Electrochemical Glucose Sensing by Glucose Equilibrium Binding to Genetically Engineered Glucose-Binding Proteins

Abstract

Aspects of the present disclosure provide devices and methods that can optimize in vivo electrochemical measurements of target molecules, such as glucose. Such aspects can include engineered binding proteins, such as engineered glucose binding proteins. The engineered binding protein can change its conformation in response to binding or dissociation to a ligand and / or an analyte, such as glucose. Such conformational changes can either expose or occlude a redox molecule bound to the binding protein. When exposed, the redox molecule can generate a redox signal depending on the conformational state of the binding protein. The redox signal can be measurable, for example, by cyclic voltammetry. The protein can be incorporated into a sensor that can be used as an implantable continuous glucose monitoring device.

Description

Technical Field

[0001] Incorporation by Reference of Priority Applications Any and all applications in which foreign or domestic priority claims are identified in the application data sheet filed with this application are hereby incorporated by reference into this specification under 37 CFR 1.57. For example, this application claims the priority of U.S. Provisional Application No. 63 / 368,760, filed on July 18, 2022, the entire content of which is hereby incorporated by reference in its entirety for all purposes and forms a part of this specification.

Background Art

[0002] Field The present disclosure relates to continuous glucose monitoring (CGM). More specifically, the present invention relates to a CGM sensor.

[0003] Description of Related Art Monitoring of blood glucose concentration levels has long been essential for the management and care of diabetes. Current blood glucose monitors involve a chemical reaction between blood or serum and a test strip and require an invasive extraction of blood via a lancet or a finger prick. Small portable monitors have been developed, allowing patients to perform this procedure anytime and anywhere. However, the inconvenience of this procedure, particularly blood extraction, the pain associated with this procedure, and the use and disposal of lancets and test strips have led to low compliance. Therefore, it is desirable to continuously monitor the concentration of glucose levels in the human body.

[0004] The first-generation continuous glucose monitoring (CGM) sensors based on electrochemical principles were developed based on Clark-type amperometric detection. Such CGM sensors measure the current generated by the electrochemical oxidation of hydrogen peroxide (H2O2) at the surface of either an activated platinum or platinum / iridium (90:10 Pt:Ir) electrode. Hydrogen peroxide (H2O2) is a byproduct of the oxidation (electron loss) of glucose by the enzyme glucose oxidase (GOx). Each molecule of glucose oxidized by GOx produces one molecule of H2O2 as a byproduct, which acts as a surrogate for measuring glucose concentration. The fraction of H2O2 that diffuses inward (towards the electrode) is electrochemically oxidized to oxygen at the Pt surface, thereby generating a current for measuring glucose concentration.

[0005]

Number

[0006] CGM sensors based on electrochemical methods may be capable of detecting glucose by the indirect measurement of molecules generated during the enzymatic reaction. The enzyme product may be, for example, hydrogen peroxide, an artificial mediator, and / or the reduced enzyme cofactor itself. The aspect common to all these measurements is to apply an electrochemical potential sufficient to (re)oxidize these molecules to generate a current.

Prior Art Documents

Non-Patent Documents

[0007]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Summary of the Invention

[0008] For the purpose of summarizing the present disclosure, certain aspects, advantages, and novel features of several devices, systems, and methods are described herein. It should be understood that not all examples of the present disclosure are necessarily disclosed herein. Thus, the devices, systems, and methods disclosed herein may be embodied or implemented to achieve or optimize one advantage or a group of advantages as taught herein without necessarily achieving other advantages that may be taught or suggested herein. [[Means for Solving the Problem]]

[0009] Aspects of the present disclosure relate to sensors. The sensor may include a first binding protein configured to have a first conformation and a second conformation, wherein when the first binding protein including a binding site is in the first conformation, electrons are transferred to the electrode surface, and whether the first binding protein is in the first conformation or the second conformation depends at least in part on whether an analyte bound to the binding site is present, and a sensing electrode. In some aspects, the sensor may include a redox mediator configured to generate an electrical signal, wherein when the first binding protein is in the first conformation, the redox mediator is inactive or partially active, and when the first binding protein is in the second conformation, the redox mediator is active or relatively more active. In some aspects, the sensor of the present disclosure may include a redox mediator configured to generate an electrical signal and an effector molecule configured to shift the activity of the redox mediator when the redox mediator and the effector molecule are in proximity, wherein when the first binding protein is in the first conformation, the redox mediator and the effector molecule are not in proximity, and when the first binding protein is in the second conformation, the redox mediator and the effector molecule are in proximity. In some aspects, the first binding protein may include a glucose binding protein. In some aspects, the binding constant of the glucose binding protein may be from 100 nM to 200 mM. In some aspects, the binding constant of the glucose binding protein may be from 2 mM to 22 mM. In some aspects, the analyte includes glucose. In some aspects, the binding site includes a glucose binding site. In some aspects, the sensor includes a second binding protein, and the first binding protein and the second binding protein do not have the same binding constant. In some aspects, the redox mediator may be a Ru(II) cofactor, porphyrin, or quinone. In some aspects, the sensing electrode includes a nanowire network. In some aspects, the sensing electrode includes a hydrogel.In some embodiments, the sensing electrode includes a polymer matrix.

[0010] Aspects of the present disclosure relate to a method for measuring the concentration of an analyte. The method includes exposing a sample fluid to a binding protein configured to have a first conformation and a second conformation, wherein whether the binding protein is in the first conformation or the second conformation depends at least in part on whether an analyte bound to the binding site of the binding protein is present, and measuring an electrical signal that depends at least in part on whether the binding protein is in the first conformation or the second conformation. In some embodiments, the step of measuring the electrical signal includes using an electrode that includes a nanowire network. In some embodiments, the analyte includes glucose. In some embodiments, the binding protein includes a glucose-binding protein. In some embodiments, the binding site includes a glucose-binding site. In some embodiments, the electrical signal may be generated by a redox mediator. In some embodiments, the redox mediator is not active when the binding protein is in the first conformation. In some embodiments, when the binding protein is in the first conformation, the redox mediator is partially active. In some embodiments, when the binding protein is in the second conformation, the redox mediator is active. In some embodiments, the mediator has higher activity in the second conformation than in the first conformation. In some embodiments, the electrical activity of the redox mediator depends at least in part on proximity to an effector molecule, and when the binding protein is in the first conformation, the redox mediator and the effector molecule are not in proximity, and when the binding protein is in the second conformation, the redox mediator and the effector molecule are in proximity.

[0011] The above aspects of the embodiments of the present disclosure, as well as other features, aspects, and advantages, will now be described in connection with various embodiments with reference to the accompanying drawings. The illustrated embodiments are merely examples and are not intended to be limiting. Throughout the drawings, like reference numerals generally identify like components unless the context otherwise indicates.

Brief Description of the Drawings

[0012]

Figure 1A

Figure 1B

Figure 2

Figure 3A

Figure 3B

Figure 4A

Figure 4B

Figure 5

Figure 6A

Figure 6B

Figure 6C

[0013] Aspects of the present disclosure are described in detail herein with reference to the drawings and various examples. However, one skilled in the art will understand that other configurations of the devices and methods disclosed herein are still within the scope of the present disclosure even if not described in the same detail. The aspects of the various configurations described do not limit the scope of the disclosure herein, which is instead defined by the claims that follow this description.

[0014] Aspects of the present disclosure provide devices and methods that can optimize in vivo electrochemical measurements of target molecules, such as glucose. Such aspects may include engineered binding proteins, such as engineered glucose-binding proteins. Engineered binding proteins can change their conformation in response to binding or dissociation to a ligand and / or an analyte, such as glucose. Such conformational changes may either expose or occlude a redox molecule bound to the binding protein. When exposed, the redox molecule can generate a redox signal depending on the conformational state of the binding protein. The redox signal may be measurable, for example, by cyclic voltammetry, chronoamperometry, and / or electrochemical impedance spectroscopy.

[0015] Definitions As used herein, common abbreviations are defined as follows. °C Celsius temperature CE Counter electrode CGM Continuous glucose monitoring DAN Diaminonaphthalene GOx Glucose oxidase H2O2 Hydrogen peroxide oPD Ortho-phenylenediamine mPD meta-phenylenediamine PBS phosphate buffered saline pPD para-phenylenediamine PmPD poly(meta-phenylenediamine) PoPD poly(ortho-phenylenediamine) PpPD poly(para-phenylenediamine) PVA polyvinyl alcohol RE reference electrode WE working electrode

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The terms "including", as well as other forms such as "include", "includes", and "included", are not limiting. The terms "having", as well as other forms such as "have", "has", and "had", are not limiting. The terms "comprising", "including", "having", etc. are synonymous and are used in an inclusive, open-ended fashion and do not exclude additional elements, features, acts, operations, etc. That is, the above terms should be construed synonymously with the phrase "at least having" or "at least including". For example, when used in the context of a process, the term "comprising" means that the process includes at least the recited steps, but may also include additional steps. When used in the context of an apparatus, the term "comprising" means that the apparatus includes at least the recited features or components, but may also include additional features or components. Also, the term "or" is used in its inclusive sense (not its exclusive sense), and thus, for example, when used to combine a list of elements, the term "or" means one, some, or all of the elements in the list. Further, the term "each" as used herein, in addition to having its ordinary meaning, can mean any subset of the set of elements to which the term "each" applies.

[0017] Conditional language such as "can", "could", "might", or "may" generally does not, unless specifically stated otherwise or understood within the context in which it is used, intend to convey that a particular embodiment includes a particular feature, element, or step while other embodiments do not. Thus, such conditional language generally does not imply that a feature, element, or step is required in any way in one or more embodiments, or that one or more embodiments necessarily include logic for determining whether these features, elements, or steps are included in or should be performed in any particular embodiment, whether using user input or prompts or not.

[0018] Connective language such as the phrase "at least one of X, Y, and Z" is understood in the context in which it is generally used to convey that an item, term, etc. may be any of X, Y, or Z, unless specifically stated otherwise. Thus, such connective language generally does not intend to imply that a particular embodiment requires the presence of at least one of X, at least one of Y, and at least one of Z.

[0019] Language indicating a degree as used herein, such as terms like "about", "approximately", "generally", and "substantially" as used herein, represents a value, quantity, or characteristic that is still close to the stated value, quantity, or characteristic that still performs the desired function or achieves the desired result. For example, the terms "about", "approximately", "generally", and "substantially" may refer to amounts within less than 10%, less than 5%, less than 1%, less than 0.1%, and less than 0.01% of the stated amount.

[0020] As used herein, the term "and / or" has the broadest meaning with the least limitation, and the present disclosure includes only A, only B, both A and B together, or alternatively A or B, without requiring both A and B, or without requiring one of A or one of B. As used herein, the phrase "at least one of A, B, and C" should be construed to mean logical A or B or C using a non-exclusive logical "or".

[0021] As used herein, the term "temperature-independent" means that the reading or measurement of the glucose level by the glucose monitoring device, or the response of the glucose sensor, is not affected or is substantially not affected by a change in temperature. In other words, the sensor is less sensitive to changes in temperature (e.g., changes in body temperature as a result of physiological conditions such as hypothermia and hyperpyrexia). In some embodiments, the temperature independence of the glucose monitoring device is maintained within the operating temperature range of the device (e.g., about 30°C to about 45°C, about 33°C to about 43°C, about 35°C to about 41°C, or about 36°C to about 40°C). In some embodiments, a change in temperature (per 1°C) results in a change of less than 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, or 0.01% in the response of the sensor, or the measurement / reading provided by the device, when all other parameters remain the same (e.g., the glucose concentration is constant).

[0022] Any method disclosed herein need not be performed in the recited order. The methods disclosed herein include specific acts performed by an operator, but they can also include, explicitly or implicitly, any third-party instructions for those acts.

[0023] Enzyme glucose sensor A specific glucose sensor implanted subcutaneously relies on an enzymatic reaction that specifically converts glucose molecules, and in the process, generates electroactive by-products such as hydrogen peroxide or artificial mediators. The by-products are then oxidized at the working electrode to generate a measurable current. As shown in FIGS. 1A and 1B, the electrons transferred from the enzymatic oxidation of glucose are "captured" by the electrochemical sensor. FIG. 1A shows an example where electrons move from glucose 112 through hydrogen peroxide 104 to electrode 102. Glucose 112 interacts with enzyme 116 and FAD 108, transferring electrons from glucose 112. This electron acquisition converts FAD 108 to FADH2 110. Glucose 112 is converted to gluconic acid 114 by enzyme 116. Enzyme 116 transfers two electrons from FADH2 110 to oxygen 106 (O2) to generate hydrogen peroxide 104. FADH2 110 is converted back to FAD 108. Electrode 102 can apply a potential to oxidize hydrogen peroxide 104 and transfer electrons from hydrogen peroxide 104. This transfer of electrons from hydrogen peroxide 104 to electrode 102 can be detected.

[0024] FIG. 1B shows a second embodiment where the transfer of electrons to electrode 102 is achieved via an artificial mediator 118. Many of the cycles are the same, but instead of transferring electrons to oxygen 106 to generate hydrogen peroxide 104, enzyme 116 transfers one or more electrons to artificial mediator 118 to convert mediator 118a to mediator 118b with a negative charge. Electrode 102 can apply a potential to mediator 118b with a negative charge, oxidize mediator 118b with a negative charge, and transfer one or more electrons to the electrode. This transfer of electrons from mediator 118b with a negative charge to electrode 102 can be detected.

[0025] However, both of the enzyme-based sensor designs shown in FIGS. 1A and 1B can have drawbacks. For example, both sensors measure the constantly changing local glucose concentration. The local glucose concentration can change, for example, due to the enzymatic action of the sensor. The consumption of the analyte measured by the enzyme can result in particularly inaccurate estimates of the glucose concentration when measuring low glucose concentrations. Furthermore, enzyme activity gradually decreases over time. In the embodiments of FIGS. 1A and 1B, this limits the lifespan of the sensor. The enzymatic conversion of glucose also depends on other molecular species. The conversion of glucose occurs only in the presence of the cofactor FAD as well as the substrates oxygen and glucose. Thus, if the concentration of the cofactor or glucose is low, the conversion of glucose can decrease. Finally, one of the by-products of the enzymatic conversion of glucose is hydrogen peroxide. Hydrogen peroxide can also be produced by physiological phenomena such as inflammation or other processes. Hydrogen peroxide is a very reactive molecule that can degrade the enzyme and / or other chemical and biochemical sensor components. Thus, the presence of hydrogen peroxide can further shorten the lifespan of the sensor.

[0026] There are several drawbacks specific to the embodiment according to FIG. 1A. For example, in subcutaneous tissue, the number of glucose molecules far exceeds the amount of oxygen molecules. For example, there can be a difference of up to about 1000-fold between the number of glucose molecules and the number of oxygen molecules. In contrast, the enzymatic conversion reaction requires both equimolar amounts of glucose and oxygen. Thus, the sensor can be designed to proportionally reduce the glucose concentration within the sensor where the enzyme is located. This can result in a low signal because relatively few glucose molecules are available to react via enzymatic conversion. For example, the signal can be in the range of 1 pA to 10 nA. Such low currents may require sensitive electronic circuits that can be expensive.

[0027] Furthermore, a signal in the form of a current is generated by oxidizing hydrogen peroxide at an electrode. The electrode may need to be held at a relatively high potential, for example, +0.6 V versus Ag / AgCl, to generate this signal in the presence of hydrogen peroxide. At this working potential, there are a number of endogenous and exogenous molecules that may also be oxidized at the electrode. These molecules are referred to herein as electrochemical interferents. Oxidation of the electrochemical interferents generates additional current, which can combine with and / or distort the signal due to glucose. To eliminate these electrochemical interferents, many strategies have been developed including, but not limited to, size-based filtering and / or charge-based filtering. However, these strategies typically require one or more additional layers to shield the working electrode. The additional layers may increase manufacturing complexity and may require extensive biocompatibility studies to demonstrate the safety of the device.

[0028] Similarly, there are several drawbacks specific to the embodiment according to FIG. 1B. For example, it can be difficult to identify a suitable redox mediator. Such a mediator may desirably have a relatively low operating potential such that the current from the electrochemical interferents is minimized. Further, the redox mediator should not decompose under constant operation at the applied potential. Decomposition of the redox mediator can result in errors in the estimation of glucose concentration due to loss of current. Further, decomposition of the redox mediator can cause safety concerns as the incorporated molecules may leach into the surrounding biological fluid.

[0029] Disclosed herein is a novel configuration of an electrochemical analyte sensor. The sensor according to the present disclosure does not require enzymatic conversion of the analyte. Rather, equilibrium binding of the analyte to a binding protein is utilized to generate a measurable signal.

[0030] Binding protein A suitable binding protein can specifically bind to a target analyte. Further, a suitable binding protein may undergo a conformational change in response to binding to the analyte.

[0031] An example of a binding protein is a glucose-binding protein. The glucose-binding protein (GGBP) from Escherichia coli (E. coli) (UniprotID: P0AEE5) is a member of a protein family mainly found in bacteria called periplasmic binding proteins (PfamID: PF18610). Members of this protein family have specific affinities for ligands and / or analytes. As an example, GGBP has specific affinity for glucose and / or galactose. The structure of GGBP202 is shown as a ribbon diagram in FIG. 2. Generally, GGBP includes two large protein domains, an N-terminal domain 204 and a C-terminal domain 206, which are linked to each other by a flexible hinge 208. Other exemplary binding proteins include vitamin D-binding protein, odorant-binding protein (derived from olfactory receptors), calcium-binding protein, or any hormone-binding protein / receptor. Additionally or alternatively, portions of such binding proteins may be suitable for inclusion in the electrochemical analyte sensors of the present disclosure. For example, a portion of a binding protein that changes conformation substantially in response to analyte binding / dissociation may be suitable for inclusion. Suitable analytes may include sugars such as glucose, galactose, lactose, specific nucleotide sequences, insulin, growth hormone, retinol, calcium, iron, or other molecules present in physiological functions. Further, suitable binding proteins can be engineered, for example, by directed evolution, rational design, or de novo design techniques. As an example, a naturally occurring binding protein or binding protein fragment, such as any of the examples described above, can be selected as a starting point for modification to generate a binding protein (referred to herein as an engineered binding protein) for inclusion in the sensors of the present disclosure. Modifications of the binding protein are discussed herein with respect to GGBP. The binding protein can be modified, for example, to change analyte binding kinetics, to change conformational changes, to change the types of analytes to which the protein can bind, or to change the size and / or shape of the binding protein (thereby potentially changing the steric effects and / or diffusion rate of the protein).

[0032] Certain aspects of GGBP are useful for inclusion in the sensors according to the present disclosure. First, GGBP binds to glucose and / or galactose molecules with very high affinity and specificity. Second, the binding of GGBP to glucose is reversible and does not chemically convert the analyte. Third, when glucose binds to GGBP, a significant conformational change occurs in the protein. This conformational change is shown in Figure 2. The unbound conformation 212 is shown without shading. The bound conformation 214 is shown shaded. Note that in the bound conformation 214, the N-terminal domain 204 and the C-terminal domain 206 are closed around the glucose 210.

[0033] The conformational change is also shown schematically in Figure 3A or 3B. Figure 3A shows the conformation of unbound GGBP 202, and Figure 3B shows the conformation of GGBP 202 with glucose bound. Glucose binds to the glucose binding site 302. When glucose binds, the N-terminal domain 204 and the C-terminal domain 206 approach each other, and the movement at the hinge 208 is restricted compared to the unbound form. In the form of GGBP with glucose bound (Figure 3B), the portion of the protein close to the hinge region may be more or less exposed than in the form with glucose unbound (Figure 3A). Structurally, the bound conformation is relatively rigid, while the unbound conformation is more mobile around the hinge. The conformational change of GGBP is described in more detail by Borrok, M. J. et al., Conformational changes of glucose / galactose binding protein illuminated by open, unliganded, and ultra-high-resolution ligand-bound structures, Protein Sci. 16, 1032-41 (2007), which is incorporated herein by reference.

[0034] As disclosed herein, a concentration-dependent biosensor capable of propagating an electrochemical signal can be fabricated by utilizing the conformational change of GGBP upon binding to glucose.

[0035] Wild-type GGBP binds to glucose with very high affinity, and the binding constant is 0.2 μM. Realistic physiological glucose concentrations range from approximately 2 mM to 22 mM. Within this range, nearly 100% of wild-type GGBP binds to glucose. As disclosed herein, GGBP can be modified to shift and / or alter its binding affinity for glucose using methods described in the art. For example, changing the alanine (A) residue at position 213 of GGBP to an arginine (R) residue (A213R) shifts the binding constant 5000-fold from 0.2 μM to 1 mM. This mutation was discussed, inter alia, by Amiss, T. J. et al., Engineering and rapid selection of a low affinity glucose / galactose binding protein for a glucose biosensor, Protein Sci. 16, 2350-59 (2007), and is incorporated herein by reference.

[0036] GGBP has 332 amino acids in its protein sequence, 16 of which are located at the glucose binding site 302. Two related strategies can enable fine-tuning of the glucose binding affinity of GGBP to better match the physiological glucose range.

[0037] The first strategy involves genetically engineering some or all of the 16 amino acid positions of the glucose binding site 302. For example, any of these 16 amino acid positions can be changed to a different amino acid from among the 20 standard amino acids. When the amino acids in these 16 binding pockets are changed, the glucose binding affinity of GGBP can be altered such that its binding constant is within or near the relevant physiological range.

[0038] The second strategy involves genetically engineering several different modified GGBPs. A plurality of mutant GGBPs having different binding constants are mixed within the sensor to achieve sensitivity over the range of glucose concentrations found in physiological functions. As an illustrative example, a series of GGBPs having binding constants of 2 mM, 4 mM, 8 mM, and 16 mM can provide an effective range for physiologically relevant glucose concentrations. Other sets of GGBPs having other binding constants may be appropriate.

[0039] For a GGBP or set of GGBPs that appropriately cover a physiological glucose concentration range, an electroactive redox molecule can be introduced at specific positions on the GGBP proteins disclosed herein. Such electroactive redox molecules are referred to herein as redox mediators. One or more redox mediators, when exposed, can generate a measurable electrical signal. The redox mediator can be introduced at a position on the GGBP that is accessible depending on the conformational state of the GGBP. In certain embodiments, when the GGBP is in a glucose-bound state, the redox mediator may be more accessible (i.e., active or relatively more active). In other embodiments, the redox mediator may be more accessible (i.e., active or relatively more active) when the GGBP is in an unbound state. FIGS. 4A and 4B show one such exemplary embodiment. FIG. 4A shows an engineered GGBP 402 in an unbound conformation having a redox mediator 404. FIG. 4B shows an engineered GGBP 402 in a glucose-bound conformation having a redox mediator 404. In this example, the redox mediator 404 binds to a portion of the engineered GGBP 402, specifically a portion of the C-terminal domain 206, that becomes inaccessible when glucose 210 binds, as shown in FIG. 4B. Thus, the glucose-bound conformation versus the unbound conformation of the engineered GGBP can be reflected in a change in the electrical signal due to the accessibility, and thus the activity, of the redox mediator.

[0040] FIG. 5 shows an exemplary scheme of conformational-dependent signal propagation to electrode 102. In the illustrated example, when the GGBP is in its unbound conformation 502, the redox mediator 404 is accessible and thus active (or relatively more active). When active (or relatively more active), the redox mediator 404 generates a signal that can be detected by the electrode 102, as indicated by the dashed arrow. However, the unbound GGBP 502 can reversibly bind to glucose 112 to form the bound GGBP 504. When the GGBP is in its bound conformation 504, the redox mediator 404 is not accessible and thus not active (or only partially active). As a result, when the GGBP is in its bound conformation 504, the redox mediator 404 may not generate a signal detectable by the electrode 102. As discussed herein, the electrode 102 can include a nanowire network.

[0041] The operated GGBP can be embedded in a conductive hydrogel and / or a polymer matrix. The conductive hydrogel or polymer matrix can also incorporate a nanowire network. The synthesis of such a nanowire network within the hydrogel or polymer matrix is discussed in detail by Shi, Q. et al., Kinetic controlled synthesis of AuPt bi-metallic aerogels and their enhanced electrocatalytic performance, J. Mater. Chem. A 5, 19626-31(2017), and Shi, Q. et al., Mesoporous Pt Nanotubes as a Novel Sensing Platform for Sensitive Detection of Intracellular Hydrogen Peroxide, Acs. Appl. Mater. Inter. 7, 24288-95(2015), which are incorporated herein by reference. FIGS. 6A-6C show examples of GGBP embedded in a conductive hydrogel 602. There is a nanowire network 604 embedded in the conductive hydrogel 602. According to the examples shown in FIGS. 4A and 4B, some of the operated GGBP are also embedded, the redox mediator 404 is exposed, but the operated GGBP 402 is not bound. FIG. 6A shows a situation where the population of GGBP is all unbound GGBP 502. Thus, all of the redox mediator 404 is exposed and the signal intensity is at its maximum.

[0042] In FIG. 6B, half of the GGBP is unbound GGBP 502 and half is bound GGBP 504. Thus, the signal intensity by the redox mediator 404 can be less than that in FIG. 6B. In FIG. 6C, all of the GGPB is bound GGBP 502. In this example, the signal intensity is the lowest.

[0043] The nanowire network can improve the sampling of conformation-dependent electrical signals generated by redox mediators on the GGBP protein. The nanowire network can also increase the signal density due to redox molecules, which may enable miniaturization of the sensor.

[0044] Such a nanowire network, when used in combination with the engineered GGBP, can be measured within the potential range of about -1 to +1 V versus Ag / AgCl, about -0.75 to +0.75 V versus Ag / AgCl, about -0.5 to +0.5 V versus Ag / AgCl, about -0.4 to +0.4 V versus Ag / AgCl, about -0.3 to +0.3 V versus Ag / AgCl, about -0.25 to +0.25 V versus Ag / AgCl, about -0.2 to +0.2 V versus Ag / AgCl, or within any value or range within or surrounded by or bounded by these ranges or values, or values outside of these ranges or values may also be used in some cases.

[0045] Addition of electroactive molecules to the binding protein In some embodiments, a single site on the binding protein is engineered to incorporate a redox mediator. For example, if the GGBP undergoes a glucose-dependent conformational change, the binding site can be selected such that the redox mediator is either occluded or accessible. Thus, the redox molecule can generate a glucose-dependent redox signal that depends on, for example, GGBP glucose binding as measured by cyclic voltammetry. Suitable redox molecules can include, but are not limited to, Ru(II) cofactors, porphyrins, and quinones. The redox molecule may be covalently attached to a binding protein, such as GGBP. Engineering of hinge-binding proteins is further discussed by Benson, D. E. et al., Design of Bioelectronic Interfaces by Exploiting Hinge-Bending Motions in Proteins, Science 293, 1641-44 (2001), which is incorporated herein by reference.

[0046] The higher the concentration of glucose, the more engineered GGBP molecules in the sensor according to the present disclosure will be in the bound state. In embodiments where the redox molecule is accessible when the engineered GGBP binds to glucose, the electrical signal measured by the nanowire network may correspondingly increase as the glucose concentration increases. In other embodiments where the redox molecule is inaccessible when the engineered GGBP binds to glucose, the electrical signal measured by the nanowire network may correspondingly decrease as the glucose concentration increases.

[0047] In certain embodiments, two electroactive molecules may be attached to the engineered GGBP. One of these molecules may be a redox mediator as discussed above. The second molecule, herein referred to as an effector molecule, can shift the activity of the redox mediator when in proximity to the redox mediator. Each of the redox mediator and the effector molecule may be attached to different amino acid sites on the GGBP. The amino acid sites can be selected such that the distance between the amino acid sites changes due to the conformational change of the GGBP upon glucose binding. For example, the two sites may be relatively close in the bound conformation but relatively far apart in the unbound conformation. In such embodiments, the effector molecule may be in proximity to the redox mediator when the GGBP binds glucose and not in proximity when the GGBP does not bind glucose. In another example, the positions can be selected such that the two sites are relatively far apart in the bound conformation but relatively close in the unbound conformation. In such embodiments, the effector molecule may be in proximity to the redox mediator when the GGBP does not bind glucose and not in proximity to the redox mediator when the GGBP binds glucose. These embodiments can shift the peak potential of the redox mediator, which can be measured, for example, by cyclic voltammetry.

[0048] Aspects of the analyte-binding sensor The sensors according to the present disclosure can address the drawbacks of enzymatic sensors. For example, such sensors can rely on binding to glucose rather than conversion of glucose, so the local physiological glucose concentration may remain substantially unchanged. In the same vein, such sensors can avoid generating reactive by-products or generate fewer reactive by-products because the GGBP simply binds glucose rather than converting glucose to a different molecule.

[0049] Compared with an enzymatic glucose sensor, the sensor according to the present disclosure may have a higher signal-to-noise ratio. For example, the sensor of the present disclosure may not require a glucose limiting layer for limiting the concentration of glucose relative to another reactant of the glucose conversion reaction, such as oxygen. The molecules involved in the GGBP binding of glucose are (1) GGBP and (2) glucose. Therefore, there is no need to limit the glucose concentration within the sensor.

[0050] Redox molecules can be selected to be outside the range of electrochemical oxidation of all, substantially all, most, or many physiological electrochemical interferents. This includes exogenous electrochemical interferents such as acetaminophen. Therefore, the sensor according to the present disclosure has minimal and / or less signal perturbation due to electrochemical interferents. The signal of the sensor may be entirely, substantially, or at least partially due to the binding of glucose to the engineered GGBP.

[0051] In related, having a higher signal-to-noise ratio and depending on the glucose-correlated signal amplitude, the sensor according to the present disclosure may be smaller than an enzymatic sensor. For example, such a sensor may have a length of about 100 μm, which is smaller than currently commercially available glucose sensors.

[0052] In certain embodiments, the sensor configuration is relatively simple and includes a nanowire network and a hydrogel with engineered GGBP. The enzyme glucose sensor may have several layers, such as a sensing electrode, an enzyme layer, a glucose limiting layer, and a shielding layer. Manufacturing a sensor according to the present disclosure can be simpler compared to manufacturing a sensor with several layers. The simple manufacturing process may result in a more reproducible and scalable production process. Since the number of components required to construct such a sensor is small, the complexity of the algorithm required to convert the measured current value to a concentration estimate can also be substantially reduced. In practice, the simplicity of the sensor design can reduce the number of parameters that the algorithm may need to address.

[0053] Sensor calibration may also be relatively simple for the sensors according to the present disclosure. For example, the absolute change due to glucose and the zero response of the sensor can be established and / or calibrated before manufacturing the sensor. This pre-manufacturing calibration can be translated to the embedded sensor. There may be no need to determine or infer unknown parameters, such as the effect of certain electrochemical interferents, after embedding, as in the case of enzyme-based electrochemical CGM sensors.

[0054] The sensors according to the present disclosure may have a longer sensing lifetime than enzyme sensors. Since the sensors according to the present disclosure require enzymatic action, there is no or substantially no reactive oxygen species (ROS) generated by sensing. Most of the measurements by such sensors can be carried out at a potential close to 0 V with respect to Ag / AgCl, for example, -0.2 to +0.2 V. The potential close to 0 V may result in fewer potential electrochemical interferents being oxidized and less ROS. Since the sensors according to the present disclosure have no enzymatic action and the measurement potential is close to 0 V, less ROS is generated, and thus the sensor lifetime can be relatively long. For example, such sensors may have a sensing lifetime of up to 30 days or more than 30 days.

Description of the Reference Numerals

[0055] 102 Electrode 104 Hydrogen peroxide 106 Oxygen 108 FAD 110 FADH2 112 Glucose 114 Gluconic acid 116 Enzyme 118a Mediator 118b Mediator with a negative charge 202 GGBP 204 N-terminal domain 206 C-terminal domain 208 Hinge 210 Glucose 212 Unbound conformation 214 Bound conformation 302 Glucose binding site 402 Engineered GGBP 404 Redox mediator 502 GGBP 504 Bound GGBP 602 Conductive hydrogel 604 Nanowire network 606

Claims

**Claim 1** A first binding protein configured to have a first conformation and a second conformation, wherein when an analyte bound to the binding site of the first binding protein is present, the first binding protein is in the first conformation, and when no analyte bound to the binding site of the first binding protein is present, the first binding protein is in the second conformation, A first binding protein that generates an electrical signal when the first binding protein including the binding site is in the first conformation, A sensing electrode A sensor comprising **Claim 2** Including a redox mediator configured to generate the electrical signal, When the first binding protein is in the first conformation, the redox mediator is inactive or partially active, When the first binding protein is in the second conformation, the redox mediator is active or relatively more active compared to the first conformation. The sensor according to claim 1. **Claim 3** Further comprising an effector molecule configured to shift the electrical activity of the redox mediator when the redox mediator and the effector molecule are in proximity, When the first binding protein is in the first conformation, the redox mediator and the effector molecule are not in proximity, When the first binding protein is in the second conformation, the redox mediator and the effector molecule are in proximity. The sensor according to claim 2. **Claim 4** The sensor according to claim 2 or 3, wherein the redox mediator is a Ru(II) cofactor, porphyrin, or quinone. **Claim 5** The sensor according to any one of claims 1 to 4, wherein the first binding protein includes a glucose binding protein. **Claim 6** The sensor according to claim 5, wherein the binding constant of the glucose binding protein is from about 100 nM to about 200 mM. **Claim 7** The sensor according to claim 5 or 6, wherein the binding constant of the glucose binding protein is from about 2 mM to about 22 mM. **Claim 8** The sensor according to any one of claims 1 to 7, wherein the analyte includes glucose. **Claim 9** The sensor according to claim 8, wherein the binding site of the first binding protein includes a glucose binding site. **Claim 10** The sensor according to any one of claims 1 to 9, further comprising a second binding protein, wherein the first binding protein and the second binding protein do not have the same binding constant.

11. The sensor according to any one of claims 1 to 10, wherein the sensing electrode comprises a nanowire network.

12. The sensing electrode according to any one of claims 1 to 11, wherein the sensing electrode comprises a hydrogel.

13. The sensing electrode according to any one of claims 1 to 11, wherein the sensing electrode comprises a polymer matrix.

14. A method for measuring the concentration of an analyte, comprising: exposing the sensor according to claim 1 to a sample fluid; measuring an electrical signal generated from the sensor; and a method comprising the steps of.

15. The method according to claim 14, wherein measuring the electrical signal comprises using an electrode comprising a nanowire network.

16. The method according to claim 14 or 15, wherein the analyte comprises glucose.

17. The method according to any one of claims 14 to 16, wherein the binding protein comprises a glucose-binding protein.

18. The method according to any one of claims 14 to 17, wherein the binding site comprises a glucose-binding site.

19. The method according to any one of claims 14 to 18, wherein the electrical signal is generated by a redox mediator.

20. When the binding protein is in the first conformation, the redox mediator is inactive or partially active. The method according to claim 19, wherein when the binding protein is in the second conformation, the redox mediator is active or relatively more active compared to the first conformation.

21. When the redox mediator and the effector molecule are in proximity, further comprising an effector molecule configured to shift the electrical activity of the redox mediator. When the binding protein is in the first conformation, the redox mediator and the effector molecule are not in proximity. The method according to claim 20, wherein when the binding protein is in the second conformation, the redox mediator and the effector molecule are in proximity.

Images