Wulff-type boronic acid-based glucose sensor

Abstract

The present invention relates to a biosensor for measuring the concentration of glucose and its use in glucose sensing, wherein the biosensor comprising a polymer comprising the moiety of formula (I). The biosensor of the present invention is particularly useful for glucose monitoring performed on a subject under intensive care as well as in the situations wherein the glucose monitoring is performed on an unconscious subject.

Description

[0001] Wulff-type boronic acid-based glucose sensor

[0002] The present application claims the benefit of priority of European patent application EP 23187710.1 , filed on July 25, 2023, which is incorporated herein by reference in its entirety.

[0003] The present invention relates to a biosensor for measuring the concentration of glucose and its use in glucose sensing. The biosensor of the present invention comprises a polymer (in particular a polymeric hydrogel) comprising a boronic acid-based glucose-binding moiety of formula (I). The biosensor of the present invention is particularly useful for glucose monitoring performed on a subject under intensive care as well as in situations wherein the glucose monitoring is performed on an unconscious subject. Accordingly, the present invention relates to the development of glucose sensors based on the measurement of osmotic pressure, incorporating a boronic acid glucose binding moiety (GBM) of formula (I) to yield a glucose responsive material with exceptional sensing properties.

[0004] In addition to glucose sensing applications, polymers capable of delivering active agents in a glucose concentrationdependent manner are sought after, in particular for the treatment of conditions characterized by pathological glucose concentration. For example, diabetes mellitus is a disorder of glucose regulation, characterized by an accumulating glucose concentration in the blood. The breakdown of glucose regulation can be attributed to the inability of the endocrine pancreas to secrete insulin or to the body's inability to properly use insulin. In the case of type 1 diabetes, the usual treatment consists in multiple daily blood glucose controls and subcutaneous injections. However, a better control of glycemia could be achieved if the insulin dose could be continuously adapted to the level of glucose in the blood, therefore avoiding glucose levels below or above the normal range, which causes detrimental complications. In this context, closed-loop delivery of insulin is highly sought-after. This can accordingly be achieved by using a glucose concentration sensing polymeric release system carrying insulin.

[0005] However, the use of glucose sensors based on polymers comprising a glucose-binding moiety (GBM) can be limited due to limited sensitivity of such measurements. Furthermore, due to pKa of boronic acids usually falling within the range of 7-9, such polymeric sensors have been shown to be heavily dependent on the pH (Skjaervold et al ANESTHESIOLOGY 2011 ; 114:18-20, Worsley et al. J Diabetes Sci Technol Vol 2, Issue 2, March 2008, Strasma et al. DOI: 10.1177 / 1932296815585872).

[0006] The article "The Development of a Continuous Intravascular Glucose Monitoring Sensor” by Crane, B.C. et al. published in Journal of Diabetes Science and Technology (https: / / doi.org / 10.1177 / 1932296815587937) discloses a fluorescence-type hydrogel for glucose sensing comprising a Wulff-type boronic acid. The document discloses the use of Wulff-type boronic acid in glucose sensing and discusses stoichiometry of binding between the boronic acid and glucose. This disclosure differs from the present invention, inter alia, in that the sensor is based on fluorescence measurement and not on change in the hydrogel's volume.

[0007] The article "A Wulff-type boronate for boronate affinity capture of cis-diol compounds at medium acidic pH condition” (Li et al., Chem. Commun., 2011,47, 8169-8171) discloses a boronate affinity column comprising a boronic acid moiety for cis-diol capture. This article, however, does not disclose glucose sensing using the provided polymers.

[0008] Kim et al. ("Polymeric Monosaccharide Receptors Responsive at Neutral pH") describe the synthesis of a styrene ADAM monomer and corresponding linear polymer by RAFT. Glucose sensitivity is determined by changes in turbidity of the polymer solutions (no hydrogel) (DOI: 10.1021 / ja905652w).

[0009] Document US 2016 / 109370 discloses a sensor containing a polymeric structure (HEAA-BIS) containing 5- acrylamido-2-((dimethylamino)methyl)phenylboronic acid units.

[0010] Document US 2008 / 214912 discloses a biosensor for detection of glucose comprising 4-aminomethyl-2-N,N'- dimethylaminomethylphenylboronic acid units in the polymeric structure.

[0011] Document ON 102 219 800 B discloses 4-amino-2-(dimethylamino methyl) phenylboronic acid incorporated into glycidyl methacrylate and polyethyleneglycol diacrylate by copolymerization.

[0012] The present invention addresses the problem of providing a glucose biosensor with improved properties. In particular the biosensors of the present invention overcome the problem of significant pH interference seen for the state of the art biosensors based on boronic acid. Furthermore, the biosensors of the present invention are, in particular embodiments of the present invention, characterized by improved selectivity for glucose over fructose, and / or by supressed mannitol interference, as well as other common interferents.

[0013] Accordingly, the present invention provides the formulation of a polymer sensor, particularly a polymeric hydrogel sensor which shrinks linearly when exposed to increasing concentrations of glucose. It has been postulated that due to the presence of an ortho-amino group as in the moiety of formula (I), the pKa of the boronic acid moiety is lowered, allowing complexation of glucose at physiological pH (which is meant as pH of 7.35 to 7.45) with very low pH dependence within said physiological range.

[0014] The problem is thus solved by the embodiments disclosed herein and as characterized by the claims.

[0015] The invention will be summarized in the following embodiments. In a first embodiment, the present invention relates to a biosensor for measuring the concentration of glucose, the biosensor comprising a polymer comprising the moiety of formula (I): wherein said moiety of formula (I) is immobilized in said polymer, wherein each R is independently C1-5 alkyl, C2-5 alkenyl or C2-5 alkynyl; wherein each Rsis independently selected from C1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, -(C0-3 alkylene)-OH, -(C0-3 alkylene)-O(Ci-5 alkyl), -(C0-3 alkylene)-O(Ci-5 alkylene)-OH, -(C0-3 alkylene)-O(Ci-5 alkylene)-O(Ci-5 alkyl), -(C0-3 alkylene)-SH, -(C0-3 alkylene)-S(Ci-5 alkyl), -(C0-3 alkylene)-S(Ci-5 alkylene)-SH, -(C0-3 alkylene)-S(Ci-5 alkylene)-S(Ci-5 alkyl), -(C0-3 alkylene)-NH2, -(C0-3 alkylene)-NH(Ci-5 alkyl), -(C0-3 alkylene)-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-3 alkylene)-NH-OH, -(C0-3 alkylene)-N(Ci_5alkyl)-OH, -(C0-3 alkylene)-NH-O(Ci.5alkyl), -(C0-3 alky lene)-N (C1-5 alkyl)-O(Ci-5 alkyl), -(C0-3 alky lene)-halogen, -(C0-3 alkylene)-(Ci-5 haloalkyl), -(C0-3 alkylene)-O-(Ci-5 haloalkyl), -(C0-3 alkylene)-CN, -(C0-3 alkylene)-NO2, -(C0-3 alkylene)-CHO, -(C0-3 alkylene)-CO-(Ci-5 alkyl), -(C0-3 alkylene)-COOH, -(C0-3 alkylene)-CO-O-(Ci-5 alkyl), -(C0-3 alkylene)-O-CO-(Ci-5 alkyl), -(C0-3 alkylene)-CO-NH2, -(C0-3 alkylene)-CO-NH(Ci-5 alkyl), -(C0-3 alkylene)-CO-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-3 alkylene)-NH-CO-(Ci-5 alkyl), -(C0-3 alkylene)-N(Ci-5 alkyl)-CO-(Ci-5 alkyl), -(C0-3 alkylene)-NH-CO-O-(Ci-5 alkyl), -(C0-3 alkylene)-N(Ci-5 alkyl)-CO-O-(Ci-5 alkyl), -(C0-3 alkylene)-O-CO-NH-(Ci-5 alkyl), -(C0-3 alkylene)-O-CO- N(CI-5 alkyl)-(Ci-5 alkyl), -(C0-3 alkylene)-SO2-NH2, -(C0-3 alkylene)-SO2-NH(Ci-5 alkyl), -(C0-3 alkylene)-SO2-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-3 alkylene)-NH-SO2-(Ci-5 alkyl), -(C0-3 alkylene)-N(Ci-5 alkyl)-SO2-(Ci-5 alkyl), -(C0-3 alkylene)-SO2-(Ci-5 alkyl), -(C0-3 alkylene)-SO-(Ci-5 alkyl), -(C0-3 alkylene)-carbocyclyl, and -(C0-3 alkylene)-heterocyclyl, wherein the carbocyclyl moiety in said -(C0-3 alkylene)-carbocyclyl and the heterocyclyl moiety in said -(C0-3 alkylene)-heterocyclyl are each optionally substituted with one or more groups independently selected from C1-4 alkyl, halogen, -CN, -NO2, -OH, -O-(Ci-4 alkyl), -SH, -S-(Ci-4 alkyl), -NH2, -NH(CI-4 alkyl), -N(CI-4 alkyl)(Ci.4 alkyl), -COCH, -COO(Ci.4alkyl), -CONH2, -CONH(CI.4alkyl), -CON(CI.4alkyl)(Ci.4alkyl), -NHCO(CI.4alkyl) and -N(CI-4 alkyl)-CO(Ci-4 alkyl); and wherein n Is O, 1, 2 or 3.

[0016] In a second embodiment, the present invention relates to the polymer comprised in the biosensor of the first embodiment, as described herein above. In a third embodiment, the present invention relates to a biosensor for measuring the concentration of glucose, as described in the first embodiment of the present invention, for use in an in vivo diagnostic method.

[0017] In a fourth embodiment, the present invention relates to a biosensor for measuring the concentration of glucose, as described in the first embodiment of the present invention, for use in an in vivo method of glucose monitoring.

[0018] In a fifth embodiment, the present invention relates to a biosensor for measuring the concentration of glucose, as described in the first embodiment of the present invention, for use in an in vivo method of diagnosing hyperglycemia or hypoglycemia.

[0019] In a sixth embodiment, the present invention relates to use of the biosensor of the first embodiment of the present invention in an in vitro diagnostic method.

[0020] In a seventh embodiment, the present invention relates to use of the biosensor of the first embodiment of the present invention for measuring the glucose concentration in a sample.

[0021] In an eighth embodiment, the present invention relates to use of the biosensor of the first embodiment of the present invention in an in vitro method of diagnosing hyperglycemia or hypoglycemia.

[0022] In a ninth embodiment, the present invention relates to use of the polymer as described in the second embodiment of the present invention for the manufacture of a reagent or a biosensor for monitoring the glucose level in a subject.

[0023] In a tenth embodiment, the present invention relates to a glucose-concentration-sensitive release formulation comprising a polymer as described in the second embodiment.

[0024] In an eleventh embodiment, the present invention relates to the biosensor of the present invention or to the glucose concentration-sensitive release formulation for use in the treatment of a condition dependent of the glucose concentration (e.g., diabetes).

[0025] The invention is further illustrated by the appended figures, which however are not to be construed as limiting.

[0026] Fig. 1 presents ADAM-3%-TMAPAA sensor length change response with different concentrations of glucose (indicated), PBS pH 7.437°C.

[0027] Fig. 2 presents comparison of pH interference of 3APB sensor (panel A) and ADAM sensor (panel B). Fig. 3 presents ADAM sensors response to glucose with increasing mol% TMAPAA (A), and ADAM sensor interference to B: 1 mM fructose, C: 5 mM mannitol, D: 38 mM NaCI, E: 10 mM lactate, F: 2 mM citrate G: pH 6.9, H: pH 7.6, at 2.2 mM Glucose with increasing mol% TMAPAA. ±0.85 mM indicates the acceptance limits of the interference. PBS pH 7.4, 37°C

[0028] Fig. 4 presents response curves of ADAM-3%-TMAPAA sensors (1-5) with glucose (indicated in mM) and various interferents at 2.2 mM glucose: FRU (1 mM fructose), MAN (5 mM mannitol), LAC (10 mM lactate), CIT (2 mM citrate), NaCI (38 mM), pH 6.9 and pH 7.6. Interferent concentrations tested are well above concentrations expected in vivo.

[0029] Fig. 5 presents impact of copolymerisation of 2APB with ADAM on interference to 5 mM mannitol, in all cases.

[0030] Fig. 6 shows (A) a Model of the Fabry-Perot interferometer. The hydrogel makes up the Fabry-Perot cavity. The refractive indexes neff, ng, nt are the effective index of the fiber, index of the hydrogel and index of the fluid, respectively. The spacing of the two boundaries is indicated as Lg, also referred to as the hydrogel length; (B) a typical interference pattern from a low finesse Fabry-Perot cavity. The wavelength of the light is converted to frequency on the x-axis. The observed 0.35 phase shift is equivalent to a hydrogel cavity length change of 100 nm.

[0031] Fig. 7 shows sensor length change with increasing concentrations of glucose for ADAM-HEAA sensors (dashed) and ADAM-acrylamide sensors (solid) (panels A and B show hydrogels including TMAPAA and without it, respectively).

[0032] Fig. 8 demonstrates effects of interference by the presence of 2 mM citrate when detecting 6 mM glucose concentration.

[0033] Fig. 9 shows in panel A glucose response (indicated in mM) of post-modified ADAM sensors, in PBS, pH 7.4 at 37 degrees C, and in panel B, glucose response (indicated in mM) of directly polymerised ADAM sensor containing 28 mol% ADAM, in PBS, pH 7.4 at 37 degrees C.

[0034] As explained above, the present invention relates to a biosensor for measuring the concentration of glucose, the biosensor comprising a polymer (preferably a polymeric hydrogel) comprising the moiety of formula (I): wherein said moiety of formula (I) is immobilized in said polymer.

[0035] In formula (I), each R is independently C1-5 alkyl, C2-5 alkenyl or C2-5 alkynyl. Preferably, each is independently C1-5 alkyl. More preferably, each R is C1-2 alkyl. Even more preferably, each R is methyl.

[0036] In formula (I), each Rsis independently selected from C1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, -(C0-3 alkylene)-OH, -(C0-3 alkylene)-O(Ci-5 alkyl), -(C0-3 alkylene)-O(Ci-5 alkylene)-OH, -(C0-3 alkylene)-O(Ci-5 alkylene)-O(Ci-5 alkyl), -(C0-3 alkylene)-SH, -(C0-3 alkylene)-S(Ci-5 alkyl), -(C0-3 alkylene)-S(Ci-5 alkylene)-SH, -(C0-3 alkylene)-S(Ci-5 alkylene)-S(Ci-5 alkyl), -(C0-3 alkylene)-NH2, -(C0-3 alkylene)-NH(Ci-5 alkyl), -(C0-3 alkylene)-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-3 alkylene)-NH-OH, -(C0-3 alkylene)-N(Ci_5alkyl)-OH, -(C0-3 alkylene)-NH-O(Ci.5alkyl), -(C0-3 alky lene)-N (C1-5 alkyl)-O(Ci-5 alkyl), -(C0-3 alky lene)-halogen, -(C0-3 alkylene)-(Ci-5 haloalkyl), -(C0-3 alkylene)-O-(Ci-5 haloalkyl), -(C0-3 alkylene)-CN, -(C0-3 alkylene)-NO2, -(C0-3 alkylene)-CHO, -(C0-3 alkylene)-CO-(Ci-5 alkyl), -(C0-3 alkylene)-COOH, -(C0-3 alkylene)-CO-O-(Ci-5 alkyl), -(C0-3 alkylene)-O-CO-(Ci-5 alkyl), -(C0-3 alkylene)-CO-NH2, -(C0-3 alkylene)-CO-NH(Ci-5 alkyl), -(C0-3 alkylene)-CO-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-3 alkylene)-NH-CO-(Ci-5 alkyl), -(C0-3 alkylene)-N(Ci-5 alkyl)-CO-(Ci-5 alkyl), -(C0-3 alkylene)-NH-CO-O-(Ci-5 alkyl), -(C0-3 alkylene)-N(Ci-5 alkyl)-CO-O-(Ci-5 alkyl), -(C0-3 alkylene)-O-CO-NH-(Ci-5 alkyl), -(C0-3 alkylene)-O-CO- N(CI-5 alkyl)-(Ci-5 alkyl), -(C0-3 alkylene)-SO2-NH2, -(C0-3 alkylene)-SO2-NH(Ci-5 alkyl), -(C0-3 alkylene)-SO2-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-3 alkylene)-NH-SO2-(Ci-5 alkyl), -(C0-3 alkylene)-N(Ci-5 alkyl)-SO2-(Ci-5 alkyl), -(C0-3 alkylene)-SO2-(Ci-5 alkyl), -(C0-3 alkylene)-SO-(Ci-5 alkyl), -(C0-3 alkylene)-carbocyclyl, and -(C0-3 alkylene)-heterocyclyl, wherein the carbocyclyl moiety in said -(C0-3 alkylene)-carbocyclyl and the heterocyclyl moiety in said -(C0-3 alkylene)-heterocyclyl are each optionally substituted with one or more groups independently selected from C1-4 alkyl, halogen, -CN, -NO2, -OH, -O-(Ci-4 alkyl), -SH, -S-(Ci-4 alkyl), -NH2, -NH(CI-4 alkyl), -N(CI-4 alkyl)(Ci.4 alkyl), -COCH, -COO(Ci.4alkyl), -CONH2, -CONH(CI.4alkyl), -CON(CI.4alkyl)(Ci_4alkyl), -NHCO(CI.4alkyl) and -N(CI-4 alkyl)-CO(Ci-4 alkyl).

[0037] Preferably, each Rsis independently selected from C1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, -(C0-3 alky lene)-OH, -(C0-3 alkylene)-O(Ci-5 alkyl), -(C0-3 alkylene)-O(Ci-5 alkylene)-OH, -(C0-3 alkylene)-O(Ci-5 alkylene)-O(Ci-5 alkyl), -(C0-3 alkylene)-SH, -(C0-3 alkylene)-S(Ci-5 alkyl), -(C0-3 alkylene)-S(Ci-5 alkylene)-SH, -(C0-3 alkylene)-S(Ci-5 alkylene)-S(Ci-5 alkyl), -(C0-3 alkylene)-NH2, -(C0-3 alkylene)-NH(Ci-5 alkyl), -(C0-3 alkylene)-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-3 alkylene)-NH-OH, -(C0-3 alkylene)-N(Ci.5alkyl)-OH, -(C0-3 alkylene)-NH-O(Ci.5alkyl), -(C0-3 alky lene)-N (Ci-5 alkyl)-0(Ci-5 alkyl), -(C0-3 alky lene)-halogen, -(C0-3 alkylene)-(Ci-5 haloalkyl), -(C0-3 alkylene)-O-(Ci-5 haloalkyl), -(C0-3 alkylene)-CN, -(C0-3 alkylene)-NO2, -(C0-3 alkylene)-CHO, -(C0-3 alkylene)-CO-(Ci-5 alkyl), -(C0-3 alkylene)-COOH, -(C0-3 alkylene)-CO-O-(Ci-5 alkyl), -(C0-3 alkylene)-O-CO-(Ci-5 alkyl), -(C0-3 alkylene)-CO-NH2, -(C0-3 alkylene)-CO-NH(Ci-5 alkyl), -(C0-3 alkylene)-CO-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-3 alkylene)-NH-CO-(Ci-5 alkyl), -(C0-3 alkylene)-N(Ci-5 alkyl)-CO-(Ci-5 alkyl), -(C0-3 alkylene)-NH-CO-O-(Ci-5 alkyl), -(C0-3 alkylene)-N(Ci-5 alkyl)-CO-O-(Ci-5 alkyl), -(C0-3 alkylene)-O-CO-NH-(Ci-5 alkyl), -(C0-3 alkylene)-O-CO- N(CI-5 alkyl)-(Ci-5 alkyl), -(C0-3 alkylene)-SO2-NH2, -(C0-3 alkylene)-SO2-NH(Ci-5 alkyl), -(C0-3 alkylene)-SO2-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-3 alkylene)-NH-SO2-(Ci-5 alkyl), -(C0-3 alkylene)-N(Ci-5 alkyl)-SO2-(Ci-5 alkyl), -(C0-3 alkylene)-SO2-(Ci-5 alkyl), and -(C0-3 alkylene)-SO-(Ci-5 alkyl).

[0038] More preferably, each Rsis independently selected from C1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, -(C0-3 alkylene)-OH, -(C0-3 alkylene)-O(Ci-5 alkyl), -(C0-3 alkylene)-O(Ci-5 alkylene)-OH, -(C0-3 alkylene)-O(Ci-5 alkylene)-O(Ci-5 alkyl), -(C0-3 alkylene)-SH, -(C0-3 alkylene)-S(Ci-5 alkyl), -(C0-3 alkylene)-S(Ci-5 alkylene)-SH, -(C0-3 alkylene)-S(Ci-5 alkylene)-S(Ci-5 alkyl), -(C0-3 alkylene)-NH2, -(C0-3 alkylene)-NH(Ci-5 alkyl), -(C0-3 alkylene)-N(Ci-5 alkyl)(Ci-5alkyl), -(C0-3 alkylene)-NH-OH, -(C0.3alkylene)-N(Ci_5alkyl)-OH, -(C0.3alkylene)-NH-O(Ci.5alkyl), -(C0-3 alky lene)-N (C1-5 alkyl)-O(Ci-5 alkyl), -(C0-3 alky lene)-halogen, -(C0-3 alkylene)-(Ci-5 haloalkyl), -(C0-3 alkylene)-O-(Ci-5 haloalkyl), -(C0-3 alkylene)-CN, -(C0-3 alkylene)-NO2, -(C0-3 alkylene)-CHO, -(C0-3 alkylene)-CO-(Ci-5 alkyl), -(C0-3 alkylene)-COOH, -(C0-3 alkylene)-CO-O-(Ci-5 alkyl), -(C0-3 alkylene)-O-CO-(Ci-5 alkyl), -(C0-3 alkylene)-CO-NH2, -(C0-3 alkylene)-CO-NH(Ci-5 alkyl), -(C0-3 alkylene)-CO-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-3 alkylene)-NH-CO-(Ci-5 alkyl), -(C0-3 alkylene)-N(Ci-5 alkyl)-CO-(Ci-5 alkyl), -(C0-3 alkylene)-NH-CO-O-(Ci-5 alkyl), -(C0-3 alkylene)-N(Ci-5 alkyl)-CO-O-(Ci-5 alkyl), -(C0-3 alkylene)-O-CO-NH-(Ci-5 alkyl), -(C0-3 alkylene)-O-CO- N(CI-5alkyl)-(Ci-5alkyl).

[0039] Even more preferably, each Rsis independently selected from C1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, -(C0-3 alkylene)-OH, -(C0-3 alkylene)-O(Ci-5 alkyl), -(C0-3 alkylene)-O(Ci-5 alkylene)-OH, -(C0-3 alkylene)-O(Ci-5 alkylene)-O(Ci-5 alkyl), -(C0-3 alkylene)-SH, -(C0-3 alkylene)-S(Ci-5 alkyl), -(C0-3 alkylene)-S(Ci-5 alkylene)-SH, -(C0-3 alkylene)-S(Ci-5 alkylene)-S(Ci-5 alkyl), -(C0-3 alkylene)-NH2, -(C0-3 alkylene)-NH(Ci-5 alkyl), -(C0-3 alkylene)-N(Ci-5 alkyl)(Ci-5alkyl), -(C0-3 alkylene)-NH-OH, -(C0-3 alkylene)-N(Ci.5alkyl)-OH, -(C0-3 alkylene)-NH-O(Ci.5alkyl), -(C0-3 alkylene)-N(Ci-5 alkyl)-O(Ci-5 alkyl), -(C0-3 alky lene)-halogen, -(C0-3 alkylene)-(Ci-5 haloalkyl), -(C0-3 alkylene)-O-(Ci-5 haloalkyl), -(C0-3 alky lene)-CN, and -(C0-3 alkylene)-NO2.

[0040] Still more preferably, each Rsis independently selected from C1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, -OH, -O(Ci-5 alkyl), -O(Ci-5 alkylene)-OH, -O(Ci-5 alkylene)-O(Ci-5 alkyl), -SH, -S(Ci-5 alkyl), -S(Ci-5 alkylene)-SH, -S(Ci-5 alkylene)-S(Ci-5alkyl), -NH2, -NH(CI.5alkyl), -N(CI.5alkyl)(Ci.5alkyl), -NH-OH, -N(CI.5alkyl)-OH, -NH-O(CI.5alkyl), -N(CI-5 alkyl)-O(Ci-5 alkyl), -halogen, C1-5 haloalkyl, -O-(Ci-5 haloalkyl), -CN, and -NO2.

[0041] Again more preferably, each Rsis independently selected from -OH, -SH, -NH2, -NH-OH, -halogen, -CN, and -NO2. In formula (I), n is 0, 1, 2 or 3. Preferably, n is O or 1. More preferably, n is O. It will be understood that the variable n indicates the number of substituents Rswhich are attached to the respective phenyl moiety. If n is 0, there are no substituents Rs, so that the corresponding phenyl ring is unsubstituted (i.e., carries hydrogen in place of Rs). Accordingly, it is preferred that the phenyl ring in the moiety of formula (I) is not substituted with Rs.

[0042] It will further be understood that the moiety of formula (I) may be attached to (or immobilized in) the polymer via any ring carbon atom of the respective phenyl ring, which is reflected by a bond that extends into the phenyl ring, whereby the wavy line (at one end of this bond) indicates the point of attachment of the moiety of formula (I).

[0043] The way in which the moiety of formula (I) is attached to the remainder of the polymer is not particularly limited, and any chemically feasible attachment is encompassed by the present invention. For example, any of these moieties may be attached via an amide linkage or an inversed amide linkage, e.g., via a group -NH-CO- or a group -CO-NH-. A particularly preferred attachment is illustrated in formulae (1-1) below; a further possible point of attachment is as shown in formula (l-b) below. Further examples of attachment points are apparent from the disclosure of the methods for preparing the polymers and the monomers used, including any of those described in the examples section.

[0044] It is preferred that the polymer of the invention comprises a moiety of formula (l-a):

[0045] (l-a) wherein R, Rsand n are as in formula (I).

[0046] However, in one embodiment of the present invention, the polymer of the invention comprises a moiety of formula (l-b): wherein R, Rsand n are as in formula (I).

[0047] It will be appreciated by the skilled person that the moiety of formula (I) may be interconvertible with a form comprising a five membered ring formed through a bonding interaction between the nitrogen atom and the boron atom in the moiety of formula (I). Both forms may be present in an equilibrium, depending on the external conditions, e.g., the surrounding medium. In particular, in an aqueous medium, these forms may be interconverted by undergoing a ring-closing reaction or a corresponding ring-opening reaction, as illustrated in the following scheme:

[0048] The polymer as provided in accordance with the present invention is not particularly limited. It is preferred that the polymer is hydrophilic, which is beneficial when the polymer is brought into contact with the blood of a subject. Preferably, the polymer forms a polymeric hydrogel, i.e., the polymer is preferably a polymeric hydrogel. A hydrogel is generally understood to be a biphasic material comprising a solid polymer, which preferably forms a porous and permeable structure, and an interstitial fluid comprised within and in contact with said solid polymer. In a hydrogel, said fluid is preferably water or an aqueous solution (e.g., a liquid composition comprising at least 90% (v / v) water). It will be understood that the polymeric hydrogel typically includes crosslinks between the individual polymer molecules forming the hydrogel. Such crosslinks may be covalent or non-covalent (which in turn may also be dynamic, i.e., may be subject to forming and unforming).

[0049] The term polymer preferably also encompasses dendrimers and polymers without crosslinks.

[0050] It is preferred that the term polymer refers to a polymer that includes crosslinks (covalent or non-covalent) between the individual polymer molecules. Preferred examples of the polymer (or the polymeric hydrogel) to be used in the biosensor of the present invention are described herein below. In general, it is preferred that the polymer does not comprise hyaluronic acid. It is accordingly preferred that the polymeric hydrogel comprised in the biosensor of the present invention does not comprise hyaluronic acid.

[0051] Preferably, the polymer of the present invention is a polyacrylamide-based polymer. The term "polyacrylamidebased polymer” preferably refers to a polymer made from (or composed of) more than 50% (w / w) of acrylamide monomers, more preferably at least 60% (w / w) of acrylamide monomers, even more preferably at least 70% (w / w) of acrylamide monomers, even more preferably at least 80% (w / w) of acrylamide monomers, or yet even more preferably at least 90% (w / w) of acrylamide monomers. Accordingly, the polyacrylamide-based polymer is preferably obtainable by polymerization of a composition of monomers wherein more than 50% (w / w) (or, with increasing preference, at least 60% (w / w), at least 70% (w / w), at least 80% (w / w), or at least 90% (w / w)) of the monomers are acrylamide monomers.

[0052] It will be understood that "acrylamide monomers” are compounds comprising an acrylamide moiety, e.g., as illustrated in the following:

[0053] Such acrylamide monomers may also be referred to as N-substituted acrylamide monomers.

[0054] In principle, further substitutions may be made to the alkenyl moiety in the above-depicted acrylamide monomer, but it is preferred that the alkenyl moiety is unsubstituted. Accordingly, reference can be made to unsubstituted polyacrylamide-based polymer, which preferably refers to a polymer made from (or composed of) more than 50% (w / w) of acrylamide monomers, wherein the alkenyl moiety is unsubstituted, more preferably at least 60% (w / w) of acrylamide monomers, wherein the alkenyl moiety is unsubstituted, even more preferably at least 70% (w / w) of acrylamide monomers, wherein the alkenyl moiety is unsubstituted, even more preferably at least 80% (w / w) of acrylamide monomers, wherein the alkenyl moiety is unsubstituted, or yet even more preferably at least 90% (w / w) of acrylamide monomers wherein the alkenyl moiety is unsubstituted. Accordingly, the unsubstituted polyacrylamide-based polymer is preferably obtainable by polymerization of a composition of monomers wherein more than 50% (w / w) (or, with increasing preference, at least 60% (w / w), at least 70% (w / w), at least 80% (w / w), or at least 90% (w / w)) of the monomers are acrylamide monomers, wherein the alkenyl moiety is unsubstituted.

[0055] The use of acrylamide monomers is advantageous, as these monomers combine the desired hydrophilic properties, which facilitate the formation of a hydrogel, with ease of functionalization, as required when constructing the polymer of the present invention. The polymer of the present invention may also be a polymethacrylamide-based polymer. The term "polymethacrylamide-based polymer” preferably refers to a polymer made from (or composed of) more than 50% (w / w) of methacrylamide monomers, more preferably at least 60% (w / w) of methacrylamide monomers, even more preferably at least 70% (w / w) of methacrylamide monomers, even more preferably at least 80% (w / w) of methacrylamide monomers, or yet even more preferably at least 90% (w / w) of methacrylamide monomers. Accordingly, the methacrylamide-based polymer is preferably obtainable by polymerization of a composition of monomers wherein more than 50% (w / w) (or, with increasing preference, at least 60% (w / w), at least 70% (w / w), at least 80% (w / w), or at least 90% (w / w)) of the monomers are methacrylamide monomers.

[0056] It will be understood that "methacrylamide monomers” are compounds comprising an methacrylamide moiety, e.g., as illustrated in the following:

[0057] Such methacrylamide monomers may also be referred to as N-substituted methacrylamide monomers.

[0058] The polymer of the present invention may also be a polyacrylate-based polymers. The term "polyacrylate-based polymer” preferably refers to a polymer made from (or composed of) more than 50% (w / w) of acrylate monomers, more preferably at least 60% (w / w) of acrylate monomers, even more preferably at least 70% (w / w) of acrylate monomers, even more preferably at least 80% (w / w) of acrylate monomers, or yet even more preferably at least 90% (w / w) of acrylate monomers. Accordingly, the polyacrylate-based polymer is preferably obtainable by polymerization of a composition of monomers wherein more than 50% (w / w) (or, with increasing preference, at least 60% (w / w), at least 70% (w / w), at least 80% (w / w), or at least 90% (w / w)) of the monomers are acrylate monomers.

[0059] It will be understood that "acrylate monomers” are compounds comprising an acrylate moiety, e.g., as illustrated in the following:

[0060] The polymer of the present invention may also be a poly methacrylate-based polymer. The term "polymethacrylate- based polymer” preferably refers to a polymer made from (or composed of) more than 50% (w / w) of methacrylate monomers, more preferably at least 60% (w / w) of methacrylate monomers, even more preferably at least 70% (w / w) of acrylate monomers, even more preferably at least 80% (w / w) of methacrylate monomers, or yet even more preferably at least 90% (w / w) of methacrylate monomers. Accordingly, the polymethacrylate-based polymer is preferably obtainable by polymerization of a composition of monomers wherein more than 50% (w / w) (or, with increasing preference, at least 60% (w / w), at least 70% (w / w), at least 80% (w / w), or at least 90% (w / w)) of the monomers are methacrylate monomers.

[0061] It will be understood that "methacrylate monomers” are compounds comprising a methacrylate moiety, e.g., as illustrated in the following:

[0062] It has been demonstrated that the polyacrylamide-based polymers, wherein the acrylamide is unsubstituted acrylamide, are better suited for use in the biosensors of the present invention compared to other polymers, in particular compared to polymers based on substituted acrylamide. In particular, it has been demonstrated that a sensor based on unsubstituted polyacrylamide outperforms a sensor based on substituted polyacrylamide, such as based on HEAA, which is N-(2-hydroxyethy l)acry lamide, at least insofar as sensitivity is concerned. Accordingly, it has been demonstrated that for the same ratios of monomers, the ADAM-acrylamide sensor length change at 20 mM glucose is around 1.6 times greater than ADAM-HEAA sensor (see Example 2 for further details).

[0063] Accordingly, it is preferred that the polymer is an unsubstituted polyacrylamide-based polymer.

[0064] As used herein, "measuring the concentration of glucose” may comprise measuring an absolute value of the concentration of glucose in a liquid (e.g., blood) which is contacted with the biosensor. However, the measuring the concentration of glucose also refers to the determination of the concentration of glucose relative to a threshold value. For example, measuring the concentration of glucose may also involve determining whether the concentration of glucose is lower than a threshold value, or higher than a threshold value, or whether said concentration falls within a reference range of concentration. This is particularly useful in clinical settings, e.g., when a dropping of the blood glucose concentration below a certain critical threshold value necessitates an intervention by a physician.

[0065] The measurement of the concentration of glucose using the biosensor of the present invention predicates on a change of volume of the polymer upon being contacted with a liquid comprising glucose, in other words, upon being contacted with glucose. The present invention is based, at least in part, on a surprising finding that the polymer of the present invention reduces its volume when contacted with glucose. Accordingly, when the polymer (or, as in the preferred case, the polymeric hydrogel) is contacted with a liquid comprising no glucose (which may also be referred to as a liquid comprising glucose below a minimum detectable concentration or below a certain threshold value), in other words in the absence of glucose, the boronic acid-based glucose-binding moiety the polymer (or the polymeric hydrogel) does not form any new interactions and the volume of the polymer (or the polymeric hydrogel) remains unchanged. In turn, when exposed to a liquid comprising glucose (i.e. , comprising a detectable concentration of glucose or comprising glucose at a concentration exceeding a certain threshold value), in other words in the presence of glucose, the boronic acid-based glucose-binding moiety binds to glucose, which results in a change of volume of the polymer. The change of volume of the polymer (or the polymeric hydrogel) which can be observed upon transition from a liquid comprising no glucose to a liquid comprising glucose, in other words upon transition from the absence of glucose to the presence of glucose, is a decrease in volume, i.e. a shrinking, of the polymer (or the polymeric hydrogel). Without being bound by the theory, it is postulated that shrinking of the polymer upon contact with glucose is due to the formation of a complex of stoichiometry 2:1 between the moiety of formula (I) and the glucose, as shown in the following scheme:

[0066] Thus preferably, in the biosensor for measuring the concentration of glucose of the present invention, the polymer shrinks when contacted with glucose.

[0067] It is preferred that the extent of shrinking is substantially linearly proportional to the concentration of glucose. Herein, the extent of shrinking is preferably understood as |AL / L|, i.e. an absolute value taken from AL / L, wherein AL is the change in linear dimensions of the polymer (or the polymeric hydrogel), and L is total linear dimension of said polymer (or said polymeric hydrogel). Accordingly, the biosensors comprising polymers as described herein preferably exhibit a substantially linear relationship, more preferably a linear relationship between the concentration of glucose and AL / L. As used herein, the term "substantially linear” preferably means within the value expected for a linear relationship, particularly with 10% tolerance (i.e., within ±10% of a linear relationship), more preferably with 5% tolerance, even more preferably with 2% tolerance.

[0068] As it is apparent to the skilled person, the dependence of the extent of shrinking on the concentration of glucose allows detection of changes in glucose concentration. For example, a reference curve of the measured extent of swelling / shrinking (AL / L) at multiple known concentrations of glucose can be prepared; by comparing a measured value of the extent of swelling / shrinking with the reference curve, the concentration of glucose can be determined.

[0069] As used herein, whenever the term "about” is employed in connection with a numerical value, it preferably refers to ± 10% of the indicated numerical value, more preferably to ± 5%, even more preferably to ± 2%, even more preferably to ± 1 % of the indicated numerical value, and most preferably to the exact numerical value indicated. If the term "about” is used in connection with the endpoints of a range, it preferably refers to the range from the lower endpoint -10% of its indicated numerical value to the upper endpoint +10% of its indicated numerical value, more preferably to the range from of the lower endpoint -5% to the upper endpoint +5%, even more preferably to the range from of the lower endpoint -2% to the upper endpoint +2%, yet even more preferably to the range from the lower endpoint -1 % to the upper endpoint +1 %, and most preferably to the range defined by the exact numerical values of the lower endpoint and the upper endpoint. It is noted that the numerical value may also be a ratio, for example, a ratio used to express the composition of the polymer of the invention, as described herein above. While a ratio may be described as a division operation on two numbers, it can also be expressed as a single number resulting from said division. For example, the ratio of 1 :2 can be otherwise expressed as a ratio of 0.5. The aforementioned deviations of ± x% can be applied to a corresponding single number (resulting from said division operation), and the endpoints of the resulting range can each be converted back into a corresponding ratio. For example, a ratio of 1 :2 ± 10% can also be expressed as a ratio of 0.5 ± 10% or as a ratio of from 0.45 to 0.55, which corresponds to a ratio of from 1 : 1.82 to 1 : 2.22.

[0070] Moreover, the determination / detection of a change of volume of the polymer can also be effected by measuring another related value, particularly a proxy (or surrogate) value which is dependent on the volume of the polymer, for example, by measuring the osmotic pressure (or a change of the osmotic pressure). Thus, the present invention also relates to a biosensor for measuring the concentration of glucose (as described herein), wherein the measurement of the concentration of glucose is based on a glucose concentration-sensitive change in the osmotic pressure within the polymer (or, as preferred in the present invention, of the polymeric hydrogel) comprised in the biosensor.

[0071] The polymer (polymeric hydrogel) volume change, i.e., swelling / shrinking in response to contact with glucose is reversible and can be measured (and consequently also real time monitored) by various signal-transducing mechanisms, including electrochemical, mechanical, and optical techniques (for example by means of Fabry-Perot- Interferometry or refractometry). The refractive index of the polymer can be changed simply as a result of the volume change (i.e. if the polymer swells it will become more rarefied and so its refractive index will fall) and / or because analyte molecules bind to the polymer chains.

[0072] The polymer, which is preferably a polymeric hydrogel, has been described herein above. In accordance with established practice in the art, the polymer can be defined according to the process of its preparation, in particular in terms of the monomers that are being polymerized. Accordingly, it is preferred that the polymer (or the polymeric hydrogel) is obtainable in a polymerization reaction of a composition comprising:

[0073] (I) an acrylamide monomer comprising the boronic acid-based glucose binding moiety of formula (I). It will be understood that the boronic acid-based glucose binding moiety in the acrylamide monomer (i) is attached to the remainder of the respective monomer through an attachment point, as shown herein above. It is to be noted that some formulae showing preferred embodiments of the boronic acid-based glucose binding moiety include an -NH-CO- moiety; in the corresponding acrylamide monomers (i) or (ii), this -NH-CO- moiety preferably forms part of the acrylamide moiety, i.e., the -CO- in said -NH-CO- moiety is preferably attached to an ethylene group (-CHOH2), thereby forming the acrylamide moiety -NH-CO-CH=CH2.

[0074] It is particularly preferred that (i) is: wherein R, Rsand n are as in formula (I).

[0075] However, in one embodiment (i) is: wherein R, Rsand n are as in formula (I). Such compounds are known from the literature (Chem. Commun., 2011, 47, 8169-8171, DOI: 10.1039 / c1cc11096a).

[0076] Preferably, the composition used in the polymerization reaction further comprises:

[0077] (ii) an acrylamide monomer free of boronic-acid moiety, and

[0078] (iii) a crosslinker acrylamide monomer.

[0079] The acrylamide monomer free of boronic-acid moiety is a monomer comprising a moiety: preferably comprising exactly one copy of such a moiety, but not comprising any boronic-acid moiety. Such monomers are not particularly limited and preferably include an unsubstituted acrylamide monomer (wherein the above-depicted moiety is attached to a hydrogen) or substituted acrylamide monomers wherein the above-depicted moiety is, e.g., attached to a hydroxylalkyl group or an ethylene glycol oligomer. Exemplary preferred monomers (ii) are selected from the following monomers:

[0080] , wherein q is an integer from 0 to 10; preferably wherein q is an integer from 2 to 5, more preferably wherein q is 2 or 5. One single type of monomer (ii) or more than one type (e.g., two or three types) of the monomers (ii) may be present in the composition whose polymerization affords the polymer of the present invention.

[0081] The crosslinker acrylamide monomer (ill) preferably comprises at least two copies of the following moiety: preferably it comprises exactly two copies of the above-depicted moiety. Moreover, it is preferably free of boronic- acid moiety.

[0082] Preferably, the crosslinker acrylamide monomer (ill) comprises (or is) a monomer selected from: wherein q is an integer from 0 to 10, preferably wherein q is an integer from 2 to 5, more preferably wherein q is 2 or 5.

[0083] Preferably, for the polymer (more preferably, the polymeric hydrogel) of the present invention, the acrylamide monomer (I) constitutes between 6 and 30 mol% of the acrylamide-based components in said composition used for the polymerization reaction. More preferably, the acrylamide monomer (I) constitutes between 10 and 14 mol% of the acrylamide-based components in said composition used for the polymerization reaction. However, the amount of the acrylamide monomer (I) may also be higher and constitute between 15 and 24 mol% of the acrylamide-based components in said composition used for the polymerization reaction, preferably between 16 and 20 mol% of the acrylamide-based components in said composition used in the polymerization reaction.

[0084] Preferably, in the polymer of the present invention, the monomer (ii) constitutes between 60 and 94 mol% of the acrylamide-based components in said composition used for the polymerization reaction, preferably between 66 and 90 mol% of the acrylamide-based components in said composition used for the polymerization reaction, more preferably between 74 and 84 mol% of the acrylamide-based components in said composition used for the polymerization reaction.

[0085] Preferably, in the polymer of the present invention, the crosslinker acrylamide monomer (ill) constitutes between 0.1 and 4 mol% of the acrylamide-based components in said composition used for the polymerization reaction. More preferably, in the polymer of the present invention, the crosslinker acrylamide monomer (ill) constitutes between 1 and 4 mol% of the acrylamide-based components in said composition used for the polymerization reaction.

[0086] In an alternative embodiment of the present invention, the polymer (or the polymeric hydrogel) is obtainable in a polymerization reaction of a composition comprising:

[0087] (i-a) a non-acrylamide monomer comprising the boronic acid-based glucose binding moiety of formula (I). Herein, non-acrylamide monomer is to be understood as a monomer not comprising the moiety -NH-CO- CH=CH2.

[0088] Accordingly, in this alternative embodiment of the present invention, the polymer (or the polymeric hydrogel) is obtainable in a polymerization reaction as described hereinabove, wherein (I) is replaced with (i-a).

[0089] In this alternative embodiment of the present invention, it is preferred that (i-a) is: wherein R, Rsand n are as in formula (I). Such monomers are known from the literature (J. Am. Chem. Soc. 2009, 131 , 13908-13909, doi: 10. 1021 / ja905652w) In a further alternative embodiment, (i-a) is an acrylate monomer.

[0090] The polymers and, as applicable, the polymeric hydrogels of the present invention can be prepared according to the methods described herein above and as illustrated in the examples section.

[0091] Accordingly, it is preferred that the moiety of formula (I) is incorporated into the monomer(s) and then copolymerized into the polymer of the invention (preferably a polymeric hydrogel) comprising said moiety. However, the present invention also encompasses embodiments wherein the moiety of formula (I) is incorporated into a previously prepared polymer through a modification of said polymer. One exemplary way of such modification is a Michael- like addition reaction of -SH group, present in a preformed polymer as thiolactone to acrylamide moiety.

[0092] The polymers of the present invention, in particular the polymeric hydrogels of the present invention, exhibit advantageous properties, as discussed above and as also demonstrated in the examples, and are therefore particularly suitable for use in the biosensors of the present invention for measuring (or sensing) the concentration of glucose.

[0093] It is particularly preferred that the extent of shrinking of the polymer (or the polymeric hydrogel) in the presence of glucose is substantially independent of the pH value in the range from about pH 6.9 to about pH 7.6. This pH range is particularly relevant in the case of application in the intensive care unit (ICU), wherein not only normal pH range of blood may be seen in the treated patient, i.e., the range from about 7.4 to about 7.6, but treated patients may also show a broader range of blood pH, including pathological pH values.

[0094] As used herein, the expression "substantially independent of the pH value” in a particular pH range preferably means that a maximum change / variation of the extent of swelling / shrinking of 15%, understood as AL / L of 15% (corresponding to the sensitivity (0-20 mM glucose)), is observed within said pH range, more preferably a maximum change / variation of 10%, even more preferably a maximum change / variation of 5%. The term "substantially independent of” also includes a specific reference to the narrower meaning of "independent of”.

[0095] The biosensors of the present invention and the polymers of the present invention may include further moieties and further monomers, which may lead to additional advantages. These are described in the specific embodiments of the biosensor of the present invention, disclosed in the following.

[0096] In a first specific embodiment of the biosensor of the present invention, the polymer further comprises a moiety that is positively charged at pH = 7.4. As understood herein, the moiety that is positively charged is a moiety whose net charge at pH = 7.4 is above 0. Accordingly, a zwitterionic moiety which includes both positive and negative charge is not considered to be positively charged as its net charge is neutral. Preferably, in this first specific embodiment, the polymer further comprises a moiety comprising an amino (or ammonium) group that is positively charged at pH = 7.4.

[0097] Examples of such a group include a primary amino group -NH2(which in its ammonium form has the structure of -N Hs+), a secondary amino group -NH(CI-5 alkyl) such as -NHCH3 (which in its ammonium form has the structure of -(NH2(CI-5 alkyl))*, for example -(NH2CH3)+), or a tertiary amino group -N(CI-5 alkyl)2, such as -N(CH3)2(which in its ammonium form has the structure of -(NH(CI-5 al ky l)2)*).

[0098] An example of a quaternary ammonium group is -N(CI-5 alky Os’1-which can for example be -N(CH3)3+.

[0099] Thus, preferably, in this first specific embodiment, the polymer further comprises a moiety comprising a group selected from -NH2, -NH3+, -NH(CI.5alkyl), -(NH2(CI.5alkyl))*, -N(Ci_5alkyl)2, -(NH(Ci_5alkyl)2)+, and -N(Ci.5alkyl)3*.

[0100] It is to be understood that said moiety with positive charge is preferably placed within a monomer used in the polymerization reaction (or, in other words, said moiety with positive charge is preferably obtained / obtainable by using a corresponding monomer, e.g. as described herein below, in the polymerization reaction). Preferably, in this specific embodiment, said monomer is selected from:

[0101] However, the present invention also encompasses embodiments wherein the above-described moiety with positive charge is introduced in a reaction that takes place after the polymerization reaction. In other words, the moiety with positive charge can also be introduced to the polymer by modification of the polymer.

[0102] Preferably, in this first specific embodiment of the biosensor of the present invention, the polymer further comprises a moiety comprising a quaternary ammonium group. Said moiety comprising a quaternary ammonium group may be a moiety of formula (II):

[0103] The moiety comprising a quaternary ammonium group, in particular the moiety of formula (II) is attached to the polymer of the present invention in the same way as described for the moiety of formula (I) hereinabove. Thus, in this first specific embodiment, the moiety comprising a quaternary ammonium group is preferably a moiety of formula (Ila):

[0104] The biosensor of the present invention comprising a moiety comprising a quaternary ammonium group has been shown to have advantageous properties in glucose sensing. Accordingly, the first specific embodiment of the present invention is based at least in part on a finding that selectivity of the biosensors of the present invention towards glucose over the common interf erents (which include, but are not limited to, fructose, mannitol, lactate, and citrate) is increasing if the polymer of the present invention further comprises a moiety comprising a quaternary ammonium group, e.g. a moiety of formula (I I). In the context of the present invention, it has been surprisingly found that a ratio of the monomer containing a quaternary ammonium group to the monomer containing a boronic acid moiety of from 1 :2 to 1 :7, preferably from 1 :3 to 1 :6, more preferably from 1 :4 to 1 :5 is particularly advantageous, as it is sufficient to substantially suppress the interference from fructose, mannitol, lactate, and citrate. Further advantageous ratio of the monomer containing a quaternary ammonium group to the monomer containing a boronic acid moiety is from 1 : 5 to 1 : 7. This is further demonstrated in Figure 3.

[0105] Accordingly, preferably the ratio of the monomer containing a moiety that is positively charged at pH = 7.4 (including any specific and preferred such moieties disclosed herein) to the monomer containing a boronic acid moiety of from 1 :2 to 1 :7, preferably from 1 :3 to 1 :6, more preferably from 1 :4 to 1 :5 is particularly advantageous, as it is sufficient to substantially suppress the interference from fructose, mannitol, lactate, and citrate. Further advantageous ratio of the monomer containing a moiety that is positively charged at pH = 7.4 (including any specific and preferred such moieties disclosed herein) to the monomer containing a boronic acid moiety is from 1 : 5 to 1 : 7.

[0106] As used herein, when the interference of a particular substance is substantially suppressed, the readout is substantially independent of the concentration of such substance. As used herein, the expression "substantially independent of the concentration” of a particular substance in its particular concentration range preferably means that a maximum change / variation of the extent of shrinking / swelling of 15%, understood as AL / L of 15% (corresponding to the sensitivity (0-20 mM glucose)), is observed within said concentration range, more preferably a maximum change / variation of 10%, even more preferably a maximum change / variation of 5%. The term "substantially independent of' also includes a specific reference to the narrower meaning of "independent of'.

[0107] Preferably, in this first specific embodiment of the present invention, the moiety of formula (II) is introduced to the polymer through a polymerization of a reaction mixture which, further to the components as described hereinabove, further comprises (iv) a monomer comprising a moiety of formula (II). Preferably, (iv) is:

[0108] Preferably, in this first specific embodiment of the present invention, for the polymer (more preferably, the polymeric hydrogel) of the present invention, the acrylamide monomer (iv) constitutes between 0.1 and 6 mol% of the acrylamide-based components in said composition / reaction mixture used for the polymerization reaction. More preferably, the acrylamide monomer (iv) constitutes between 2 and 4 mol% of the acrylamide-based components in said composition used for the polymerization reaction.

[0109] It has been demonstrated that the effect of suppressing the interference as described herein, is dependent on the presence of positive charge in the monomer. The addition of TMAPAA has beneficial effects on the resulting glucose sensor, by lowering the interference to fructose, mannitol and citrate. It has been further demonstrated that the effect originates from the positive charge on the molecule, and thus similar cationic monomers would give similar benefits as TMAPAA.

[0110] In a second specific embodiment of the present invention, the polymer further comprises a moiety of formula (III):

[0111] Accordingly, this second specific embodiment of the present invention is based, at least in part, on a surprising finding that the biosensors of the present invention exhibit suppressed sensitivity to mannitol if, further to the moiety of formula (I), a moiety of formula (II) is present. Preferably, the ratio of the moiety of formula (III) to the moiety of formula (I) is from 1 :10 to 1 :5, preferably from 1 :8 to 1 :6.

[0112] Preferably, in this second specific embodiment, the moiety of formula (III) is introduced to the polymer through a polymerization of a reaction mixture which, further to the components as described hereinabove, further comprises (v) a monomer comprising a moiety of formula (III). Preferably, the monomer (v) is a compound of the following formula:

[0113] Preferably, in this second specific embodiment of the present invention, for the polymer (more preferably, the polymeric hydrogel) of the present invention, the acrylamide monomer (v) constitutes between 1 and 5 mol% of the acrylamide-based components in said composition used for the polymerization reaction. More preferably, the acrylamide monomer (v) constitutes between 2 and 4 mol% of the acrylamide-based components in said composition used for the polymerization reaction. Even more preferably, the acrylamide monomer (v) constitutes about 3 mol% of the acrylamide-based components in said composition used for the polymerization reaction. In this second specific embodiment of the present invention, preferably the acrylamide monomer (i) and (v), respectively, constitute about 25 mol% and about 3 mol%, respectively.

[0114] The following definitions apply throughout the present specification and the claims, unless specifically indicated otherwise.

[0115] The term "hydrocarbon group” refers to a group consisting of carbon atoms and hydrogen atoms.

[0116] The term "alicyclic” is used in connection with cyclic groups and denotes that the corresponding cyclic group is non-aromatic.

[0117] As used herein, the term "alkyl” refers to a monovalent saturated acyclic (i.e. , non-cyclic) hydrocarbon group which may be linear or branched. Accordingly, an "alkyl” group does not comprise any carbon-to-carbon double bond or any carbon-to-carbon triple bond. A “C1-5 alkyl” denotes an alkyl group having 1 to 5 carbon atoms. Preferred exemplary alkyl groups are methyl, ethyl, propyl (e.g., n-propyl or isopropyl), or butyl (e.g., n-butyl, isobutyl, sec-butyl, or tert-butyl). Unless defined otherwise, the term "alkyl” preferably refers to C1-4 alkyl, more preferably to methyl or ethyl, and even more preferably to methyl. As used herein, the term "alkenyl” refers to a monovalent unsaturated acyclic hydrocarbon group which may be linear or branched and comprises one or more (e.g., one or two) carbon-to-carbon double bonds while it does not comprise any carbon-to-carbon triple bond. The term "C2-5 alkenyl” denotes an alkenyl group having 2 to 5 carbon atoms. Preferred exemplary alkenyl groups are ethenyl, propenyl (e.g., prop-1 -en-1-yl, prop-1 -en-2-yl, or prop-2- en-1-yl), butenyl, butadienyl (e.g., buta-1,3-dien-1-yl or buta-1,3-dien-2-yl), pentenyl, or pentadienyl (e.g., isoprenyl). Unless defined otherwise, the term "alkenyl” preferably refers to C2-4 alkenyl.

[0118] As used herein, the term “alkynyl” refers to a monovalent unsaturated acyclic hydrocarbon group which may be linear or branched and comprises one or more (e.g., one or two) carbon-to-carbon triple bonds and optionally one or more (e.g., one or two) carbon-to-carbon double bonds. The term "C2-5 alkynyl” denotes an alkynyl group having 2 to 5 carbon atoms. Preferred exemplary alkynyl groups are ethynyl, propynyl (e.g., propargyl), or butynyl. Unless defined otherwise, the term "alkynyl” preferably refers to C2-4 alkynyl.

[0119] As used herein, the term "alkylene” refers to an alkanediyl group, i.e. a divalent saturated acyclic hydrocarbon group which may be linear or branched. A “C1-5 alkylene” denotes an alkylene group having 1 to 5 carbon atoms, and the term "C0-3 alkylene” indicates that a covalent bond (corresponding to the option "Co alkylene”) or a C1-3 alkylene is present. Preferred exemplary alkylene groups are methylene (-CH2-), ethylene (e.g., -CH2-CH2- or -CH(-CH3)-), propylene (e.g., -CH2-CH2-CH2-, -CH(-CH2-CH3)-, -CH2-CH(-CH3)-, or -CH(-CH3)-CH2-), or butylene (e.g., -CH2- CH2-CH2-CH2-). Unless defined otherwise, the term "alkylene” preferably refers to C1-4 alkylene (including, in particular, linear C1-4 alkylene), more preferably to methylene or ethylene, and even more preferably to methylene.

[0120] As used herein, the term "carbocyclyl” refers to a hydrocarbon ring group, including monocyclic rings as well as bridged ring, spiro ring and / or fused ring systems (which may be composed, e.g., of two or three rings), wherein said ring group may be saturated, partially unsaturated (i.e., unsaturated but not aromatic) or aromatic. Unless defined otherwise, "carbocyclyl” preferably refers to aryl, cycloalkyl or cycloalkenyl.

[0121] As used herein, the term “heterocyclyl” refers to a ring group, including monocyclic rings as well as bridged ring, spiro ring and / or fused ring systems (which may be composed, e.g., of two or three rings), wherein said ring group comprises one or more (such as, e.g., one, two, three, or four) ring heteroatoms independently selected from 0, S and N, and the remaining ring atoms are carbon atoms, wherein one or more S ring atoms (if present) and / or one or more N ring atoms (if present) may optionally be oxidized, wherein one or more carbon ring atoms may optionally be oxidized (i.e., to form an oxo group), and further wherein said ring group may be saturated, partially unsaturated (i.e., unsaturated but not aromatic) or aromatic. For example, each heteroatom-containing ring comprised in said ring group may contain one or two 0 atoms and / or one or two S atoms (which may optionally be oxidized) and / or one, two, three or four N atoms (which may optionally be oxidized), provided that the total number of heteroatoms in the corresponding heteroatom-containing ring is 1 to 4 and that there is at least one carbon ring atom (which may optionally be oxidized) in the corresponding heteroatom-containing ring. Unless defined otherwise,

[0122] "heterocyclyl” preferably refers to heteroaryl, heterocycloalkyl or heterocycloalkenyl.

[0123] As used herein, the term "aryl” refers to an aromatic hydrocarbon ring group, including monocyclic aromatic rings as well as bridged ring and / or fused ring systems containing at least one aromatic ring (e.g., ring systems composed of two or three fused rings, wherein at least one of these fused rings is aromatic; or bridged ring systems composed of two or three rings, wherein at least one of these bridged rings is aromatic). If the aryl is a bridged and / or fused ring system which contains, besides one or more aromatic rings, at least one non-aromatic ring (e.g., a saturated ring or an unsaturated alicyclic ring), then one or more carbon ring atoms in each non-aromatic ring may optionally be oxidized (i.e., to form an oxo group). "Aryl” may, e.g., refer to phenyl, naphthyl, dialinyl (i.e., 1,2-dihydronaphthyl), tetralinyl (i.e., 1 ,2,3,4-tetrahydronaphthyl), indanyl, indenyl (e.g., 1 H-indenyl), anthracenyl, phenanthrenyl, 9H- fluorenyl, or azulenyl. Unless defined otherwise, an "aryl” preferably has 6 to 14 ring atoms, more preferably 6 to 10 ring atoms, even more preferably refers to phenyl or naphthyl, and most preferably refers to phenyl.

[0124] As used herein, the term "heteroaryl” refers to an aromatic ring group, including monocyclic aromatic rings as well as bridged ring and / or fused ring systems containing at least one aromatic ring (e.g., ring systems composed of two or three fused rings, wherein at least one of these fused rings is aromatic; or bridged ring systems composed of two or three rings, wherein at least one of these bridged rings is aromatic), wherein said aromatic ring group comprises one or more (such as, e.g., one, two, three, or four) ring heteroatoms independently selected from 0, S and N, and the remaining ring atoms are carbon atoms, wherein one or more S ring atoms (if present) and / or one or more N ring atoms (if present) may optionally be oxidized, and further wherein one or more carbon ring atoms may optionally be oxidized (i.e., to form an oxo group). For example, each heteroatom-containing ring comprised in said aromatic ring group may contain one or two 0 atoms and / or one or two S atoms (which may optionally be oxidized) and / or one, two, three or four N atoms (which may optionally be oxidized), provided that the total number of heteroatoms in the corresponding heteroatom-containing ring is 1 to 4 and that there is at least one carbon ring atom (which may optionally be oxidized) in the corresponding heteroatom-containing ring. "Heteroaryl” may, e.g., refer to thienyl (i.e., thiophenyl), benzo[b]thienyl, naphtho[2,3-b]thienyl, thianthrenyl, furyl (i.e., furanyl), benzofuranyl, isobenzofuranyl, chromanyl, chromenyl (e.g., 2H-1 -benzopyranyl or 4H-1 -benzopyranyl), isochromenyl (e.g., 1 H-2-benzopyranyl), chromonyl, xanthenyl, phenoxathiinyl, pyrrolyl (e.g., 1 H-pyrrolyl), imidazolyl, pyrazolyl, pyridyl (i.e., pyridinyl; e.g., 2-pyridyl, 3-pyridyl, or 4-pyridyl), pyrazinyl, pyrimidinyl, pyridazinyl, indolyl (e.g., 3H-indolyl), isoindolyl, indazolyl, indolizinyl, purinyl, quinolyl, isoquinolyl, phthalazinyl, naphthyridinyl, quinoxalinyl, cinnolinyl, pteridinyl, carbazolyl, p-carbolinyl, phenanthridinyl, acridinyl, perimidinyl, phenanthrolinyl (e.g., [1, 10]phenanthrolinyl, [1,7]phenanthrolinyl, or [4,7]phenanthrolinyl), phenazinyl, thiazolyl, isothiazolyl, phenothiazinyl, oxazolyl, isoxazolyl, oxadiazolyl (e.g., 1 ,2,4-oxadiazolyl, 1,2,5-oxadiazolyl (i.e., furazanyl), or 1,3,4- oxadiazolyl), thiadiazolyl (e.g., 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, or 1,3,4-thiadiazolyl), phenoxazinyl, pyrazolo[1 ,5-a]pyrimidinyl (e.g., pyrazolo[1 ,5-a]pyrimidin-3-yl), 1,2-benzoisoxazol-3-yl, benzothiazolyl, benzothiadiazolyl, benzoxazolyl, benzisoxazolyl, benzimidazolyl, benzo[b]thiophenyl (i.e., benzothienyl), triazolyl (e.g., 1 H-1,2,3-triazolyl, 2H-1 ,2,3-triazolyl, 1 H-1,2,4-triazolyl, or 4H-1,2,4-triazolyl), benzotriazolyl, 1 H-tetrazolyl, 2H-tetrazolyl, triazinyl (e.g., 1 ,2,3-triazinyl, 1 ,2,4-triazinyl, or 1 ,3,5-triazinyl), furo[2,3-c]pyridinyl, dihydrofuropyridinyl (e.g., 2,3-dihydrofuro[2,3-c]pyridinyl or 1,3-dihydrofuro[3,4-c]pyridinyl), imidazopyridinyl (e.g., imidazo[1,2- a]pyridinyl or imidazo[3,2-a]pyridinyl), quinazolinyl, thienopyridinyl, tetrahydrothienopyridinyl (e.g., 4, 5,6,7- tetrahydrothieno[3,2-c]pyridinyl), dibenzofuranyl, 1 ,3-benzodioxolyl, benzodioxanyl (e.g., 1,3-benzodioxanyl or

[0125] 1.4-benzodioxanyl), or coumarinyl. Unless defined otherwise, the term "heteroaryl” preferably refers to a 5 to 14 membered (more preferably 5 to 10 membered) monocyclic ring or fused ring system comprising one or more (e.g., one, two, three or four) ring heteroatoms independently selected from 0, S and N, wherein one or more S ring atoms (if present) and / or one or more N ring atoms (if present) are optionally oxidized, and wherein one or more carbon ring atoms are optionally oxidized; even more preferably, a "heteroaryl” refers to a 5 or 6 membered monocyclic ring comprising one or more (e.g., one, two or three) ring heteroatoms independently selected from 0, S and N, wherein one or more S ring atoms (if present) and / or one or more N ring atoms (if present) are optionally oxidized, and wherein one or more carbon ring atoms are optionally oxidized.

[0126] As used herein, the term "cycloalkyl” refers to a saturated hydrocarbon ring group, including monocyclic rings as well as bridged ring, spiro ring and / or fused ring systems (which may be composed, e.g., of two or three rings; such as, e.g., a fused ring system composed of two or three fused rings). "Cycloalkyl” may, e.g., refer to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, decalinyl (i.e., decahydronaphthyl), or adamantyl. Unless defined otherwise, "cycloalkyl” preferably refers to a C3-11 cycloalkyl, and more preferably refers to a C3-7 cycloalkyl. A particularly preferred "cycloalkyl” is a monocyclic saturated hydrocarbon ring having 3 to 7 ring members (e.g., cyclopropyl or cyclohexyl).

[0127] As used herein, the term "heterocycloalkyl” refers to a saturated ring group, including monocyclic rings as well as bridged ring, spiro ring and / or fused ring systems (which may be composed, e.g., of two or three rings; such as, e.g., a fused ring system composed of two or three fused rings), wherein said ring group contains one or more (such as, e.g., one, two, three, or four) ring heteroatoms independently selected from 0, S and N, and the remaining ring atoms are carbon atoms, wherein one or more S ring atoms (if present) and / or one or more N ring atoms (if present) may optionally be oxidized, and further wherein one or more carbon ring atoms may optionally be oxidized (i.e., to form an oxo group). For example, each heteroatom-containing ring comprised in said saturated ring group may contain one or two 0 atoms and / or one or two S atoms (which may optionally be oxidized) and / or one, two, three or four N atoms (which may optionally be oxidized), provided that the total number of heteroatoms in the corresponding heteroatom-containing ring is 1 to 4 and that there is at least one carbon ring atom (which may optionally be oxidized) in the corresponding heteroatom-containing ring. "Heterocycloalkyl” may, e.g., refer to aziridinyl, azetidinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, piperidinyl, piperazinyl, azepanyl, diazepanyl (e.g.,

[0128] 1.4-diazepanyl), oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolidinyl, morpholinyl (e.g., morpholin-4-yl), thiomorpholinyl (e.g., thiomorpholin-4-yl), oxazepanyl, oxiranyl, oxetanyl, tetrahydrofuranyl, 1 ,3-dioxolanyl, tetrahydropyranyl, 1 ,4-dioxanyl, oxepanyl, thiiranyl, thietanyl, tetrahydrothiophenyl (i.e., thiolanyl), 1 ,3-dithiolanyl, thianyl, 1,1-dioxothianyl, thiepanyl, decahydroquinolinyl, decahydroisoquinolinyl, or 2-oxa-5-aza-bicyclo[2.2.1]hept- 5-yl. Unless defined otherwise, "heterocycloalkyl” preferably refers to a 3 to 11 membered saturated ring group, which is a monocyclic ring or a fused ring system (e.g., a fused ring system composed of two fused rings), wherein said ring group contains one or more (e.g., one, two, three, or four) ring heteroatoms independently selected from 0, S and N, wherein one or more S ring atoms (if present) and / or one or more N ring atoms (if present) are optionally oxidized, and wherein one or more carbon ring atoms are optionally oxidized; more preferably, "heterocycloalkyl” refers to a 5 to 7 membered saturated monocyclic ring group containing one or more (e.g., one, two, or three) ring heteroatoms independently selected from 0, S and N, wherein one or more S ring atoms (if present) and / or one or more N ring atoms (if present) are optionally oxidized, and wherein one or more carbon ring atoms are optionally oxidized.

[0129] As used herein, the term "halogen” refers to fluoro (-F), chloro (-CI), bromo (-Br), or iodo (-I). The terms "halogen” and "halo” may be used interchangeably.

[0130] As used herein, the term “haloalky I” refers to an alkyl group substituted with one or more (preferably 1 to 6, more preferably 1 to 3) halogen atoms which are selected independently from fluoro, chloro, bromo and iodo, and are preferably all fluoro atoms. It will be understood that the maximum number of halogen atoms is limited by the number of available attachment sites and, thus, depends on the number of carbon atoms comprised in the alkyl moiety of the haloalkyl group. “Haloalkyl” may, e.g., refer to -CF3, -CHF2, -CH2F, -CF2-CH3, -CH2-CF3, -CH2-CHF2, -CH2-CF2-CH3, -CH2-CF2-CF3, or -CH(CF3)2. A particularly preferred "haloalkyl” group is -CF3.

[0131] The terms "bond” and "covalent bond” are used herein synonymously, unless explicitly indicated otherwise or contradicted by context.

[0132] As used herein, the terms "optional”, "optionally” and "may” denote that the indicated feature may be present but can also be absent. Whenever the term "optional”, "optionally” or "may” is used, the present invention specifically relates to both possibilities, i.e., that the corresponding feature is present or, alternatively, that the corresponding feature is absent. For example, the expression "X is optionally substituted with Y” (or "X may be substituted with Y”) means that X is either substituted with Y or is unsubstituted. Likewise, if a component of a composition is indicated to be "optional”, the invention specifically relates to both possibilities, i.e., that the corresponding component is present (contained in the composition) or that the corresponding component is absent from the composition.

[0133] Various groups are referred to as being "optionally substituted” in this specification. Generally, these groups may carry one or more substituents, such as, e.g., one, two, three or four substituents. It will be understood that the maximum number of substituents is limited by the number of attachment sites available on the substituted moiety. Unless defined otherwise, the "optionally substituted” groups referred to in this specification carry preferably not more than two substituents and may, in particular, carry only one substituent. Moreover, unless defined otherwise, it is preferred that the optional substituents are absent, i.e. that the corresponding groups are unsubstituted.

[0134] A skilled person will appreciate that the substituent groups comprised in the compounds of the present invention may be attached to the remainder of the respective compound via a number of different positions of the corresponding specific substituent group. Unless defined otherwise, the preferred attachment positions for the various specific substituent groups are as illustrated in the examples.

[0135] As used herein, unless explicitly indicated otherwise or contradicted by context, the terms "a”, "an” and "the” are used interchangeably with "one or more” and "at least one”.

[0136] It is to be understood that wherever numerical ranges are provided / disclosed herein, all values and subranges encompassed by the respective numerical range are meant to be encompassed within the scope of the invention. Accordingly, the present invention specifically and individually relates to each value that falls within a numerical range disclosed herein, including in particular each individual integer value that falls within a corresponding numerical range, as well as each subrange encompassed by a numerical range disclosed herein.

[0137] As used herein, the term "comprising” (or "comprise”, "comprises”, "contain”, "contains”, or "containing”), unless explicitly indicated otherwise or contradicted by context, has the meaning of "containing, inter alia”, i.e., "containing, among further optional elements, In addition thereto, this term also includes the narrower meanings of "consisting essentially of' and "consisting of'. For example, the term "A comprising B and C” has the meaning of "A containing, inter alia, B and C”, wherein A may contain further optional elements (e.g., "A containing B, C and D” would also be encompassed), but this term also includes the meaning of "A consisting essentially of B and C” and the meaning of "A consisting of B and C” (i.e., no other components than B and C are comprised in A).

[0138] As used herein, the term "subject” or "patient” refers to an animal, preferably a mammal (e.g., a human or a nonhuman mammal). Most preferably, the "subject” or "patient” is a human (e.g., a male human or a female human).

[0139] The measurement of the glucose concentration using the biosensor of the present invention typically requires a measurement of the polymer (or the polymeric hydrogel), particularly of the change in volume detected through the measurement of the change in length of the polymer / polymeric hydrogel. A corresponding measurement can be conducted using the method described in US 7,602,498 B2, which is incorporated by reference in its entirety. In particular, in order to measure the length of the polymeric hydrogel, the reflected interferometric spectrum from the hydrogel can be used to monitor changes in hydrogel length induced by varying glucose concentrations. The hydrogel is located at the tip of a cleaved single mode fiber. The relative length change of the hydrogel is monitored continuously against an initial absolute length Lg(t = measurement. The hydrogel length is measured from the center of the cleave, along a straight vertical path relative to the cleave surface and to the tip of the hydrogel. The reflected spectrum of the hydrogel approximates a sinewave with a DC-component, amplitude, period, and a phase term. The stepwise change of the refractive index between the silica fiber, hydrogel and fluid can be modelled as two weakly reflecting mirrors depicted by the boundary between the fiber and the hydrogel, T , and the hydrogel and surrounding fluid, r2. The system represents a low finesse Fabry-Perot (FP) cavity. The model is shown in Figure 6.

[0140] The reflection coefficients are T and r2. The secondary and weakest reflection, at boundary r2, interferes with the reflected light at boundary T . This interaction creates a sinusoidal interference pattern in the reflected intensity as a function of wavelength. The FP interferogram is described by: where k = is the wavenumber of the light inside the hydrogel cavity and A is the wavelength of the light. An FP reflection spectrum from a hydrogel cavity of 39 nm long and ng= 1.35 is shown in Figure 6. The absolute length of the hydrogel is determined by finding the period (free spectral range) of the sinus and length changes are determined by finding the phase-shift between spectra in time.

[0141] The present invention relates to specific uses of the biosensor for measuring the concentration of glucose. Accordingly, the biosensor of the present invention is suitable for use in an in vivo diagnostic method. Said method preferably comprises a step of determining the glucose concentration in the blood of a subject (e.g., a human subject) using the biosensor of the invention, as described herein above. It is thus to be understood that the biosensor of the present invention is suitable for use in a method of determining the glucose concentration in the blood of a subject.

[0142] Preferably, the measurement can be performed on a continuous basis or repeated in certain time intervals, so that not only a single measurement point, which provides limited information on the state of the subject / patient, can be provided, but also a trend in blood glucose concentration over time or a series of measurements can be obtained, which are very important when monitoring the state of the patient over a longer time, for example when monitoring post-operative recovery of the patient or monitoring said patient in an intensive care setup. Such repeated or continued measurement may also be referred to as glucose monitoring. Hence, the present invention also relates to an in vivo method of glucose monitoring in a subject, the method comprising repeated measurements of the glucose concentration in the blood of a subject by using the biosensor of the present invention. Accordingly, the biosensor for measuring the concentration of glucose of the present invention is provided for use in an in vivo method of glucose monitoring.

[0143] The measurement of the blood glucose concentration is informative of conditions, diseases and disorders that are characterized by pathological glucose blood concentration, i.e. a concentration that is different from the concentration range considered to be normal. If the blood glucose concentration is lower than normal, typically lower than 70 mg / dL (or lower than 3.9 mmol / L), this may be referred to as hypoglycemia. If the blood glucose concentration is higher than normal, typically higher than 200 mg / dL (or higher than 11.1 mmol / L), this may be referred to as hyperglycemia. Such conditions can be determined or diagnosed by a direct measurement of the glucose concentration in the blood. Thus, the present invention further relates to a method of diagnosing hyperglycemia or hypoglycemia in a subject, the method comprising a step of measuring the blood glucose concentration using the biosensor of the present invention. Accordingly, the present invention provides a biosensor for measuring the concentration of glucose of the first embodiment of the present invention for use in an in vivo method of diagnosing hyperglycemia or hypoglycemia. In addition, the present invention also relates to use of the biosensor of the present invention in an in vitro method of diagnosing hyperglycemia or hypoglycemia, e.g., by measuring the glucose concentration in a blood sample obtained from a subject.

[0144] Thus, the present invention also provides a diagnostic method (particularly an in vitro diagnostic method), wherein the biosensor of the present invention is used, comprising a step of measuring the blood glucose concentration in a blood sample from a subject using said biosensor. Accordingly, the biosensor of the present invention may also be used outside of a patient's body for measuring the concentration of glucose in a sample obtained from the patient. Said method may also be referred to as an in vitro diagnostic method. In other words, the present invention provides the biosensor of the present invention for use in an in vitro diagnostic method. The method itself is not particularly limited, as long as it includes the necessary step of determining the glucose concentration in the blood sample. The measurement can be performed as described herein above, for example by measuring the change in volume of the polymer, occurring in a glucose concentration-dependent manner.

[0145] The measurements of the glucose concentration using the biosensor of the present invention are not limited to measurements in blood. Accordingly, the present invention generally relates to use of the biosensor of the invention for measuring the glucose concentration in a sample, including also a non-blood sample, such as, e.g., urine. Preferably, however, the sample is a blood sample (e.g., a whole blood sample, a serum sample, or a plasma sample).

[0146] The biosensor of the present invention can be configured for accessing the blood of a subject through an indwelling arterial catheter. As the sensor can be applied without disrupting the catheter's use, the sensor of the present invention requires no new catheters and displaces no other equipment.

[0147] As further provided herein, the present invention relates to the use of the polymer as provided by the present invention for the manufacture of a reagent or a biosensor for monitoring the glucose level in a subject. It is to be understood that changes in the properties of the polymer upon contact with glucose at different concentrations allow the measurements to be performed. As discussed herein above, the biosensor of the present invention may also be used therapeutically, e.g., when incorporated in a glucose-concentration dependent release formulation. Thus, the present invention further provides a glucose-concentration-sensitive release formulation comprising the polymer as described herein. A corresponding glucose-sensitive release formulation may further comprise one or more pharmaceutically acceptable carriers, and an active substance / therapeutic agent (e.g., insulin) to be delivered in a glucose concentration-dependent manner.

[0148] Such formulations can be prepared by techniques known in the art, such as the techniques published in "Remington: The Science and Practice of Pharmacy”, Pharmaceutical Press, 22ndedition. As the release into blood, upon measurement of the blood glucose concentration, is preferred, the formulations can be formulated as dosage forms for parenteral administration, such as intramuscular, intravenous, subcutaneous, intraarterial, or intracardial administration. Dosage forms for parenteral administration include, e.g., solutions, emulsions, suspensions, dispersions and powders and granules for reconstitution. Emulsions are a preferred dosage form for parenteral administration.

[0149] Accordingly, if the formulations are administered parenterally, then examples of such administration include one or more of: intravenously, intraarterially, intraperitoneally, intrathecally, intraventricularly, intraurethrally, intrasternally, intracardially, intracranially, intramuscularly or subcutaneously administering the formulations, and / or by using infusion techniques. For parenteral administration, the formulations comprising the polymers of the invention are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or saccharides to make the solution isotonic with blood. The aqueous solutions should be suitably buffered (preferably to a physiological pH), if necessary. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques.

[0150] The formulation of the present invention may also be formulated as a sustained release system, which may include semi permeable polymer matrices in the form of shaped articles, e.g., films or microcapsules. Sustained-release matrices may include, e.g., polylactides, copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, poly(2- hydroxyethyl methacrylate), ethylene vinyl acetate, or poly-D-(— )-3-hydroxybutyric acid.

[0151] It is preferred that a condition / disease / disorder related to pathological blood concentration of glucose is to be treated. Accordingly, in one embodiment, the present invention relates to the biosensor of the present invention or to the glucose concentration-sensitive release formulations for use in the treatment of a condition / disease / disorder dependent of the blood glucose concentration. An example of such a condition is diabetes mellitus, for example type I diabetes. As known in the art, diabetes is typically treated by dosing the patient with insulin. Thus, the formulation of the present invention may further comprise insulin. However, it is preferred that the biosensor of the present invention is not intended for therapeutic application and / or that the biosensor of the present invention does not comprise insulin.

[0152] The invention will be illustrated by the following examples, which serve solely illustrative purposes and are not to be construed as limiting the scope of the claims.

[0153] EXAMPLES

[0154] Names and abbreviations

[0155] 3APB: 3-Acrylamidophenylboronic acid

[0156] 2APB: 2-Acrylamidophenylboronic acid

[0157] ADAM: 5-Acrylamido-2-((dimethylamino)methyl)phenylboronic acid

[0158] TMAPAA: (3-Acrylamidopropyl)trimethylammonium chloride PBS: Phosphate buffered saline

[0159] GBM: Glucose binding molecule

[0160] The structural formulas of certain compounds discussed in the experimental section are provided in the following.

[0161] Acrylamide TMAPAA

[0162]

[0163] 2APB 3APB Thiolactone

[0164] Materials and methods

[0165] Chemicals

[0166] Acrylamide, methylene bis acrylamide, N-Hydroxyethyl acrylamide (HEAA), 1 -hydroxycyclohexyl phenyl ketone, 3-(trimethoxysilyl)propyl methacrylate, squalane were purchased from Sigma-Aldrich. 5-Acrylamido-2- ((dimethylamino)methyl)phenylboronic acid (ADAM) and 2-Acrylamidophenylboronic acid (2APB) were purchased from Combi-Blocks Inc. 3-Acrylamidophenylboronic acid (3APB), (3-Acrylamidopropyl)trimethylammonium chloride (TMAPAA) and N-(3-Aminopropyl)methacrylamide hydrochloride (APMA) were purchased from Sigma-Aldrich. N-(3-(Dimethylamino)propyl)acrylamide (DMAPAA) was purchased from Tokyo Chemical Industry Co., Ltd. D-(-)- Fructose, D-Mannitol, Sodium L-lactate and Sodium citrate tribasic dihydrate were purchased from Sigma-Aldrich. Dimethyl sulfoxide was purchased from Sigma-Aldrich.

[0167] D,L-homocysteine thiolactone acrylamide was purchased from Specific Polymers. Ethanolamine was purchased from Sigma-Aldrich.

[0168] 0.01 M PBS pH 7.4 (0.132 M NaCI) was prepared in-house, di-sodium hydrogen phosphate was purchased from Alfa Aesar, sodium phosphate monobasic monohydrate was purchased from Sigma-Aldrich and sodium chloride was purchased from Sigma-Aldrich.

[0169] All chemicals were used without further purification. Fabrication of the hydrogel sensor

[0170] In a typical formulation of the hydrogel sensor the monomers and molar % are as follows: Boronic acid acrylamide 20 %, acrylamide 75%, methylene bis acrylamide 1-2%. Exemplary compositions are shown in Table 1.

[0171] Monomers are diluted either in deionized (DI) water, 1 M fructose or mannitol solution in PBS (pH 7.4, 0.132 M NaCI), to give a final total monomer concentration of 1.0 or 1.5 M. 1 -hydroxycyclohexyl phenyl ketone (Photoinitiator) is added at a concentration of 1 .5 mM.

[0172] Table 1. Exemplary sensor formulations discussed herein.

[0173] Targeted mol % in the pre-gel

[0174] Fabrication of the hydrogel sensor follows that described in WO 2007 / 104974 (which is incorporated herein by reference). Firstly, optical glass fibers are stripped and cleaved to prepare a uniform cleave. Fiber cleaves are then silanized to provide covalent attachment of the hydrogel to the glass surface. Silanization involves firstly submerging fiber cleaves in hydrochloric acid (1.0 M) for 15 minutes, followed by washing with DI water and then submerging in ethanol containing 3-(trimethoxysilyl)propyl methacrylate (84 mM) for 10 minutes. Excess 3- (trimethoxysilyl)propyl methacrylate is removed by washing fiber cleaves under flowing ethanol. A droplet of the pregel solution resembling a dome is deposited onto the fiber cleave using a pipette while both cleave and pregel are inside a larger droplet of squalane oil. The oil firstly serves to maintain the stability of the pregel droplet and secondly contains an excess of dissolved photoinitiator 1 -hydroxycyclohexyl phenyl ketone (132 mM). The excess photoinitiator in the oil is necessary to enable the polymerization to occur without oxygen-free conditions whereby radicals generated firstly react with dissolved oxygen in the oil. The higher concentration in the oil compared to the pregel droplet also ensures that photoinitiator does not significantly leach out of the pregel droplet into the oil droplet.

[0175] Polymerisation of the pregel occurs via irradiation (5 minutes) of the pregel by a 340 nm light source positioned directly in front of the pregel droplet and inside the droplet of oil. After polymerization, sensors are washed briefly in pentane to remove the oil and then further washed in 50% ethanol in DI water for 15 minutes to remove unreacted monomers. At this point sensors are placed in PBS pH 6.0 until testing.

[0176] Sensor experiment setup

[0177] In a typical experiment, test solutions are prepared using 0.01 M PBS containing 132 mM NaCI at pH 7.4 at 37 degrees Celsius. Sensors are submersed in test solutions and allowed to equilibrate for at least 10 minutes, after which time, the gel cavity signal is locked to give an initial absolute length of the hydrogel and the experiment is started. At timepoints, portions of glucose in PBS from a 1 M stock solution which has been left for at least 6 hours to reach mutarotation equilibrium, are added to achieve the desired concentrations while the change in gel length is continuously monitored. Length changes in response to pH are measured by moving sensors between two test solutions at a constant glucose concentration but a different pH.

[0178] Example 1

[0179] Results and discussion

[0180] Glucose sensors based on mono boronic acids often suffer from poor selectivity to glucose over other monosaccharides (for example fructose) and from a significant change in glucose binding with small variations in pH. Here a sensor has been developed which exhibits good selectivity for glucose over a range of common interferents while also being minimally influenced by changes in pH.

[0181] One of the most used mono boronic acids for glucose sensing is 3-Acrylamidophenylboronic acid (3APB), which has been incorporated into materials such as polymers to give glucose-responsive properties. Herein, 3APB will be used as a reference example.

[0182] The boron center of 3APB has a pKa of around 8.83and therefore predominantly exists in the trigonal planar sp2 form at physiological pH which is not favored for binding glucose.4’5As a result of the relatively high pKa, small changes to higher or lower pH from pH 7.4 significantly increases or decreases the extent of glucose binding respectively and thus would significantly impact a sensor reading requiring complicated pH correction.6’7This pH sensitivity makes the use of 3APB unsuitable for in-vivo glucose sensing, where in extreme cases the pH of blood can reach a value as low as 6.9 and as high as 7.6. The glucose response of sensors possessing 3APB and a positively charged comonomer (as described in various publications7’8’9’10) was tested at pH 6.9, 7.4 and 7.6 (Fig. 2A) and a large deviation in sensor vs reference glucose readings was observed. To address the pH interference the Wulff-type2boronic acid 5-Acrylamido-2-((dimethylamino)methyl)phenylboronic acid (ADAM) was chosen as the GBM to replace 3APB. Wulff-type boronic acids possess an amino group ortho to the boronic acid which lowers the pKa of the boronic acid to around 5.2,2 11favoring glucose binding at physiological pH. While Wulff-type boronic acids have been successfully employed in fluorescent glucose sensors12 13, the exact mechanism of interaction between the amino group and the boron is still debated.14 15

[0183] Despite the lowered pKa Wulff-type boronic acids are reported to suffer from poorer affinity to glucose compared with other mono boronic acids11 16possibly as a result of steric hindrance.5Indeed, sensors constructed with ADAM were found to require between 2.5 to 3.5 times the mol % of the GBM to yield similar glucose response than with 3APB. Despite this, with proper optimization of ADAM concentration, a high and linear change in length (shrinking) of the ADAM hydrogel sensor was achieved, suitable for glucose detection within the physiological range (Fig 3A). Sensors with ADAM did not require any addition of cationic monomer to yield linear shrinking with glucose (Fig. 3), in contrast to 3APB sensors.9

[0184] Importantly, the ADAM sensor displayed a significantly lower pH dependence compared with the 3APB sensor (Fig. 2), demonstrating the suitability of the ADAM sensor for in-vivo glucose monitoring.

[0185] Selectivity of the ADAM sensor towards glucose over other common I nterferents was found to be increased by the addition of a quaternary ammonium comonomer, TMAPAA (Fig. 3). Other groups have reported a similar effect when using cationic comonomers with 3APB, where the positive charge is needed to achieve an acceptable glucose response with 3APB at physiological pH.17’18 10

[0186] Compared with other examples, a much smaller ratio of cationic monomer to boronic acid was required to almost completely suppress the interference from fructose (Fig. 2B).17’18’10

[0187] Addition of TMAPAA did not significantly impact the sensor response with glucose (Fig. 3A), and importantly, a low mol % of cationic groups in the hydrogel is crucial to avoid unwanted interference to salts (Fig. 3D). It was found that 3% TMAPAA and 20% ADAM sensors displayed negligible interference to fructose, mannitol, lactate, citrate as well as further reducing interference to changes in pH from 6.9 to 7.6 despite concentrations of interferents tested well exceeding those expected in vivo (Fig. 3, Fig. 4), demonstrating a selectivity not usually observed for monoboronic acids. The observed selectivity is hypothesized to be partially driven by the ability of the ADAM hydrogel to form a 2: 1 complex with glucofuranose.1

[0188] 2APB, has been recently investigated as a GBM, offering significantly lower pH interference than 3APB due to internal coordination of the boron with adjacent carbonyl group.19’20’21Sensors constructed with 2APB exhibited slow kinetics with glucose, resulting in a modest contraction at 6 and 20 mM glucose but extremely low interference to lactate, citrate and changes in pH . Unexpectedly, 2APB sensors displayed a large and fast contraction with 5 mM mannitol (Fig. 4). The mannitol response for 2APB sensors was significantly larger with respect to glucose or fructose compared to ADAM or 3APB sensors (Table 2). The origin of this response is not known but may be due to crosslinking between 2APB and mannitol.22Given 2APB sensors contract with mannitol and ADAM sensors swell with mannitol, it was hypothesized that adding 2APB at a certain mol % to ADAM sensors could selectively suppress the mannitol interference. By varying the molar ratio of ADAM and 2APB it was found that the interference of mannitol at 2.2 mM glucose could be suppressed when 3% 2APB and 25% ADAM was used (Fig. 5).

[0189] Table 2. Length change response of 3APB, ADAM and 2APB-basd sensors to glucose, fructose and mannitol.

[0190] Example 2

[0191] Sensors comprising the following polymeric hydrogels have been tested for their sensitivity to glucose in this Example, following the methods outlined hereinabove, as summarized in the following Table 3.1 :

[0192] Table 3.1. Hydrogels obtained in Example 2.

[0193] The results are shown in Figure 7A. It is concluded that for the same ratios of monomers, the AD AM-acryl amide sensor length change at 20 mM glucose is around 1.6 times greater than ADAM-HEAA sensor. Accordingly, sensors with acrylamide exhibit greater sensitivity to glucose. Of note, in both cases (acrylamide and HEAA) the sensor is shrinking with glucose.

[0194] The experiments were repeated for the polymeric hydrogels not including TMAPAA, which were obtained following the methods outlined hereinabove and as summarized in the following Table 3.2 Table 3.2. Further hydrogels obtained in Example 2. mol% mol % ADAM Acrylamide / HEAA mol% BIS mol % TMAPAA Total monomer concentration (M)

[0195] ADAM-acrylamide 28 70 1.4 0 1.4

[0196] ADAM-HEAA 28 70 1.4 0 1.4

[0197] The results are shown in Figure 7B. It is concluded that for the same ratios of monomers, the ADAM-Acrylamide sensor length change at 20 mM glucose is roughly 2 times greater than ADAM-HEAA sensor. Accordingly, sensors with acrylamide exhibit greater sensitivity to glucose. Of note, in both cases (acrylamide and HEAA) the sensor is shrinking with glucose.

[0198] Example 3

[0199] Three different cationic monomers were introduced into the polymeric gel formulation and the resulting biosensors were tested for their ability to withstand the interference effects coming from the presence of citrate. The introduced monomers are shown below:

[0200] TMAPAA Quaternary amine

[0201] D APAA Tertiary amine

[0202] The obtained hydrogels are characterized in the following Table 4. Total monomer concentration in all formulations is 1.5 M.

[0203] Table 4. Polymeric hydrogels tested in Example 3. The results of swelling measurements performed with these hydrogels are summarized in Figure 8. Sensors without a cationic comonomer swell to a large degree with citrate, which is indicative of citrate interference, which is undesirable for clinically relevant measurements. The cationic monomers TMAPAA, DMAPAA and APMA were found to reduce the interference to citrate without introducing a significant change in the sensor's response to glucose.

[0204] Example 4

[0205] A polymeric hydrogel comprising ADAM moiety has been prepared according to the method of obtaining a polymeric hydrogel and subsequently modified to obtain a polymeric hydrogel of the invention.23

[0206] A monomer solution containing 16 or 28 mol% thiolactone acrylamide was photopolymerized to the corresponding hydrogel. The hydrogel sensors were then incubated in a solution of 1 :1 DMSO and PBS with 26 mM of the alkene (ADAM) and after a short period of time ethanolamine was added (final concentration 5 M) to start the reaction. The reaction was left overnight at room temperature with stirring and after this time the sensors were washed with 50% ethanol in DI water for 15 minutes before testing.

[0207] The obtained hydrogels are characterized in the following Table 5. Table 5. Polymeric hydrogels tested in Example 4.

[0208] The results are summarized in Figure 9. ADAM sensor may also be constructed via a thiol-ene type post polymerisation modification. As shown in Figure 9, greater contraction with glucose is observed for post-modified sensors (Figure 9A) compared with directly polymerised sensors (Figure 9B). It is however reiterated that very similar behaviour of both types of sensors is observed.

[0209] References

[0210] 1. Eggert, H., Frederiksen, J., Morin, C. & Norrild, J. C. A new glucose-selective fluorescent bisboronic acid. First report of strong o-furanose complexation in aqueous solution at physiological pH. J. Org. Chem. 64, 3846-3852 (1999).

[0211] 2. Wulff, G. Selective binding to polymers via covalent bonds, the construction of chiral cavities as specific receptor sites. PureAppl. Chem. 54, 2093-2102 (1982).

[0212] 3. Springsteen, G. & Wang, B. A detailed examination of boronic acid-diol complexation. Tetrahedron 58, 5291-5300 (2002).

[0213] 4. Bosch, L. I., Fyles, T. M. & James, T. D. Binary and ternary phenylboronic acid complexes with saccharides and Lewis bases. Tetrahedron 60, 11175-11190 (2004).

[0214] 5. Yan, J., Springsteen, G., Deeter, S. & Wang, B. The relationship among pK a, pH, and binding constants in the interactions between boronic acids and diols - It is not as simple as it appears. Tetrahedron 60, 11205— 11209 (2004).

[0215] 6. Worsley, G. J. et al. Measurement of glucose in blood with a phenylboronic acid optical sensor. J. Diabetes Sci. Techno / . 2, 213-220 (2008).

[0216] 7. Tierney, S., Hasle Falch, B. M., Hjelme, D. R. & Stokke, B. T. Determination of glucose levels using a functionalized hydrogel-optical fiber biosensor: Toward continuous monitoring of blood glucose in vivo. Anal. Chem. 81, 3630-3636 (2009).

[0217] 8. Horkay, F. et al. Thermodynamic analysis of the selectivity enhancement obtained by using smart hydrogels that are zwitterionic when detecting glucose with boronic acid moieties. Sensors Actuators, B Chem. 160, 1363-1371 (2011).

[0218] 9. Lin, G. et al. Osmotic swelling pressure response of smart hydrogels suitable for chronically implantable glucose sensors. Sensors Actuators, B Chem. 144, 332-336 (2010).

[0219] 10. Horgan, A. M. et al. Crosslinking of phenylboronic acid receptors as a means of glucose selective holographic detection. Biosens. Bioelectron. 21, 1838-1845 (2006). 11. Brooks, W. L. A., Deng, C. C. & Sumerlin, B. S. Structure-Reactivity Relationships in Boronic Acid-Diol Complexation. ACS Omega 3, 17863-17870 (2018).

[0220] 12. Crane, B. C. et al. The development of a continuous intravascular glucose monitoring sensor. J. Diabetes Sci. Techno / . 9, 751-761 (2015).

[0221] 13. Mortellaro, M. & DeHennis, A. Performance characterization of an abiotic and fluorescent-based continuous glucose monitoring system in patients with type 1 diabetes. Biosens. Bioelectron. 61, 227-231 (2014).

[0222] 14. Sun, X. et al. The mechanisms of boronate ester formation and fluorescent turn-on in ortho- aminomethylphenylboronic acids. Nat. Chem. 11, 768-778 (2019).

[0223] 15. Ortega-Valdovinos, L. R. & Yatsimirsky, A. K. Probing the Role of the Bridging Nitrogen in the Signaling Mechanism of an Anthracene-Boronic Acid Sugar Sensor and a Different Version of the PET-Based Mechanism. J. Org. Chem. (2023) doi:10.1021 / acs.joc.3c00129.

[0224] 16. Dowlut, M. & Hall, D. G. An improved class of sugar-binding boronic acids, soluble and capable of complexing glycosides in neutral water. J. Am. Chem. Soc. 128, 4226-4227 (2006).

[0225] 17. Nguyen, T., Magda, J. J. & Tathireddy, P. Manipulation of the isoelectric point of polyampholytic smart hydrogels in order to increase the range and selectivity of continuous glucose sensors. Sensors Actuators, B Chem. 255, 1057-1063 (2018).

[0226] 18. Tierney, S., Volden, S. & Stokke, B. T. Glucose sensors based on a responsive gel incorporated as a Fabry- Perot cavity on a fiber-optic readout platform. Biosens. Bioelectron. 24, 2034-2039 (2009).

[0227] 19. Tang, Z., Guan, Y. & Zhang, Y. The synthesis of a contraction-type glucose-sensitive microgel working at physiological temperature guided by a new glucose-sensing mechanism. Polym. Chem. 9, 1012-1021 (2018).

[0228] 20. Zhang, C., Losego, M. D. & Braun, P. V. Hydrogel-based glucose sensors: Effects of phenylboronic acid chemical structure on response. Chem. Mater. 25, 3239-3250 (2013).

[0229] 21. Yang, X., Pan, X., Blyth, J. & Lowe, C. R. Towards the real-time monitoring of glucose in tear fluid: Holographic glucose sensors with reduced interference from lactate and pH. Biosens. Bioelectron. 23, 899— 905 (2008).

[0230] 22. Lopalco, A., Marinara, W. A., Day, V. W. & Stella, V. J. Isolation, Solubility, and Characterization of D- Mannitol Esters of 4-Methoxybenzeneboronic Acid. J. Pharm. Sci. 106, 601-610 (2017).

[0231] 23. Espeel, P., Goethals, F., Stamenovic, M. M., Petton, L. & Du Prez, F. E. Double modular modification of thiolactone-containing polymers: Towards polythiols and derived structures. Polym. Chem. 3, 1007-1015 (2012).

Claims

CLAIMS1 . A biosensor for measuring the concentration of glucose, the biosensor comprising a polymer comprising the moiety of formula (I):wherein said moiety of formula (I) is immobilized in said polymer, wherein each R is independently C1-5 alkyl, C2-5 alkenyl or C2-5 alkynyl, wherein each Rsis independently selected from C1-5 alkyl, C2-5 alkenyl, C2-5 alkynyl, -(C0-3 alkylene)-OH, -(C0-3 alkylene)-O(Ci-5 alkyl), -(C0-3 alkylene)-O(Ci-5 alkylene)-OH, -(C0-3 alkylene)-O(Ci-5 alkylene)-O(Ci-5 alkyl), -(C0-3 alkylene)-SH, -(C0-3 alkylene)-S(Ci-5 alkyl), -(C0-3 alkylene)-S(Ci-5 alkylene)-SH, -(C0-3 alkylene)-S(Ci-5 alkylene)-S(Ci-5 alkyl), -(C0-3 alkylene)-NH2, -(C0-3 alkylene)-NH(Ci-5 alkyl), -(C0-3 alkylene)-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-3 alkylene)-NH-OH, -(C0-3 alkylene)-N(Ci-5 alkyl)-OH, -(C0-3 alkylene)-NH-O(Ci-5 alkyl), -(C0-3 alkylene)-N(Ci-5 alkyl)-O(Ci-5 alkyl), -(C0-3 alkylene)-halogen, -(C0-3 alkylene)-(Ci-5 haloalkyl), -(C0-3 alkylene)-O-(Ci-5 haloalkyl), -(C0-3 alkylene)-CN, -(C0-3 alkylene)-NO2, -(C0-3 alkylene)-CHO, -(C0-3 alkylene)-CO-(Ci-5 alkyl), -(C0-3 alkylene)-COOH, -(C0-3 alkylene)-CO-O-(Ci-5 alkyl), -(C0-3 alkylene)-O-CO-(Ci-5 alkyl), -(C0-3 alkylene)-CO-NH2, -(C0-3 alkylene)-CO-NH(Ci-5 alkyl), -(C0-3 alkylene)-CO-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-3 alkylene)-NH-CO-(Ci-5 alkyl), -(C0-3 alkylene)-N(Ci-5 alkyl)-CO-(Ci-5 alkyl), -(C0-3 alkylene)-NH-CO-O-(Ci-5 alkyl), -(C0-3 alkylene)-N(Ci-5 alkyl)-CO-O-(Ci-5 alkyl), -(C0-3 alkylene)-O-CO-NH-(Ci-5 alkyl), -(C0-3 alkylene)-O-CO-N(Ci-5 alkyl)-(Ci-5 alkyl), -(C0-3 alkylene)-SO2-NH2, -(C0-3 alkylene)-SO2-NH(Ci-5 alkyl), -(C0-3 alkylene)-SO2-N(Ci-5 alkyl)(Ci-5 alkyl), -(C0-3 alkylene)-NH-SO2-(Ci-5 alkyl), -(C0-3 alkylene)-N(Ci-5 alkyl)-SO2-(Ci-5 alkyl), -(C0-3 alkylene)-SO2-(Ci-5 alkyl), -(C0-3 alkylene)-SO-(Ci-5 alkyl), -(C0-3 alkylene)-carbocyclyl, and -(C0-3 alkylene)-heterocyclyl, wherein the carbocyclyl moiety in said -(C0-3 alkylene)-carbocyclyl and the heterocyclyl moiety in said -(C0-3 alkylene)-heterocyclyl are each optionallysubstituted with one or more groups independently selected from C1-4 alkyl, halogen, -CN, -NO2, -OH, -0- (C1.4 alkyl), -SH, -S-(Ci.4alkyl), -NH2, -NH(Ci.4alkyl), -N(CI.4alkyl)(Ci_4alkyl), -COOH, -COO(Ci.4alkyl), - CONH2, -CONH(CI.4alkyl), -CON(CI.4alkyl)(Ci_4alkyl), -NHCO(CI.4alkyl) and -N(Ci.4alkyl)-CO(Ci_4alkyl); and wherein n Is O, 1, 2 or 3.

2. The biosensor of claim 1 , wherein each R is methyl .

3. The biosensor of claim 1 or 2, wherein n is 0 or 1, preferably wherein n is 0.

4. The biosensor of any one of claims 1 to 3, wherein the polymer comprises a moiety of formula (la):wherein R, Rsand n are as in any one of claims 1 to 3.

5. The biosensor of any one of claim 1 to 4, wherein the polymer is a polymeric hydrogel.

6. The biosensor of any one of claims 1 to 5, wherein the polymer is an unsubstituted polyacrylamide-based polymer.

7. The biosensor of any one of claims 1 to 6, wherein the polymer further comprises a moiety that is positively charged at pH = 7.4.

8. The biosensor of any one of claims 1 to 6, wherein the polymer further comprises a moiety comprising a group selected from -NH2, -NH3T -NH(CI-5 alkyl), -(NH2(CI-5 alkyl))+, -N(CI-5 alkyl)2, -(NH(CI-5 alkyl)2)+, and -N(CI-5 alkyl)3+.

9. The biosensor of any one of claims 1 to 6, wherein the polymer further comprises a moiety of formula (II):

10. The biosensor of any one of claims 1 to 9, wherein the polymer further comprises a moiety of formula (III):11 . The biosensor of any one of claims 1 to 10, wherein the polymer is obtainable in a polymerization reaction of a composition comprising:(I) acrylamide monomer comprising a boronic acid-based glucose binding moiety of formula (I), preferably wherein (I) is:wherein R, Rsand n are as in any one of claims 1 to 10.

12. The biosensor of claim 11, wherein the composition used in the polymerization reaction further comprises(ii) acrylamide monomer free of boronic-acid moiety, and(ill) crosslinker acrylamide monomer, preferably wherein (ii) comprises a monomer selected from:, wherein q is an integer from 1 to 10; and / or wherein (ill) comprises a monomer selected from13. The biosensor of any one of claims 1 to 12, wherein the polymer contracts upon the presence of glucose, preferably wherein the rate of contraction is substantially linearly proportional to the concentration of glucose.

14. The biosensor of claim 1, wherein each R is methyl, n is 0, and the polymer is an unsubstituted polyacrylamide-based polymer.

15. The biosensor of claim 1, wherein each R is methyl, n Is O, and the polymer further comprises a moiety that is positively charged at pH = 7.4.

16. The biosensor of claim 1, wherein each R is methyl, n is 0, and the polymer further comprises a moiety comprising a group selected from -NH2, -NH3T -NH(CI-5 alkyl), -(NH2(CI-5 alkyl))+, -N(CI-5 alkyl)2 , -(NH(CI-5 alkyl)2)+, and -N(CI-5 alkyl)3+.

17. The biosensor of any one of claims 1 to 16, wherein the rate of contraction dependent on glucose is substantially independent of pH, preferably substantially independent of pH for pH in the range from 6.9 to 7.6, preferably in the range from 7.4 to 7.6.

18. The biosensor of any one of claims 1 to 17 for use in diagnostics.

19. The biosensor of any one of claims 1 to 17 for use in glucose level monitoring.

20. The biosensor for use of claim 19, wherein the glucose level monitoring is performed on a subject in an intensive care and / or wherein the glucose level monitoring is performed on an unconscious subject.

21. A polymer as defined in any one of claims 1 to 16.

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