a spheroid, and wherein said contacting is for a time sufficient for internalization of the compound into the spheroid.

29. The method of any one of claims 23 to 28, wherein said concentration is in a range between 0.0001 and 10 mM; and wherein said cell further comprises a pharmaceutically active ingredient.

30. The method of any one of claims 23 to 29, wherein the complex is an intracellular complex.

31. The method of any one of claims 23 to 29, wherein said medium is devoid of glucose.

32. An extracellular glucose indicator including any salt, any hydrate, or any solvate thereof, wherein said extracellular glucose indicator is represented by Formula 4:wherein each wavy bond represents an attachment point to a polymeric material or H, and at least one wavy bond is the attachment point; each a is an integer being independently between 3 and 20; each R2 independently is selected from halo, haloalkyl, a cyano group, carboxy, amide, carbonyl, anhydride, carbonate ester, carbamate, a sulfonyl group, a sulfonate group, a sulfinyl group, a sulfonamide group, an azo group, a guanidine group, and ammonium group, or any combination thereof;X represents ethyl, CNR’2, NH, O, S, -CONH-, -CONR’-, -C(=NH)NR’-, -C(=S)NR’-, - NC(=O)-, -NC(=O)O-, -NC(=O)N-, -NC(=S)O-, -NC(=S)N-, -C(=O)-, -C(=O)O-, -0C(=0)0-, -OC(=O)N-, -OC(=S)O-, -OC(=S)N-,, or phosphate; wherein each R is selected from H and Cl -CIO alkyl; and A represents any of cycloalkyl, aryl, and heteroaryl, a fused aryl, or a fused cycloalkyl or any combination thereof.

33. The extracellular glucose indicator of claim 32, wherein R2 is haloalkyl, wherein X is -CONH-; and wherein A is phenyl.

34. The extracellular glucose indicator of claim 32 or 33, represented by Formula 5:wherein the a is between 5 and 10.

35. The extracellular glucose indicator of claim 34, wherein each a is 6, and wherein each wavy bond represents said attachment point.

36. A method for determining a concentration of glucose both in a cell and in a cell medium, the method comprising:(i) contacting the cell attached to a cell support with the compound of any one of claims 4 to 9, to obtain a first complex comprising the compound bound to glucose within said cell; wherein said cell support is bound to the extracellular glucose indicator of any one of claims 32 to 35; and wherein said extracellular glucose indicator is configured for binding glucose within said cell medium to obtain a second complex;(ii) irradiating said cell at a first wavelength suitable for excitation of said first complex, measuring a first fluorescence to obtain a first fluorescence value, and determining a concentration of glucose within said cell based on said first fluorescence value; and(iii) irradiating said cell support at a second wavelength suitable for excitation of said second complex, measuring a second fluorescence to obtain a second fluorescence value, and determining a concentration of glucose within said cell medium based on said second fluorescence value.

37. The method of claim 36, wherein said step (iii) is performed while the cell support is immersed within the cell medium.

38. The method of claim 36 or 37, wherein said first fluorescence is at a wavelength between 410 and 450; and wherein said second fluorescence is at a wavelength between 460 and 510.

39. The method of any one of claims 36 to 38, wherein said step (ii) and said step (iii) are performed simultaneously or subsequently.

40. The method of claim 39, wherein said measuring the first fluorescence is by a confocal microscope.

41. The method of any one of claims 36 to 40, wherein said first wavelength is in a range between 300 and 400 nm; and wherein said second wavelength is in a range between 400 and 500 nm.

42. The method of any one of claims 36 to 41, wherein said cell is an aggregate of cells.

43. The method of claim 42, wherein said aggregate comprises a spheroid.

44. The method of any one of claims 36 to 43, wherein said concentration of glucose within said cell is in a range between 0.0001 and 10 mM; and wherein the concentration of glucose within said cell medium is in a range between 0.01 and 500 mM; and wherein said cell, said cell medium or both further comprises a pharmaceutically active ingredient.

45. The method of any one of claims 36 to 44, wherein said first fluorescence is measured at a wavelength of between about 420 and about 430nm; and wherein said second fluorescence is measured at a wavelength of between about 480 and about 490nm.

46. A method for determining glucose concentration within an aqueous liquid, the method comprising: (i) contacting said aqueous liquid with the compound of any one of claims 10 to 19, to obtain a obtain a complex comprising the compound bound to glucose;(ii) irradiating said aqueous liquid at a wavelength suitable for excitation of said complex;(iii) measuring fluorescence emitted by complex to obtain a fluorescence value, and (iv) determining glucose concentration within the aqueous liquid based on said fluorescence value.

47. The method of claim 46, wherein said aqueous liquid is selected from an aqueous solution, a biological fluid, a cell culture medium or a cell culture medium in contact with a cell.

48. The compound of claim 1 to 9, for use in delivery of glucose into a cell.

49. The compound for use of claim 48, wherein said delivery comprises in-vivo, ex- vivo and in-vitro; and wherein said cell is a human cell.

50. The compound for use of claim 48 or 49, wherein said cell is an aggregate of cells.

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