Detection of analytes by protein switches

Abstract

To provide a protein switch that can be used to determine a level of an analyte that is diagnostic of health and / or well-being of a subject.SOLUTION: A protein switch comprises at least one non-naturally occurring polypeptide including: (a) at least one analyte-binding domain capable of binding to at least one analyte; and (b) at least one oxidase domain or dehydrogenase domain having oxidase activity or dehydrogenase activity and capable of binding to or reacting with at least one reactant, where (i) the analyte that binds to the analyte-binding domain is different from the reactant that binds to or reacts with the oxidase domain or dehydrogenase domain; and (ii) the oxidase activity or dehydrogenase activity is altered when the analyte binds to the analyte-binding domain.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Patent Application No. 62 / 909,411, filed October 2, 2019, the contents of which are incorporated herein by reference.

[0002] Incorporation by Reference of Electronically Submitted Materials This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy created on October 1, 2020 is named "37810-601_ST25_ST25.txt" and is 46,815 bytes in size.

[0003] Field The present disclosure relates to compositions, methods, devices, systems, and apparatus, such as protein switches and their use in in vivo sensors, for detecting and / or monitoring analytes. The protein switches can be used to determine the level of one or more analytes that are diagnostic and / or prognostic indicia for the health and / or well-being of a subject. [Background technology]

[0004] Detection or monitoring of target analytes is beneficial to the health and / or well-being of certain individuals. Many disease states involve analytes that can be measured to diagnose and / or monitor the disease state in an individual. For example, vital organs such as the brain, heart, kidneys, and liver, as well as the nervous system and endocrine system, can be monitored by such analytes.

[0005] Key requirements for analyte detection and monitoring are assay specificity and sensitivity. These requirements are particularly important when the target analyte is present in a fluid or biological sample in small or limited amounts or concentrations. Generally, assay specificity and / or sensitivity are provided by the capture and / or detection antibodies used to detect one or more analytes of interest. Diagnostic assays utilizing this approach are well known and widely used, generally in enzyme-linked immunosorbent assay sandwich assays (ELISAs) or immunoassays. It can be difficult (and expensive) to generate antibodies that provide sufficient specificity and / or sensitivity for some analytes.

[0006] Current assays for detecting target analytes for prognostic, diagnostic, and / or monitoring purposes have several limitations that limit their widespread application in clinical, non-clinical (e.g., wearable), and point-of-care settings. In addition, many of these assays typically require a significant level of expertise and panels of expensive and specialized reagents (e.g., antibodies), along with sophisticated biomedical equipment housed in specialized laboratory environments. Summary of the Invention [Problem to be solved by the invention]

[0007] As a result, there is a need for compositions, devices, systems and methods capable of detecting target analytes in a subject for prognostic, diagnostic and / or monitoring purposes that are relatively inexpensive to manufacture, are capable of measuring small or limited amounts or concentrations of analytes in fluids or biological samples, and are suitable for a wide range of applications in clinical, non-clinical and point-of-care settings. [Means for solving the problem]

[0008] (Summary of the Invention) In one embodiment, the present disclosure relates to a protein switch. The protein switch of the present disclosure includes at least one non-naturally occurring polypeptide having (a) at least one analyte-binding domain capable of binding to at least one analyte; and (b) at least one oxidase or dehydrogenase domain having oxidase or dehydrogenase activity and capable of binding to or reacting with at least one reactive agent, wherein (i) the analyte that binds to the analyte-binding domain is different from the reactive agent that binds to or reacts with the oxidase or dehydrogenase domain; and (ii) the oxidase or dehydrogenase activity is altered upon binding of the analyte to the analyte-binding domain. In a further aspect, the oxidase is glucose oxidase or lactate oxidase. In another aspect, the dehydrogenase is glucose dehydrogenase or lactate dehydrogenase.

[0009] In further aspects, in the above protein switches, when the analyte-binding domain binds to the analyte, the activity of the oxidase or dehydrogenase decreases. In still further aspects, in the above protein switches, when the analyte-binding domain binds to the analyte, the activity of the oxidase or dehydrogenase increases. In still yet further aspects, when the analyte-binding domain binds to the analyte, the activity of the oxidase or dehydrogenase increases or decreases as a result of competitive inhibition, uncompetitive, or non-competitive inhibition. In still yet another aspect, when the analyte-binding domain binds to the analyte, the activity of the oxidase or dehydrogenase decreases as a result of competitive inhibition.

[0010] In yet another aspect, in any of the above protein switches, the analyte is warfarin, cortisol, methotrexate, or triiodothyronine.

[0011] In still yet a further aspect, in any of the above protein switches, the reactant is glucose or lactate.

[0012] In another embodiment, the present disclosure relates to a protein switch comprising at least seven mutations at amino acid positions 96, 155, 156, 159, 170, 198 and 252 of SEQ ID NO:20.

[0013] In some embodiments, the protein switch comprises at least the following mutations: (a) a cysteine, phenylalanine, methionine, tryptophan, or tyrosine at amino acid position 96 of SEQ ID NO:20; (b) an alanine, glycine, isoleucine, leucine, or valine at amino acid position 155 of SEQ ID NO:20; (c) a threonine or serine at amino acid position 156 of SEQ ID NO:20; (d) a threonine or serine at amino acid position 159 of SEQ ID NO:20; (e) a lysine, arginine, or histidine at amino acid position 170 of SEQ ID NO:20; (f) a glutamic acid or aspartic acid at amino acid position 198 of SEQ ID NO:20; and (g) an alanine, glycine, isoleucine, leucine, or valine at amino acid position 252 of SEQ ID NO:20.In other embodiments, in addition to the mutations (a) through (g) above, the protein switch comprises any of the following mutations: (1) glycine, isoleucine, leucine, or valine at amino acid position 11 of SEQ ID NO:20; (2) glycine, isoleucine, leucine, or valine at amino acid position 22 of SEQ ID NO:20; (3) asparagine or glutamine at amino acid position 45 of SEQ ID NO:20; (4) alanine, glycine, isoleucine, or leucine at amino acid position 48 of SEQ ID NO:20; (5) aspartic acid or glutamic acid at amino acid position 55 of SEQ ID NO:20; (6) alanine, glycine, isoleucine, leucine, or valine at amino acid position 98 of SEQ ID NO:20; (7) phenylalanine, tryptophan, or tyrosine at amino acid position 137 of SEQ ID NO:20; (8) alanine, glycine, leucine, or valine at amino acid position 141 of SEQ ID NO:20; (9) alanine, glycine, isoleucine, or leucine at amino acid position 149 of SEQ ID NO:20; (10) alanine, glycine, isoleucine, or valine at amino acid position 154 of SEQ ID NO:20; (11) threonine or serine at amino acid position 166 of SEQ ID NO:20; (12) glycine, isoleucine, leucine, or valine at amino acid position 173 of SEQ ID NO:20; (13) threonine or serine at amino acid position 184 of SEQ ID NO:20; (14) histidine, leucine, or arginine at amino acid position 195 of SEQ ID NO:20; (15) threonine or serine at amino acid position 219 of SEQ ID NO:20; (16) asparagine or glutamine at amino acid position 240 of SEQ ID NO:20; and / or (17) alanine, glycine, isoleucine, leucine, or valine at amino acid position 251 of SEQ ID NO:20.

[0014] In yet another embodiment, the protein switch comprises seven mutations at amino acid positions 96, 155, 156, 159, 170, 198, and 252 of SEQ ID NO: 20. In these embodiments, the protein switch comprises a cysteine ​​or phenylalanine at amino acid position 96, an alanine at amino acid position 155, a serine at amino acid position 156, a tyrosine at amino acid position 159, a lysine at amino acid position 170, a glutamic acid at amino acid position 198, and a lysine or valine at amino acid position 252 of SEQ ID NO:20.

[0015] In another embodiment, the protein switch comprises eight mutations at amino acid positions 96, 98, 155, 156, 159, 170, 198, and 252 of SEQ ID NO: 20. In these embodiments, the protein switch comprises a cysteine ​​or phenylalanine at amino acid position 96, a leucine at amino acid position 98, an alanine at amino acid position 155, a serine at amino acid position 156, a tyrosine at amino acid position 159, a lysine at amino acid position 170, a glutamic acid at amino acid position 198, and a lysine or valine at amino acid position 252 of SEQ ID NO:20.

[0016] In another embodiment, the protein switch comprises eight mutations at amino acid positions 96, 155, 156, 159, 170, 198, 219, and 252 of SEQ ID NO: 20. In these embodiments, the protein switch comprises a cysteine ​​or phenylalanine at amino acid position 96, an alanine at amino acid position 155, a serine at amino acid position 156, a tyrosine at amino acid position 159, a lysine at amino acid position 170, a glutamic acid at amino acid position 198, a threonine at amino acid position 219, and a lysine or valine at amino acid position 252 of SEQ ID NO:20.

[0017] In another embodiment, the protein switch comprises eight mutations at amino acid positions 96, 155, 156, 159, 170, 195, 198, and 252 of SEQ ID NO: 20. In these embodiments, the protein switch comprises a cysteine ​​or phenylalanine at amino acid position 96, an alanine at amino acid position 155, a serine at amino acid position 156, a tyrosine at amino acid position 159, a lysine at amino acid position 170, an arginine at amino acid position 195, a glutamic acid at amino acid position 198, and a lysine or valine at amino acid position 252 of SEQ ID NO:20.

[0018] In another embodiment, the protein switch comprises eight mutations at amino acid positions 96, 141, 155, 156, 159, 170, 198, and 252 of SEQ ID NO: 20. In these embodiments, the protein switch comprises a cysteine ​​or phenylalanine at amino acid position 96, a valine at amino acid position 141, an alanine at amino acid position 155, a serine at amino acid position 156, a tyrosine at amino acid position 159, a lysine at amino acid position 170, a glutamic acid at amino acid position 198, and a lysine or valine at amino acid position 252 of SEQ ID NO:20.

[0019] In another embodiment, the protein switch comprises eight mutations at amino acid positions 96, 155, 156, 159, 170, 173, 198, and 252 of SEQ ID NO: 20. In these embodiments, the protein switch comprises a cysteine ​​or phenylalanine at amino acid position 96, an alanine at amino acid position 155, a serine at amino acid position 156, a tyrosine at amino acid position 159, a lysine at amino acid position 170, a glycine at amino acid position 173, a glutamic acid at amino acid position 198, and a lysine or valine at amino acid position 252 of SEQ ID NO:20.

[0020] In another embodiment, the protein switch comprises eight mutations at amino acid positions 55, 96, 155, 156, 159, 170, 198, and 252 of SEQ ID NO: 20. In these embodiments, the protein switch comprises a glutamic acid at amino acid position 55, a cysteine ​​or phenylalanine at amino acid position 96, an alanine at amino acid position 155, a serine at amino acid position 156, a tyrosine at amino acid position 159, a lysine at amino acid position 170, a glutamic acid at amino acid position 198, and a lysine or valine at amino acid position 252 of SEQ ID NO:20.

[0021] In another embodiment, the protein switch comprises eight mutations at amino acid positions 96, 137, 155, 156, 159, 170, 198, and 252 of SEQ ID NO: 20. In these embodiments, the protein switch comprises a cysteine ​​or phenylalanine at amino acid position 96, a tyrosine at amino acid position 137, an alanine at amino acid position 155, a serine at amino acid position 156, a tyrosine at amino acid position 159, a lysine at amino acid position 170, a glutamic acid at amino acid position 198, and a lysine or valine at amino acid position 252 of SEQ ID NO:20.

[0022] In another embodiment, the protein switch comprises eight mutations at amino acid positions 48, 96, 155, 156, 159, 170, 198, and 252 of SEQ ID NO: 20. In these embodiments, the protein switch comprises an alanine at amino acid position 48, a cysteine ​​or phenylalanine at amino acid position 96, an alanine at amino acid position 155, a serine at amino acid position 156, a tyrosine at amino acid position 159, a lysine at amino acid position 170, a glutamic acid at amino acid position 198, and a lysine or valine at amino acid position 252 of SEQ ID NO:20.

[0023] In another embodiment, the protein switch comprises eight mutations at amino acid positions 96, 155, 156, 159, 170, 184, 198, and 252 of SEQ ID NO: 20. In these embodiments, the protein switch comprises a cysteine ​​or phenylalanine at amino acid position 96, an alanine at amino acid position 155, a serine at amino acid position 156, a tyrosine at amino acid position 159, a lysine at amino acid position 170, a serine at amino acid position 184, a glutamic acid at amino acid position 198, and a lysine or valine at amino acid position 252 of SEQ ID NO:20.

[0024] In another embodiment, the protein switch comprises eight mutations at amino acid positions 96, 155, 156, 159, 166, 170, 198, and 252 of SEQ ID NO: 20. In these embodiments, the protein switch comprises a cysteine ​​or phenylalanine at amino acid position 96, an alanine at amino acid position 155, a serine at amino acid position 156, a tyrosine at amino acid position 159, a serine at amino acid position 166, a lysine at amino acid position 170, a glutamic acid at amino acid position 198, and a lysine or valine at amino acid position 252 of SEQ ID NO:20.

[0025] In another embodiment, the protein switch comprises eight mutations at amino acid positions 96, 155, 156, 159, 170, 198, 240, and 252 of SEQ ID NO: 20. In these embodiments, the protein switch comprises a cysteine ​​or phenylalanine at amino acid position 96, an alanine at amino acid position 155, a serine at amino acid position 156, a tyrosine at amino acid position 159, a lysine at amino acid position 170, a glutamic acid at amino acid position 198, a glutamine at amino acid position 240, and a lysine or valine at amino acid position 252 of SEQ ID NO:20.

[0026] In still yet other embodiments, the protein switch comprises nine mutations at amino acid positions 45, 96, 149, 155, 156, 159, 170, 198, and 252 of SEQ ID NO: 20. In these embodiments, the protein switch comprises an asparagine at amino acid position 45, a cysteine ​​or phenylalanine at amino acid position 96, an alanine at amino acid position 149, an alanine at amino acid position 155, a serine at amino acid position 156, a tyrosine at amino acid position 159, a lysine at amino acid position 170, a glutamic acid at amino acid position 198, and a lysine or valine at amino acid position 252 of SEQ ID NO:20.

[0027] In still yet other embodiments, the protein switch comprises nine mutations at amino acid positions 22, 96, 154, 155, 156, 159, 170, 198, and 252 of SEQ ID NO: 20. In these embodiments, the protein switch comprises a glycine at amino acid position 22, a cysteine ​​or phenylalanine at amino acid position 96, a glycine at amino acid position 154, an alanine at amino acid position 155, a serine at amino acid position 156, a tyrosine at amino acid position 159, a lysine at amino acid position 170, a glutamic acid at amino acid position 198, and a lysine or valine at amino acid position 252 of SEQ ID NO:20.

[0028] In yet another embodiment, the protein switch comprises nine mutations at amino acid positions 96, 141, 154, 155, 156, 159, 170, 198, and 252 of SEQ ID NO: 20. In these embodiments, the protein switch comprises a cysteine ​​or phenylalanine at amino acid position 96, a valine at amino acid position 141, a glycine at amino acid position 154, an alanine at amino acid position 155, a serine at amino acid position 156, a tyrosine at amino acid position 159, a lysine at amino acid position 170, a glutamic acid at amino acid position 198, and a lysine or valine at amino acid position 252 of SEQ ID NO:20.

[0029] In still yet further embodiments, the protein switch comprises ten mutations at amino acid positions 12, 96, 155, 156, 159, 170, 195, 198, 251, and 252 of SEQ ID NO: 20. In these embodiments, the protein switch comprises a lysine at amino acid position 12, a cysteine ​​or phenylalanine at amino acid position 96, an alanine at amino acid position 155, a serine at amino acid position 156, a tyrosine at amino acid position 159, a lysine at amino acid position 170, an arginine at amino acid position 195, a glutamic acid at amino acid position 198, a lysine at amino acid position 251, and a lysine or valine at amino acid position 252 of SEQ ID NO:20.

[0030] In another embodiment, the present disclosure relates to a protein switch comprising an amino acid sequence having at least 80% identity to SEQ ID NO:11.

[0031] In one embodiment, the protein switch comprises an amino acid sequence having at least 85% identity to SEQ ID NO:11.

[0032] In yet another embodiment, the protein switch comprises an amino acid sequence having at least 90% identity to SEQ ID NO:11.

[0033] In still yet another embodiment, the protein switch comprises an amino acid sequence having at least 95% identity to SEQ ID NO:11.

[0034] In still yet another embodiment, the protein switch comprises an amino acid sequence having at least 96% identity to SEQ ID NO:11.

[0035] In still yet another embodiment, the protein switch comprises an amino acid sequence having at least 97% identity to SEQ ID NO:11.

[0036] In still yet another embodiment, the protein switch comprises an amino acid sequence having at least 98% identity to SEQ ID NO:11.

[0037] In still yet another embodiment, the protein switch comprises an amino acid sequence having at least 99% identity to SEQ ID NO:11.

[0038] In still yet another embodiment, the protein switch comprises an amino acid sequence having at least 100% identity to SEQ ID NO:11.

[0039] In still yet another embodiment, the protein switch comprises the amino acid sequence of any one of SEQ ID NOs: 1-10 or 12-19.

[0040] In yet another embodiment, the present disclosure relates to a composition or kit comprising at least one of the above protein switches and at least one reactive agent. In one aspect, the reactive agent in the composition or kit is glucose or lactate.

[0041] In yet another embodiment, the present disclosure relates to a method for detecting an analyte, the method comprising: a. providing a protein switch comprising at least one polypeptide having (a) at least one analyte binding domain capable of binding to at least one analyte; and (b) at least one oxidase or dehydrogenase domain having oxidase or dehydrogenase activity and capable of binding to or reacting with at least one reactive agent, wherein (i) the analyte that binds to the analyte binding domain is different from the reactive agent that binds to or reacts with the oxidase or dehydrogenase domain; and (ii) the oxidase or dehydrogenase activity is altered upon binding of the analyte to the analyte binding domain; b. contacting the protein switch with a fluid containing a reactive agent specific for the protein switch, wherein the analyte binding domain binds to the analyte in the fluid, thereby altering the oxidase activity or the dehydrogenase activity; and c. detecting a change in the rate of degradation of the reactive agent by the oxidase domain or dehydrogenase domain of the protein switch. Includes.

[0042] In one embodiment of the above method, the oxidase is glucose oxidase or lactate oxidase.

[0043] In another embodiment of the above method, the dehydrogenase is glucose dehydrogenase or lactate dehydrogenase.

[0044] In still yet another aspect of the above method, the reactant is glucose or lactic acid.

[0045] In still yet another embodiment of the above method, the analyte is warfarin, cortisol, methotrexate, or triiodothyronine.

[0046] In yet another embodiment, the present disclosure relates to a method for detecting an analyte, the method comprising: a. providing one of the protein switches described above; b. contacting the protein switch with a fluid containing a reactive agent specific for the protein switch, wherein the analyte binding domain binds to the analyte in the fluid, thereby altering the oxidase activity or the dehydrogenase activity; and c. detecting a change in the rate of degradation of the reactive agent by the oxidase domain or dehydrogenase domain of the protein switch. Includes.

[0047] In one embodiment of the above method, the protein switch comprises an amino acid sequence having at least 80% identity to SEQ ID NO:11.

[0048] In one embodiment of the above method, the protein switch comprises an amino acid sequence having at least 85% identity to SEQ ID NO:11.

[0049] In another embodiment of the above method, the protein switch comprises an amino acid sequence having at least 90% identity to SEQ ID NO:11.

[0050] In yet another embodiment of the above method, the protein switch comprises an amino acid sequence having at least 95% identity to SEQ ID NO:11.

[0051] In still yet another embodiment of any of the above methods, the protein switch comprises an amino acid sequence having at least 96% identity to SEQ ID NO:11.

[0052] In still yet another embodiment of any of the above methods, the protein switch comprises an amino acid sequence having at least 97% identity to SEQ ID NO:11.

[0053] In still yet another embodiment of the above method, the protein switch comprises an amino acid sequence having at least 98% identity to SEQ ID NO:11.

[0054] In still yet another embodiment of any of the above methods, the protein switch comprises an amino acid sequence having at least 99% identity to SEQ ID NO:11.

[0055] In still yet another embodiment of the above method, the protein switch comprises an amino acid sequence having at least 100% identity to SEQ ID NO:11.

[0056] In still another embodiment of the above method, the protein switch comprises the amino acid sequence of any one of SEQ ID NOs: 1-10 or 12-19.

[0057] In still yet another embodiment of the above method, the oxidase is glucose oxidase or lactate oxidase.

[0058] In still yet another embodiment of the above method, the dehydrogenase is glucose dehydrogenase or lactate dehydrogenase.

[0059] In still yet another aspect of the above method, the reactant is glucose or lactic acid.

[0060] In still yet another embodiment of the above method, the analyte is warfarin, cortisol, methotrexate, or triiodothyronine.

[0061] In yet another embodiment, the present disclosure relates to a system for detecting or monitoring an analyte concentration, the system including a sensor control device and a signal detection device, where the sensor control device includes at least one sensor that includes one of the protein switches described above.

[0062] In one embodiment, the oxidase in the protein switch used in the sensor is glucose oxidase or lactate oxidase.

[0063] In another embodiment, the dehydrogenase in the protein switch used in the sensor is glucose dehydrogenase or lactate dehydrogenase.

[0064] In yet another embodiment, the present disclosure relates to an analyte monitoring system, comprising a sensor including a substrate, one or more working electrodes, and one of the protein switches described above, at least a portion of the sensor adapted for implantation and in intimate contact with a bodily fluid, the sensor constructed and arranged to provide a signal representative of a level of an analyte in the bodily fluid; and a signal detection device for receiving the signal, the signal generated by contact of the analyte with the protein switch.

[0065] In one embodiment, the oxidase in the protein switch used in the analyte monitoring system is glucose oxidase or lactate oxidase.

[0066] In another embodiment, the dehydrogenase in the protein switch used in the analyte monitoring system is glucose dehydrogenase or lactate dehydrogenase.

[0067] In still further embodiments, the analyte detected in the analyte monitoring system is warfarin, cortisol, methotrexate, or triiodothyronine. [Brief explanation of the drawings]

[0068] [Figure 1] FIG. 1 shows a schematic diagram of an exemplary sensing system incorporating a sensor comprising at least one protein switch of the present disclosure. [Figure 2A] FIG. 1 shows a schematic of an exemplary two-electrode sensor configuration having a single working electrode. [Figure 2B] FIG. 1 shows a schematic of an exemplary three-electrode sensor configuration with a single working electrode. [Figure 2C] FIG. 1 shows a schematic of an exemplary three-electrode sensor configuration with a single working electrode. [Figure 3]FIG. 1 shows a schematic of an exemplary sensor configuration having two working electrodes, a reference electrode, and a counter electrode. [Figure 4] FIG. 1 shows an exemplary sensor configuration in which two distinct active regions are disposed on the surface of a single working electrode. [Figure 5A] FIG. 1 shows a schematic of an exemplary working electrode in which a first active region is disposed directly on the surface of the working electrode and a second active region is separated from the working electrode by a membrane. [Figure 5B] FIG. 1 shows a schematic of an exemplary working electrode in which a first active region is disposed directly on the surface of the working electrode and a second active region is separated from the working electrode by a membrane. [Figure 6] FIG. 1 shows an exemplary schematic diagram of a portion of a sensor having two working electrodes and featuring a bilayer membrane overcoating one of the two working electrodes. [Figure 7] 1 shows the linear sensitivity response to increasing glucose concentrations of sensors containing the protein switches GDH-2004, GDH-2007, GDH-2008, GDH-2009, GDH-2010, GDH-2011, GDH-2015, GDH-2018, GDH-2019, GDH-2005, GDH-2013, GDH-2016, and GDH-2024. The sensor containing GDH-105 is a control. [Figure 8-1] Figure 1 shows the in-beaker (long-term) stability of sensors containing the protein switches GDH-2004, GDH-2007, GDH-2008, GDH-2009, GDH-2010, GDH-2011, GDH-2015, GDH-2018, GDH-2019, GDH-2005, GDH-2013, GDH-2016, and GDH-2024. A sensor containing GDH-105 serves as a control. [Figure 8-2]Figure 1 shows the in-beaker (long-term) stability of sensors containing the protein switches GDH-2004, GDH-2007, GDH-2008, GDH-2009, GDH-2010, GDH-2011, GDH-2015, GDH-2018, GDH-2019, GDH-2005, GDH-2013, GDH-2016, and GDH-2024. A sensor containing GDH-105 serves as a control. [Figure 8-3] Figure 1 shows the in-beaker (long-term) stability of sensors containing the protein switches GDH-2004, GDH-2007, GDH-2008, GDH-2009, GDH-2010, GDH-2011, GDH-2015, GDH-2018, GDH-2019, GDH-2005, GDH-2013, GDH-2016, and GDH-2024. A sensor containing GDH-105 serves as a control. [Figure 9A] FIG. 1 shows the inhibition of sensors containing the protein switch, GDH-2016, to increasing concentrations of warfarin, compared to sensors containing GDH-105 (control), which were unaffected. [Figure 9B] FIG. 1 shows the inhibition of sensors containing the protein switch, GDH-2016, to increasing concentrations of warfarin, compared to sensors containing GDH-105 (control), which were unaffected. [Figure 10] FIG. 1 shows the linear sensitivity response of sensors containing GDH-105 (control) and proteins switches GDH-2025, GDH-2026, GDH-2027 and GDH-2028 based on in-beaker calibration. [Figure 11] 1 shows the in-beaker (long-term) stability of sensors containing the protein switches GDH-2025, GDH-2026, GDH-2027, and GDH-2028. A sensor containing GDH-105 serves as a control. [Figure 12A]1 shows the inhibition of the protein switches GDH-2016-containing sensors, GDH-2025, GDH-2026, GDH-2027, and GDH-20288 with increasing concentrations of warfarin. The sensor containing GDH-105 is a control. [Figure 12B] 1 shows the inhibition of the protein switches GDH-2016-containing sensors, GDH-2025, GDH-2026, GDH-2027, and GDH-20288 with increasing concentrations of warfarin. The sensor containing GDH-105 is a control. [Figure 13] 1 shows sensors containing inhibitors GDH-2016, GDH-2025, GDH-2026, GDH-2027, and GDH-20288 in response to increasing concentrations of warfarin. Sensors containing GDH-105 are controls. [Figure 14-1] FIG. 1 shows the sequences of SEQ ID NOs: 1 to 20. [Figure 14-2] FIG. 1 shows the sequences of SEQ ID NOs: 1 to 20. DETAILED DESCRIPTION OF THE INVENTION

[0069] Exemplary embodiments of compositions, methods, devices (e.g., sensors), systems, and apparatus for detecting and / or monitoring are presented herein. It is to be understood that the teachings of the present disclosure are not limited to the particular embodiments described and, as such, can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0070] By way of example and not limitation, a protein switch can be designed to recognize, identify, and / or quantify one or more target analytes in a bodily fluid or biological sample. The exact nature and configuration of the protein switch can vary. The protein switch can have an analyte-binding element for detection of the target analyte and an enzymatic element that generates a product (e.g., a signal) or a change in the generated product (e.g., a change in the signal) upon binding of the analyte. The protein switch can be utilized under a variety of conditions and configurations, including in sensors for measuring the level of an analyte in a subject. The configuration of such sensors can depend on the analyte being measured and the bodily fluid the device is monitoring for the analyte. The sensor can be configured to detect and / or measure the analyte in vivo in a subject. An in vivo sensor can include an insertion tip that can be placed below the surface of the skin, for example, penetrating through the skin into the dermal or subcutaneous region. The sensor can interrogate the analyte in intradermal fluid, interstitial fluid, subcutaneous fluid, or blood (e.g., capillary blood).

[0071] A sensor can include one or more protein switches that bind to an analyte (at an analyte-binding element or moiety) and then generate a product (e.g., a signal from an enzymatic element or moiety) that can be detected by the sensor. The protein switch within the sensor can have one level of activity (e.g., low activity or high activity) when the analyte is not bound and a different activity (e.g., high activity or low activity) when the analyte is bound. For example, analyte binding by the analyte-binding portion of the protein switch can decrease enzymatic activity. Alternatively, analyte binding by the analyte-binding portion of the protein switch can increase enzymatic activity. The change in enzymatic activity can be detected by the sensor (directly or indirectly) as a change in signal based on the amount of product produced by the enzyme. The signal detected by the sensor can be correlated to the amount of analyte, which can be prognostic or diagnostic for the health and / or well-being of a patient. Alternatively, the signal detected by the sensor may be correlated to the amount of the analyte, which is used to monitor the health and / or well-being of the patient. The amount of the analyte measured may also be correlated to and / or converted to the amount in blood or other bodily fluids.

[0072] A.Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In the case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below; however, methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this disclosure. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and are not intended to be limiting.

[0073] As used herein, the terms "comprise," "having," "having," "may," "containing," and variations thereof are intended to be open-ended transitional phrases, terms, or phrases that do not exclude additional acts or structures. Unless the context clearly indicates otherwise, the singular forms "a," "an," and "the" include plural referents. Whether expressly stated or not, the present disclosure also "comprising," "consisting of," "consisting essentially of," or "consisting essentially of" any aspect or element presented herein. Other embodiments are envisioned.

[0074] For the recitation of numerical ranges herein, each intervening number is expressly contemplated with the same degree of precision. For example, for the range of 6 to 9, the numbers 7 and 8 are also contemplated in addition to 6 and 9, and for the range of 6.0 to 7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are also expressly contemplated.

[0075] As used herein, "affimer" generally refers to a peptide that specifically or selectively binds to a target (e.g., an analyte, target tissue, target molecule, target cell, etc.). In general, affimers can be small peptides or proteins, generally having a molecular weight of less than 12 kDa. Although affimers can have the ability to recognize a specific epitope or antigen and do so with a binding affinity that can approach that of an antibody (e.g., in the low nanomolar to picomolar range), the term "affimer" as used herein does not encompass antibodies, immunoglobulins, the Fab region of an antibody, or the Fc region of an antibody. Affimers can have the advantage of the same specificity as antibodies, but can be small, may be chemically synthesized or chemically modified, and may have the advantage of being free of cell culture contaminants.

[0076] As used interchangeably herein, a "sensor," "in vivo sensor," or "sensor" refers to a device configured to detect the presence and / or measure the level of one or more (e.g., multiple) analytes in a sample via electrochemical oxidation and reduction reactions on the sensor. These reactions are converted into an electrical signal that can be correlated to the amount, concentration, or level of the analyte or enzymatic activity in the sample.

[0077] "Enzyme" refers to a protein or fragment thereof that has activity (e.g., catalytic activity, enzymatic activity) toward one or more reactants (e.g., enzyme substrates). Examples of the one or more reactants (e.g., enzyme substrates) are glucose, lactate, glutamate, ascorbic acid, cholesterol, choline, acetylcholine, hypoxanthine, norepinephrine, 5-hydroxytryptamine, phenylethylamine, and e / e-methylhistamine, polyphenols, ethanol, aldehydes, or malate.

[0078] As used interchangeably herein, "fluid," "body fluid," "sample," or "biological sample" refers to intradermal, interstitial, or subcutaneous fluid, or blood (e.g., capillary blood, etc.) obtained from a subject or patient. In one embodiment, the fluid or sample is intradermal fluid. In one embodiment, the fluid or sample is interstitial fluid. In yet another embodiment, the fluid is interstitial fluid. In yet another embodiment, the fluid or sample is blood (e.g., capillary blood).

[0079] As used herein in the context of two or more polypeptide or polynucleotide sequences, "identical," "identity," or "sequence identity" can mean that the sequences have a specified percentage of the same residues over a specified region, as determined using standard algorithms and considering the extent to which the sequences are identical over the comparison region. Identity or sequence identity can be determined using computer algorithms, such as the GAP, BESTFIT, FASTA, and BLAST family of programs disclosed by Altschul et al., 1997, Nucl. Acids Res., 25, 3389. A detailed discussion of sequence analysis can be found in Unit 19.3 of "CURRENT PROTOCOLS IN MOLECULAR BIOLOGY," edited by Ausubel et al. (John Wiley & Sons Inc., NY, 1995-1999). Sequence identity is suitably measured over the entire length of SEQ ID NOs: 1-20.

[0080] As used herein, an "isolated polynucleotide" can mean a polynucleotide that, by virtue of its origin, is not associated with all or part of the polynucleotide with which it is found in nature (e.g., a polynucleotide of genomic, cDNA, or synthetic origin, or a combination thereof).

[0081] As used herein, "sensing layer" refers to a component of a sensor that includes a constituent that promotes the electronic oxidation or electronic reduction of a compound, either directly at an electrode or via one or more electron transfer agents, one or more cofactors, or a combination of one or more electron transfer agents and one or more cofactors. In some sensor embodiments, the sensing layer is disposed adjacent to or on the working electrode.

[0082] As used herein, "sensing region" refers to the active chemical area of ​​a sensor.

[0083] "Subject" or "patient," as used interchangeably herein, refers to any vertebrate, including, but not limited to, mammals (e.g., cows, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats and mice, non-human primates (e.g., monkeys such as cynomolgus or rhesus monkeys, chimpanzees), and humans). In some embodiments, the subject can be human or non-human. In some embodiments, the subject is human. The subject or patient may be undergoing one or more forms of treatment.

[0084] The terms "variant protein," "protein variant," or "variant," used interchangeably herein, refer to a protein that differs from a parent protein by at least one amino acid modification. The term "protein variant" may refer to the protein itself, a composition comprising the protein, or the amino acid sequence encoding it. In some embodiments, a protein variant has at least one amino acid modification compared to a parent or reference protein, e.g., about one to about 50 amino acid modifications compared to the parent protein. In some embodiments, a protein variant has about one to about 40 amino acid modifications compared to the parent protein. In some embodiments, a protein variant has about one to about 30 amino acid modifications compared to the parent protein. In some embodiments, a protein variant has about one to about 20 amino acid modifications compared to the parent protein. In some embodiments, a protein variant has about one to about 10 amino acid modifications compared to the parent protein. In some embodiments, a protein variant has about one to about five amino acid modifications compared to the parent protein. In some embodiments, a protein variant sequence herein retains at least about 80% identity with the parent protein sequence. In other embodiments, the protein variant sequences herein share at least about 90% identity, hi still other embodiments, the protein variant sequences share at least about 95%, 96%, 97%, 98%, or 99% identity.

[0085] In this disclosure, references are made to amino acids. In addition to the names of amino acids, three-letter and one-letter codes are also used herein. For clarity, the amino acids referred to in this disclosure are referred to as follows: alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamine (Qln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V).

[0086] Unless otherwise specified herein, scientific and technical terms used in connection with this disclosure shall have the same meaning as commonly understood by those skilled in the art. For example, the terminology and techniques used in connection with cell and tissue culture methods, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are well-known terminology and techniques commonly used in the art. The meaning and scope of terms shall be clear, but in the event of any potential ambiguity, the definitions provided herein shall take precedence over any dictionary or external definitions. Furthermore, unless otherwise required by context, singular terms shall include the plural, and plural terms shall include the singular.

[0087] B. Protein Switch In some embodiments, the present disclosure relates to compositions that can include one or more protein switches. Specifically, a protein switch can include, but is not limited to, at least two elements: at least one analyte-binding element (or portion) and at least one enzymatic element (or portion). The analyte-binding element modulates the activity of the enzymatic element of the protein switch. As discussed in more detail herein, when an analyte binds to the analyte-binding element, the activity of the enzymatic element is altered (e.g., increased or decreased), for example, as a result of competitive inhibition, uncompetitive inhibition, or non-competitive inhibition. In some embodiments, when an analyte binds to the analyte-binding element, the activity of the enzymatic element increases (compared to the absence of the analyte). In other embodiments, when an analyte binds to the analyte-binding element, the activity of the enzymatic element decreases (compared to the absence of the analyte). Such modulation of enzymatic activity can result in the detection of the analyte.

[0088] The protein switch of the present disclosure comprises at least one non-naturally occurring polypeptide having at least two distinct domains or portions. In some embodiments, the at least two distinct domains or portions may overlap with each other in terms of their sequences (e.g., overlapping, containing one or more nucleic acid sequences or amino acid sequences) or their spatial orientation. The first domain or portion is an analyte-binding element (or binding portion) comprising at least one analyte-binding domain. The at least one analyte-binding domain binds to or is capable of binding to one or more analytes of interest. The second domain or portion is an enzymatic element comprising at least one oxidase or dehydrogenase domain having enzymatic activity (e.g., catalytic activity). In other words, the second domain or portion is catalytically active. Additionally, the oxidase or dehydrogenase domain may bind to, react with, be capable of binding to, and / or react with one or more reactants and optionally one or more cofactors that create or result in a detectable product (e.g., a signal), as discussed in more detail herein.

[0089] In one embodiment, in the presence of an analyte (e.g., when at least one analyte binds to the analyte-binding domain), the oxidase or dehydrogenase domain binds to and / or reacts with at least one reactive agent, and the activity of the enzyme (e.g., oxidase or dehydrogenase) increases. Specifically, the amount of product (e.g., signal) produced increases. The activity of the enzyme is about 0.1%, about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53% (compared to the wild-type enzyme). %, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100%.

[0090] In still yet another embodiment, in the presence of an analyte (e.g., when the analyte-binding region binds to one or more analytes), the oxidase domain or dehydrogenase domain may not bind to or react with one or more reactive agents, or may exhibit reduced (e.g., inhibition) binding and / or reaction with the reactive agent, and the activity of the enzyme (e.g., oxidase or dehydrogenase) may be decreased. Specifically, the amount of product (e.g., signal) produced may be decreased. The activity of the enzyme (e.g., oxidase or dehydrogenase) may be reduced by about 0.1%, about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, or more (compared to the wild-type enzyme). ,approximately 19%,approximately 20%,approximately 21%,approximately 22%,approximately 23%,approximately 24%,approximately 25%,approximately 26%,approximately 27%,approximately 28%,approximately 29%,approximately 30%,approximately 40%,approximately 41%,approximately 42%,approximately 43%,approximately 44%,approximately 45%,approximately 46%,approximately 47%,approximately 48%,approximately 49%,approximately 50%,approximately 51%,approximately 52%,approximately 53%,approximately 54%,approximately 55%, approximately 56%, approximately 57%, approximately 58%, approximately 59%, approximately 60%, approximately 61%, approximately 62%, approximately 63%, approximately 64%, approximately 65%, approximately 66%, approximately 67%, approximately 68%, approximately 69%, approximately 70%, approximately 71%, approximately 72%, approximately 73%, approximately 74%, approximately 75%, approximately 76%, approximately 77%, approximately 78%, approximately 79%, approximately 80%, approximately 81%, approximately 82% %, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% (e.g., complete inhibition of enzyme (e.g., oxidase or dehydrogenase) activity).

[0091] In one embodiment, the analyte-binding domain can be derived from any protein or polypeptide that binds or is capable of binding to one or more analytes of interest. For example, in one embodiment, the analyte-binding domain can be derived from one or more affimers, antibodies, peptides, receptors (including full-length receptors and single-chain receptors), small molecules, artificial binding proteins (such as artificial binding proteins generated using scaffolding technologies, display technologies, etc.), or any functional fragment or variant thereof. In another embodiment, the analyte-binding domain can be derived from a polypeptide or protein (such as an oxidase or dehydrogenase) that has been mutated and / or modified, such as to contain one or more amino acid mutations, deletions, substitutions, and / or truncations. In some embodiments, the binding affinity of the analyte-binding domain for the analyte enables the protein switch to detect one or more analytes at physiological levels.

[0092] The length of the analyte binding domain is not critical, provided that the analyte binding domain binds or is capable of binding to one or more analytes of interest. For example, the analyte binding domain may be 3 consecutive amino acids, 4 consecutive amino acids, 5 consecutive amino acids, 6 consecutive amino acids, 7 consecutive amino acids, 8 consecutive amino acids, 9 consecutive amino acids, 10 consecutive amino acids, 11 consecutive amino acids, 12 consecutive amino acids, 13 consecutive amino acids, 14 consecutive amino acids, 15 consecutive amino acids, 16 consecutive amino acids, 17 consecutive amino acids, 18 consecutive amino acids, 19 consecutive amino acids, 20 consecutive amino acids, 21 consecutive amino acids, 22 consecutive amino acids, 23 consecutive amino acids, 24 consecutive amino acids, 25 consecutive amino acids, 26 consecutive amino acids, 27 consecutive amino acids, 28 consecutive amino acids, 29 consecutive amino acids, 30 consecutive amino acids, 31 consecutive amino acids, 32 consecutive amino acids, 33 consecutive amino acids, 34 consecutive amino acids, 35 consecutive amino acids, 36 consecutive amino acids, 37 consecutive amino acids, 38 consecutive amino acids, 39 consecutive amino acids, or the like. The length may be 40 contiguous amino acids, 41 contiguous amino acids, 42 contiguous amino acids, 43 contiguous amino acids, 44 contiguous amino acids, 45 contiguous amino acids, 46 contiguous amino acids, 47 contiguous amino acids, 48 ​​contiguous amino acids, 49 contiguous amino acids, 50 contiguous amino acids, 75 contiguous amino acids, 80 contiguous amino acids, 85 contiguous amino acids, 90 contiguous amino acids, 95 contiguous amino acids, 100 contiguous amino acids, 125 contiguous amino acids, 150 contiguous amino acids, 175 contiguous amino acids, 200 contiguous amino acids, 225 contiguous amino acids, 250 contiguous amino acids, 275 contiguous amino acids, 300 contiguous amino acids, 325 contiguous amino acids, 350 contiguous amino acids, 375 contiguous amino acids, 400 contiguous amino acids, 425 contiguous amino acids, 450 contiguous amino acids, 475 contiguous amino acids, or 500 contiguous amino acids.

[0093] In another aspect, at least one oxidase domain or portion or dehydrogenase domain or portion comprises an amino acid sequence encoding at least one oxidase and / or at least one dehydrogenase having enzymatic activity (e.g., catalytic activity) and capable of binding to, reacting with, or binding and / or reacting with at least one reactant and optional cofactor to produce a product (e.g., a signal). The amino acid sequence encoding the oxidase or dehydrogenase can be a naturally occurring (wild-type) sequence derived, obtained, and / or synthesized from one or more microorganisms, such as bacteria, viruses, or fungi, or mammalian cells. The amino acid sequence can encode the entire enzyme or a functional fragment thereof, provided that the functional fragment has enzymatic activity. Alternatively, the amino acid sequence of the oxidase or dehydrogenase domain can be a variant of a naturally occurring (wild-type) sequence encoding an oxidase or dehydrogenase, wherein the variant has enzymatic activity. As with the analyte binding domain, the length of the oxidase or dehydrogenase domain is not critical, provided that it encodes an enzymatic (e.g., whole enzyme), functional fragment, or variant thereof that has enzymatic (e.g., oxidase or dehydrogenase) activity.For example, the oxidase domain or dehydrogenase domain may be 3 consecutive amino acids, 4 consecutive amino acids, 5 consecutive amino acids, 6 consecutive amino acids, 7 consecutive amino acids, 8 consecutive amino acids, 9 consecutive amino acids, 10 consecutive amino acids, 11 consecutive amino acids, 12 consecutive amino acids, 13 consecutive amino acids, 14 consecutive amino acids, 15 consecutive amino acids, 16 consecutive amino acids, 17 consecutive amino acids, 18 consecutive amino acids, 19 consecutive amino acids, 20 consecutive amino acids, 21 consecutive amino acids, 22 consecutive amino acids, 23 consecutive amino acids, 24 consecutive amino acids, 25 consecutive amino acids, 26 consecutive amino acids, 27 consecutive amino acids, 28 consecutive amino acids, 29 consecutive amino acids, 30 consecutive amino acids, 31 consecutive amino acids, 32 consecutive amino acids, 33 consecutive amino acids, 34 consecutive amino acids, 35 consecutive amino acids, 36 consecutive amino acids, 37 consecutive amino acids, 38 consecutive amino acids, 39 consecutive amino acids, 40 consecutive amino acids, 41 consecutive amino acids, 42 consecutive amino acids, 43 consecutive amino acids, 44 consecutive amino acids, 45 consecutive amino acids, 46 consecutive amino acids, 47 consecutive amino acids, 48 ​​consecutive amino acids, 49 consecutive amino acids, 50 consecutive amino acids, 51 consecutive amino acids, 52 consecutive amino acids, 53 consecutive amino acids, 54 consecutive amino acids, 55 consecutive amino acids, 56 consecutive amino acids, 57 consecutive amino acids, 58 consecutive amino acids, 59 consecutive amino acids, 60 consecutive amino acids, 61 consecutive amino acids, 62 consecutive amino acids, 63 consecutive amino acids, 64 consecutive amino acids, 65 consecutive amino acids, 66 consecutive amino acids, 67 consecutive amino acids, 68 consecutive amino acids, 69 consecutive amino acids, 70 consecutive amino acids, 71 consecutive amino acids, 72 consecutive amino acids, 73 consecutive amino acids, 74 consecutive amino The length may be 39 contiguous amino acids, 40 contiguous amino acids, 41 contiguous amino acids, 42 contiguous amino acids, 43 contiguous amino acids, 44 contiguous amino acids, 45 contiguous amino acids, 46 contiguous amino acids, 47 contiguous amino acids, 48 ​​contiguous amino acids, 49 contiguous amino acids, 50 contiguous amino acids, 75 contiguous amino acids, 80 contiguous amino acids, 85 contiguous amino acids, 90 contiguous amino acids, 95 contiguous amino acids, 100 contiguous amino acids, 125 contiguous amino acids, 150 contiguous amino acids, 175 contiguous amino acids, 200 contiguous amino acids, 225 contiguous amino acids, 250 contiguous amino acids, 275 contiguous amino acids, 300 contiguous amino acids, 325 contiguous amino acids, 350 contiguous amino acids, 375 contiguous amino acids, 400 contiguous amino acids, 425 contiguous amino acids, 450 contiguous amino acids, 475 contiguous amino acids, or 500 contiguous amino acids.

[0094] Enzymes that may be used in at least one oxidase domain or dehydrogenase domain include (i) one or more oxidases, such as, for example, glucose oxidase or lactate oxidase; (ii) one or more dehydrogenases, such as, for example, glucose dehydrogenase or lactate dehydrogenase; or (iii) any combination of (i) and (ii). In some embodiments, the oxidase is glucose oxidase. In other embodiments, the oxidase is lactate oxidase. In some embodiments, the oxidase is glutamate oxidase. In still further embodiments, the dehydrogenase is glucose dehydrogenase. In still further embodiments, the dehydrogenase is lactate dehydrogenase. In yet other embodiments, the dehydrogenase is glutamate dehydrogenase.

[0095] The oxidases and / or dehydrogenases used in the oxidase or dehydrogenase domains are derived from or encoded by microorganisms such as bacteria, viruses, or fungi. Examples of sources for oxidase or dehydrogenase domains used in the protein switches described herein are provided in Tables 1 and 2 below.

[0096] [Table 1]

[0097] [Table 2]

[0098] Methods for using the oxidases and dehydrogenases in Tables 1 and 2 in combination with one or more reactants (and one or more cofactors, such as, for example, pyroquinoline quinone (PQQ), flavin adenine dinucleotide (FAD), nicotinamide adenine dinucleotide (NAD), flavin mononucleotide (FMN), etc.) to result in or generate a detectable product (e.g., a signal) are well known in the art. By way of example, disclosed herein are methods for generating a detectable product with glucose oxidase and glucose dehydrogenase.

[0099] The enzyme glucose oxidase is composed of two identical protein subunits and a cofactor (i.e., flavin adenine dinucleotide (FAD)) in its active site. Together with FAD, glucose oxidase utilizes molecular oxygen as an electron acceptor to catalyze the oxidation of its reactant, glucose, at its primary hydroxyl group to produce the products gluconolactone and hydrogen peroxide. The resulting hydrogen peroxide product can be detected using conventional techniques known in the art (e.g., electrochemical oxidation at an electrode and detecting electron transfer). The reaction is as follows: Glucose + glucose oxidase (GOx)-FAD + → Gluconolactone + GOx-FADH2 GOx-FADH2+O2→GOx-FAD+H2O2 H2O2→2H + +O2+2e - can be summarized as shown in

[0100] Alternatively, oxygen consumption can be measured.

[0101] Glucose dehydrogenase can utilize a number of different cofactors (e.g., NAD, PQQ, etc.). When NAD is used as a cofactor, glucose dehydrogenase catalyzes the oxidation of glucose to produce gluconolactone and NADH. At the electrode, NADH is electrochemically oxidized and the number of electrons transferred is detected. The reaction is as follows: Glucose + glucose dehydrogenase (GDH) - NAD + → Gluconolactone + GDH-NADH NADH → NAD + +H + +2e - can be summarized as shown in

[0102] The target analyte detected by the protein switch may be prognostic or diagnostic for the health and / or well-being of a patient. Alternatively, or additionally, the target analyte may be used to monitor the health and / or well-being of a patient. For example, the analyte may be procalcitonin (PCT), cardiac troponin (such as cardiac troponin I or cardiac troponin T), creatine, urea, guanidinosuccinic acid, para-cresol sulfate, indicator symptoms, dimethylamine, CMPF, pseudouridine, oxalate, glyoxal, 2-oxoglutarate, glucose, lactate, cerebrospinal fluid glucose, glutamate, malate, acetylcarnitine, hypoxanthine, glycerophosphocholine, acetylneuraminic acid, inosine, pseudouridine, hydroxyphenyllactic acid, hexanoylcarnitine, neuropeptide Y, orexin A, calcitonin gene-related peptide, serotonin, brain-derived neurotrophic factor (BDNF), gamma-aminobutyric acid (GABA), dopamine, N-methyl-D-aspartic acid (NMDA), orthophosphate (ATP), orthophosphate (ATP). DA), docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), lysophosphatidylcholine (22:6 and 20:5), lysophosphatidylethanolamine (22:6 and 20:5), 3-carboxy-40-methyl-5-propyl-2-furanopropanoic acid (CMPF), acetylcholine, cortisol, estrogen, estriol, estrone, progesterone, oxytocin, follicle-stimulating hormone (FSH), luteinizing hormone (LH), thyroid-stimulating hormone (TSH), triiodothyronine (T3), thyroxine (T4), triiodothyroxine, human growth hormone, cytokines, chemokines, interleukins, procalcitonin, blood coagulation factors, C-reactive protein (CRP), procalcitonin, sTREM-1 (soluble These may be triggering receptor expressed on myeloid cells-1), pancreatic stone protein (PSP), circulating complement (C3 and C4), ferritin, cholesterol, albumin, and neutrophil gelatinase-associated lipocalin.Analyte diagnostics for cardiac function or disease include, for example, cardiac troponin, 2-oxoglutarate, creatine kinase (CK-MB), glycogen phosphorylase isoenzyme BD, BNP, myoglobin, ischemia-modified albumin, cardiac natriuretic peptide, and / or lactate dehydrogenase isoenzymes. Analytes can also be nucleic acids (e.g., microRNAs, CpG islands, and other nucleic acid markers in plasma or serum), polypeptides, metabolites, lipids, carbohydrates, or other molecules found in a subject and can be diagnostic for the subject's health and / or well-being. Analyte diagnostics for kidney function or disease include, for example, creatine and urea. Analyte diagnostics for liver function or disease include, for example, glucose, urea, albumin, and creatine. Analyte diagnostics for neurological function or disease include GFAP, UCH-L1, S100B, and NF-L. Analytes diagnostic for infectious diseases and / or sepsis include, for example, lactate, cerebrospinal fluid glucose, glutamate, malate, acetylcarnitine, hypoxanthine, glycerophosphocholine, acetylneuraminic acid, inosine, pseudouridine, hydroxyphenyllactic acid, hexanoylcarnitine, C-reactive protein (CRP), procalcitonin, sTREM-1 (soluble triggering receptor expressed on myeloid cells-1), pancreatic stone protein (PSP), circulating complement (C3 and C4), ferritin, cholesterol, albumin, cortisol, and neutrophil gelatinase-associated lipocalin. Further analytes include drugs or drug metabolites, such as warfarin, methotrexate, cyclosporin A, methotrexate, and vasopressin. In one embodiment, the analyte is warfarin, cortisol, methotrexate, triiodothyronine, cyclosporin A, GFAP, UCH-L1, S100B, NF-L, or a cardiac troponin. In yet another embodiment, the analyte is warfarin. In yet another embodiment, the analyte is cortisol. In yet a further embodiment, the analyte is methotrexate. In yet a further embodiment, the analyte is triiodothyronine.In still further embodiments, the analyte is cyclosporin A. In still further embodiments, the analyte is GFAP. In still further embodiments, the analyte is UCH-L1. In still further embodiments, the analyte is S100B. In still even further embodiments, the analyte is NF-L. In still further embodiments, the analyte is a cardiac troponin (e.g., troponin I or troponin T). In still further embodiments, the analyte is cardiac troponin I.

[0103] The analyte that binds to the analyte binding domain of the protein switch must be different from (e.g., cannot be the same as) the reactive agent that binds to and / or reacts with the oxidase or dehydrogenase domain. For example, if the analyte that binds to the analyte binding domain is glucose, the reactive agent that binds to or reacts with the oxidase or dehydrogenase domain cannot be glucose but must be a different reactive agent, such as lactate. Alternatively, if the analyte that binds to the analyte binding domain of the protein switch is warfarin, the reactive agent that binds to or reacts with the oxidase or dehydrogenase can be glucose or lactate.

[0104] As previously discussed herein, when an analyte binds to the analyte-binding domain, the oxidase or dehydrogenase activity is altered. In some embodiments, when an analyte binds to the analyte-binding domain, the oxidase or dehydrogenase activity increases (e.g., the amount of product produced increases). In other embodiments, when an analyte binds to the analyte-binding domain, the oxidase or dehydrogenase activity decreases (e.g., the amount of product produced decreases).

[0105] Any reactive agent known in the art for pairing with an enzyme is suitable for use with the protein switches described herein. Examples of reactive agents that can be used include glucose or lactate. In some embodiments, the reactive agent is glucose. In other embodiments, the reactive agent is lactate.

[0106] In another embodiment, the protein switch of the present disclosure is a glucose dehydrogenase polypeptide. In yet another embodiment, the protein switch has the amino acid sequence:

[0107] [ka] A glucose dehydrogenase polypeptide having at least seven mutations within the

[0108] In one embodiment, the protein switch comprises at least seven mutations at amino acid positions 96, 155, 156, 159, 170, 198, and 252 of SEQ ID NO: 20. Mutations that can be made at amino acid positions 96, 155, 156, 159, 170, 198, and 252 of SEQ ID NO: 20 are shown in Table 3A below.

[0109] [Table 3]

[0110] In another embodiment, the protein switch comprises mutations at amino acid positions 96, 155, 156, 159, 170, 198, and 252 of SEQ ID NO:20, as shown in Table 3A above, as well as at least one mutation at amino acid positions 11, 22, 45, 48, 55, 98, 137, 141, 149, 154, 166, 173, 184, 195, 219, 240, and / or 251 of SEQ ID NO:20, as shown in Table 3B below.

[0111] [Table 4]

[0112] In yet another embodiment, the protein switch comprises seven mutations at amino acid positions 96, 155, 156, 159, 170, 198 and 252 of SEQ ID NO:20.

[0113] In another embodiment, the protein switch comprises eight mutations at amino acid positions 96, 98, 155, 156, 159, 170, 198, and 252 of SEQ ID NO: 20. In another embodiment, the protein switch comprises eight mutations at amino acid positions 96, 155, 156, 159, 170, 198, 219, and 252 of SEQ ID NO: 20. In another embodiment, the protein switch comprises eight mutations at amino acid positions 96, 155, 156, 159, 170, 195, 198, and 252 of SEQ ID NO: 20. In another embodiment, the protein switch comprises eight mutations at amino acid positions 96, 141, 155, 156, 159, 170, 198, and 252 of SEQ ID NO: 20. In another embodiment, the protein switch comprises eight mutations at amino acid positions 96, 155, 156, 159, 170, 173, 198, and 252 of SEQ ID NO: 20. In another embodiment, the protein switch comprises eight mutations at amino acid positions 55, 96, 155, 156, 159, 170, 198, and 252 of SEQ ID NO: 20. In another embodiment, the protein switch comprises eight mutations at amino acid positions 96, 137, 155, 156, 159, 170, 198, and 252 of SEQ ID NO: 20. In another embodiment, the protein switch comprises eight mutations at amino acid positions 48, 96, 155, 156, 159, 170, 198, and 252 of SEQ ID NO: 20. In another embodiment, the protein switch comprises eight mutations at amino acid positions 96, 155, 156, 159, 170, 184, 198, and 252 of SEQ ID NO: 20. In another embodiment, the protein switch comprises eight mutations at amino acid positions 96, 155, 156, 159, 166, 170, 198, and 252 of SEQ ID NO: 20. In another embodiment, the protein switch comprises eight mutations at amino acid positions 96, 155, 156, 159, 170, 198, 240, and 252 of SEQ ID NO: 20.

[0114] In yet another embodiment, the protein switch comprises nine mutations at amino acid positions 45, 96, 149, 155, 156, 159, 170, 198, and 252 of SEQ ID NO: 20. In yet another embodiment, the protein switch comprises nine mutations at amino acid positions 22, 96, 154, 155, 156, 159, 170, 198, and 252 of SEQ ID NO: 20. In yet another embodiment, the protein switch comprises nine mutations at amino acid positions 96, 141, 154, 155, 156, 159, 170, 198, and 252 of SEQ ID NO: 20.

[0115] In still yet a further embodiment, the protein switch comprises 10 mutations at amino acid positions 12, 96, 155, 156, 159, 170, 195, 198, 251 and 252 of SEQ ID NO:20.

[0116] In yet another embodiment, the protein switch comprises seven mutations at amino acid positions 96, 155, 156, 159, 170, 198, and 252 of SEQ ID NO: 20. In these embodiments, the protein switch comprises a cysteine ​​or phenylalanine at amino acid position 96, an alanine at amino acid position 155, a serine at amino acid position 156, a tyrosine at amino acid position 159, a lysine at amino acid position 170, a glutamic acid at amino acid position 198, and a lysine or valine at amino acid position 252 of SEQ ID NO:20.

[0117] In another embodiment, the protein switch comprises eight mutations at amino acid positions 96, 98, 155, 156, 159, 170, 198, and 252 of SEQ ID NO: 20. In these embodiments, the protein switch comprises a cysteine ​​or phenylalanine at amino acid position 96, a leucine at amino acid position 98, an alanine at amino acid position 155, a serine at amino acid position 156, a tyrosine at amino acid position 159, a lysine at amino acid position 170, a glutamic acid at amino acid position 198, and a lysine or valine at amino acid position 252 of SEQ ID NO:20.

[0118] In another embodiment, the protein switch comprises eight mutations at amino acid positions 96, 155, 156, 159, 170, 198, 219, and 252 of SEQ ID NO: 20. In these embodiments, the protein switch comprises a cysteine ​​or phenylalanine at amino acid position 96, an alanine at amino acid position 155, a serine at amino acid position 156, a tyrosine at amino acid position 159, a lysine at amino acid position 170, a glutamic acid at amino acid position 198, a threonine at amino acid position 219, and a lysine or valine at amino acid position 252 of SEQ ID NO:20.

[0119] In another embodiment, the protein switch comprises eight mutations at amino acid positions 96, 155, 156, 159, 170, 195, 198, and 252 of SEQ ID NO: 20. In these embodiments, the protein switch comprises a cysteine ​​or phenylalanine at amino acid position 96, an alanine at amino acid position 155, a serine at amino acid position 156, a tyrosine at amino acid position 159, a lysine at amino acid position 170, an arginine at amino acid position 195, a glutamic acid at amino acid position 198, and a lysine or valine at amino acid position 252 of SEQ ID NO:20.

[0120] In another embodiment, the protein switch comprises eight mutations at amino acid positions 96, 141, 155, 156, 159, 170, 198, and 252 of SEQ ID NO: 20. In these embodiments, the protein switch comprises a cysteine ​​or phenylalanine at amino acid position 96, a valine at amino acid position 141, an alanine at amino acid position 155, a serine at amino acid position 156, a tyrosine at amino acid position 159, a lysine at amino acid position 170, a glutamic acid at amino acid position 198, and a lysine or valine at amino acid position 252 of SEQ ID NO:20.

[0121] In another embodiment, the protein switch comprises eight mutations at amino acid positions 96, 155, 156, 159, 170, 173, 198, and 252 of SEQ ID NO: 20. In these embodiments, the protein switch comprises a cysteine ​​or phenylalanine at amino acid position 96, an alanine at amino acid position 155, a serine at amino acid position 156, a tyrosine at amino acid position 159, a lysine at amino acid position 170, a glycine at amino acid position 173, a glutamic acid at amino acid position 198, and a lysine or valine at amino acid position 252 of SEQ ID NO:20.

[0122] In another embodiment, the protein switch comprises eight mutations at amino acid positions 55, 96, 155, 156, 159, 170, 198, and 252 of SEQ ID NO: 20. In these embodiments, the protein switch comprises a glutamic acid at amino acid position 55, a cysteine ​​or phenylalanine at amino acid position 96, an alanine at amino acid position 155, a serine at amino acid position 156, a tyrosine at amino acid position 159, a lysine at amino acid position 170, a glutamic acid at amino acid position 198, and a lysine or valine at amino acid position 252 of SEQ ID NO:20.

[0123] In another embodiment, the protein switch comprises eight mutations at amino acid positions 96, 137, 155, 156, 159, 170, 198, and 252 of SEQ ID NO: 20. In these embodiments, the protein switch comprises a cysteine ​​or phenylalanine at amino acid position 96, a tyrosine at amino acid position 137, an alanine at amino acid position 155, a serine at amino acid position 156, a tyrosine at amino acid position 159, a lysine at amino acid position 170, a glutamic acid at amino acid position 198, and a lysine or valine at amino acid position 252 of SEQ ID NO:20.

[0124] In another embodiment, the protein switch comprises eight mutations at amino acid positions 48, 96, 155, 156, 159, 170, 198, and 252 of SEQ ID NO: 20. In these embodiments, the protein switch comprises an alanine at amino acid position 48, a cysteine ​​or phenylalanine at amino acid position 96, an alanine at amino acid position 155, a serine at amino acid position 156, a tyrosine at amino acid position 159, a lysine at amino acid position 170, a glutamic acid at amino acid position 198, and a lysine or valine at amino acid position 252 of SEQ ID NO:20.

[0125] In another embodiment, the protein switch comprises eight mutations at amino acid positions 96, 155, 156, 159, 170, 184, 198, and 252 of SEQ ID NO: 20. In these embodiments, the protein switch comprises a cysteine ​​or phenylalanine at amino acid position 96, an alanine at amino acid position 155, a serine at amino acid position 156, a tyrosine at amino acid position 159, a lysine at amino acid position 170, a serine at amino acid position 184, a glutamic acid at amino acid position 198, and a lysine or valine at amino acid position 252 of SEQ ID NO:20.

[0126] In another embodiment, the protein switch comprises eight mutations at amino acid positions 96, 155, 156, 159, 166, 170, 198, and 252 of SEQ ID NO: 20. In these embodiments, the protein switch comprises a cysteine ​​or phenylalanine at amino acid position 96, an alanine at amino acid position 155, a serine at amino acid position 156, a tyrosine at amino acid position 159, a serine at amino acid position 166, a lysine at amino acid position 170, a glutamic acid at amino acid position 198, and a lysine or valine at amino acid position 252 of SEQ ID NO:20.

[0127] In another embodiment, the protein switch comprises eight mutations at amino acid positions 96, 155, 156, 159, 170, 198, 240, and 252 of SEQ ID NO: 20. In these embodiments, the protein switch comprises a cysteine ​​or phenylalanine at amino acid position 96, an alanine at amino acid position 155, a serine at amino acid position 156, a tyrosine at amino acid position 159, a lysine at amino acid position 170, a glutamic acid at amino acid position 198, a glutamine at amino acid position 240, and a lysine or valine at amino acid position 252 of SEQ ID NO:20.

[0128] In still yet other embodiments, the protein switch comprises nine mutations at amino acid positions 45, 96, 149, 155, 156, 159, 170, 198, and 252 of SEQ ID NO: 20. In these embodiments, the protein switch comprises an asparagine at amino acid position 45, a cysteine ​​or phenylalanine at amino acid position 96, an alanine at amino acid position 149, an alanine at amino acid position 155, a serine at amino acid position 156, a tyrosine at amino acid position 159, a lysine at amino acid position 170, a glutamic acid at amino acid position 198, and a lysine or valine at amino acid position 252 of SEQ ID NO:20.

[0129] In still yet other embodiments, the protein switch comprises nine mutations at amino acid positions 22, 96, 154, 155, 156, 159, 170, 198, and 252 of SEQ ID NO: 20. In these embodiments, the protein switch comprises a glycine at amino acid position 22, a cysteine ​​or phenylalanine at amino acid position 96, a glycine at amino acid position 154, an alanine at amino acid position 155, a serine at amino acid position 156, a tyrosine at amino acid position 159, a lysine at amino acid position 170, a glutamic acid at amino acid position 198, and a lysine or valine at amino acid position 252 of SEQ ID NO:20.

[0130] In yet another embodiment, the protein switch comprises nine mutations at amino acid positions 96, 141, 154, 155, 156, 159, 170, 198, and 252 of SEQ ID NO: 20. In these embodiments, the protein switch comprises a cysteine ​​or phenylalanine at amino acid position 96, a valine at amino acid position 141, a glycine at amino acid position 154, an alanine at amino acid position 155, a serine at amino acid position 156, a tyrosine at amino acid position 159, a lysine at amino acid position 170, a glutamic acid at amino acid position 198, and a lysine or valine at amino acid position 252 of SEQ ID NO:20.

[0131] In still yet further embodiments, the protein switch comprises ten mutations at amino acid positions 12, 96, 155, 156, 159, 170, 195, 198, 251, and 252 of SEQ ID NO: 20. In these embodiments, the protein switch comprises a lysine at amino acid position 12, a cysteine ​​or phenylalanine at amino acid position 96, an alanine at amino acid position 155, a serine at amino acid position 156, a tyrosine at amino acid position 159, a lysine at amino acid position 170, an arginine at amino acid position 195, a glutamic acid at amino acid position 198, a lysine at amino acid position 251, and a lysine or valine at amino acid position 252 of SEQ ID NO:20.

[0132] In yet another embodiment, the protein switch of the present disclosure has the amino acid sequence of SEQ ID NO: 11 with at least one of the following mutations (single) or combinations of mutations listed in Table 3C below and in FIG. 14:

[0133] [Table 5]

[0134] In another aspect, the protein switch of the present disclosure is a protein switch having an amino acid sequence selected from the group consisting of any of SEQ ID NOS: 1-19, at least 60% sequence identity to any one of SEQ ID NOS: 1-19, at least 65% sequence identity to any one of SEQ ID NOS: 1-19, at least 70% sequence identity to any one of SEQ ID NOS: 1-10 and 12-19, at least 75% sequence identity to any one of SEQ ID NOS: 1-19, at least 80% sequence identity to any one of SEQ ID NOS: 1-19, at least 85% sequence identity to any one of SEQ ID NOS: 1-19, The protein switch has an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1 to 19, at least 95% sequence identity to any one of SEQ ID NOs: 1 to 19, at least 96% sequence identity to any one of SEQ ID NOs: 1 to 19, at least 97% sequence identity to any one of SEQ ID NOs: 1 to 19, at least 98% sequence identity to any one of SEQ ID NOs: 1 to 19, at least 99% sequence identity to any one of SEQ ID NOs: 1 to 19, or at least 100% sequence identity to any one of SEQ ID NOs: 1 to 19. The protein switch comprising the amino acid sequence of SEQ ID NOs: 1 to 19 binds to the analyte warfarin.

[0135] In another embodiment, the protein switch of the disclosure has an amino acid sequence of any of SEQ ID NOs: 1, 7, 12 or 15, at least 60% sequence identity to any one of SEQ ID NOs: 1, 7, 12 or 15, at least 65% sequence identity to any of SEQ ID NOs: 1, 7, 12 or 15, at least 70% sequence identity to any one of SEQ ID NOs: 1, 7, 12 or 15, at least 75% sequence identity to any one of SEQ ID NOs: 1, 7, 12 or 15, at least 80% sequence identity to any one of SEQ ID NOs: 1, 7, 12 or 15, at least 85% sequence identity to any one of SEQ ID NOs: 1, 7, 12 or 15, The present invention relates to an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1, 7, 12 or 15, at least 95% sequence identity to any one of SEQ ID NOs: 1, 7, 12 or 15, at least 96% sequence identity to any one of SEQ ID NOs: 1, 7, 12 or 15, at least 97% sequence identity to any one of SEQ ID NOs: 1, 7, 12 or 15, at least 98% sequence identity to any one of SEQ ID NOs: 1, 7, 12 or 15, at least 99% sequence identity to any one of SEQ ID NOs: 1, 7, 12 or 15, or 100% sequence identity to any one of SEQ ID NOs: 1, 7, 12 or 15.

[0136] In yet another embodiment, the protein switch of the present disclosure comprises any of the enzymes of Examples 1 or 2.

[0137] As discussed in more detail herein, one or more protein switches of the present disclosure can be incorporated into one or more sensors using conventional techniques known in the art for use in sensing systems to detect one or more analytes in a sample.

[0138] C. How to Make a Protein Switch In some embodiments, the present disclosure relates to methods for identifying, creating, and / or preparing protein switches. In certain embodiments, the protein switches of the present disclosure can be created by screening a non-naturally occurring mutant polypeptide expression library with one or more analytes of interest using biochemical methods known in the art. For example, a mutant polypeptide expression library encoding an oxidase (e.g., glucose oxidase or lactate oxidase) or a dehydrogenase (e.g., glucose dehydrogenase or lactate dehydrogenase) can be screened with one or more analytes of interest. In one embodiment, the rate of formation of NADH, NADPH, FADH2, or other reduced cofactors can be monitored for an increase or decrease in absorbance or fluorescence, and polypeptides exhibiting such an increase or decrease in enzymatic activity are selected. In some embodiments, screening with the analytes of interest results in the selection of polypeptides that exhibit a decrease in enzymatic activity. In other embodiments, screening with the analytes of interest results in the selection of polypeptides that exhibit an increase in enzymatic activity.

[0139] Once an oxidase or dehydrogenase variant that exhibits increased or decreased enzymatic activity has been identified, mutant and / or evolved protein switches (e.g., enzymes) with increased or decreased oxidase or dehydrogenase activity can be readily created using routine techniques known in the art. See, e.g., Ling et al., Anal. Biochem., 254(2):157-78 (1997); Dale et al., Methods Mol. Biol., 57:369-74 (1996); Smith, Ann. Rev. Genet., 19:423-462 (1985); Botstein et al., Science, 229:1193-1201 (1985); Carter, "Site-directed mutagenesis," Biochem. J., 237:1-7 (1986); Kramer et al., Cell, 38:879-887 (1984); Wells et al., Gene, 34:315-323 (1985); Current Opinion in Chemical Biology, 3:284-290 (1999); Christians et al., Nature Biotechnology, 17:259-264 (1999); Crameri et al., Nature, 391:288-291; Crameri et al., Nature Biotechnology, 15:436-438 (1997); Zhang et al., Proceedings of the National Academy of Sciences USA, 94:4504-4509; Crameri et al., Nature Biotechnology, 14:315-319 (1996); Stemmer, Nature, 370:389-391 (1994); Stemmer, Proceedings of the National Academy of Sciences See U.S.A., 91:10747-10751 (1994); WO95 / 22625; WO97 / 0078; WO97 / 35966; WO98 / 27230; WO00 / 42651; WO01 / 75767 and U.S. Patent No. 6,537,746. To maximize any diversity, several of the techniques described above may be used sequentially.Typically, one mutagenesis or evolution method creates a library of variant polynucleotides, and their expression products are screened to find polypeptides with increased or decreased oxidase or dehydrogenase activity. A second mutagenesis or evolution method is then applied to the polynucleotides encoding the greatest or least enzyme activity to create a second library, which is then screened for oxidase or dehydrogenase activity by the same technique. The process of mutation and screening, including the insertion of point mutations, can be repeated as many times as necessary to arrive at a polynucleotide encoding a protein switch with the desired activity, thermostability, cofactor preference, or other characteristics.

[0140] D. Devices and Sensor Systems In other aspects, the present disclosure relates to one or more sensors that use and / or contain one or more (e.g., multiple) protein switches described herein for detecting and / or monitoring at least one analyte. As discussed in more detail herein, such sensors can be prepared using techniques conventional in the art. Such sensors can then be used in one or more sensor systems. A general description of suitable sensor configurations and sensor systems that use these sensors that utilize the protein switches of the present disclosure is presented. However, this description should be understood as a non-limiting example of the embodiments disclosed herein, and alternative sensors and systems remain within the scope of the present disclosure.

[0141] FIG. 1 presents a schematic of an exemplary sensing system incorporating sensors including one or more protein switches of the present disclosure. As shown, sensing system 100 includes a sensor control device 102 and a reader device 120 (e.g., a signal detection device) configured to communicate with each other via a local communication path or link, which may be wired, wireless, unidirectional, bidirectional, encrypted, or unencrypted. The reader device 120 may provide an output medium for reviewing analyte concentrations and alerts or notifications determined by the sensor / sensors 104 or their associated processors, as well as for enabling one or more user inputs. The reader device 120 may be a general-purpose smartphone or a dedicated electronic reader device. While only one reader device 120 is shown, in certain cases, multiple reader devices 120 may be present. The reader device 120 may also communicate with a remote terminal 170 and / or a trusted computer system 180 via communication paths / links 141 and / or 142, respectively, which may also be wired, wireless, unidirectional, bidirectional, encrypted, or unencrypted. Reader device 120 may also, or alternatively, be in contact with network 150 (e.g., a cellular network, the Internet, or a cloud server) via communication path / link 151. Network 150 may be further communicatively coupled with remote terminal 170 via communication path / link 152 and / or with trusted computer system 180 via communication path / link 153. Alternatively, sensor 104 may communicate directly with other signal detection devices, such as remote terminal 170 and / or trusted computer system 180, without the intervening reader device 120 present. For example, as described in U.S. Patent Application Publication No. 2011 / 0213225, which is incorporated herein by reference in its entirety, according to some embodiments, sensor 104 may communicate with remote terminal 170 and / or trusted computer system 180 via a direct communication link with network 150.Any suitable electronic communication protocol may be used for each of the communication paths or links, such as near field communication (NFC), radio frequency identification (RFID), BLUETOOTH® or BLUETOOTH® Low Energy protocols, Wi-Fi, etc. The remote terminal 170 and / or trusted computer system 180 may, according to some embodiments, be accessible by individuals other than the primary user who have an interest in the user's analyte levels. The reader device 120 may include a display 122 and an optional input component 121. According to some embodiments, the display 122 may include a touchscreen interface.

[0142] The sensor control device 102 includes a sensor housing 103, which may house circuitry and a power source for operating the sensor 104. Optionally, the power source and / or active circuitry may be omitted. A processor (not shown) may be communicatively coupled to the sensor 104, the processor physically located within the sensor housing 103 or within the reader device 120. The sensor 104 protrudes from the bottom surface of the sensor housing 103 and extends via an adhesive layer 105. According to some embodiments, the sensor housing 103 is adapted for adhesion to a tissue surface, such as skin.

[0143] The sensor 104 is adapted to be at least partially inserted into a tissue of interest, such as into the intradermal or subcutaneous layer of skin. The sensor 104 may include a sensor tail of sufficient length for insertion to a desired depth within a given tissue. The sensor tail may include at least one working electrode and one or more active regions (sensing layers or sensing regions / spots) disposed on the at least one working electrode and active for sensing one or more analytes of interest. Collectively, the one or more active regions may include one or more protein switches. The active region may include a polymeric material to which at least a portion of one or more of the protein switches is covalently attached. In various embodiments, an analyte may be monitored in any biological fluid of interest, such as intradermal fluid, interstitial fluid, subcutaneous fluid, or blood (e.g., venous blood or capillary blood). In certain embodiments, the sensor may be adapted for assaying intradermal or interstitial fluid.

[0144] In some embodiments, the sensor 104 may automatically transfer data to the reader device 120 (e.g., by transmitting a signal, etc.). For example, analyte concentration data (e.g., a signal representing the level of one or more analytes) may be communicated automatically and periodically, such as at a certain frequency or after a certain amount of time has passed since data was acquired, and the data is stored in memory until transfer (e.g., every 1 minute, 5 minutes, or other predetermined time). In other embodiments, the sensor 104 may communicate with the reader device 120 according to a set schedule rather than automatically. For example, if the sensor's electronics are integrated within the communication range of the reader device 120, data may be communicated from the sensor 104 using RFID technology. The data may remain stored in the sensor's 104's memory until communicated to the reader device 120. Thus, the patient may upload data at a convenient time without having to remain near the reader device 120 at all times. In still other embodiments, a combination of automatic and non-automatic data transfer may be implemented. For example, data transfer may continue automatically until the reader device 120 is no longer within communication range of the sensor 104 .

[0145] An introducer may be temporarily present to facilitate the introduction of the sensor 104 into the tissue. In some embodiments, the introducer may include a needle or similar sharp object. It should be recognized that in alternative embodiments, other types of introducers, such as a sheath or blade, may also be present. More specifically, the needle or other introducer may be temporarily present in proximity to the sensor 104 prior to insertion into the tissue and then withdrawn later. While present, the needle or other introducer may facilitate the insertion of the sensor 104 into the tissue by opening an access path for the sensor 104 to follow. For example, the needle may facilitate penetration of the epidermis as an access path to the dermis to allow implantation of the sensor 104. After opening the access path, the needle or other introducer may be withdrawn so as not to pose a sharp hazard. In other embodiments, suitable injection needles may be solid or hollow, beveled or non-beveled, and / or may be circular or non-circular in cross section. In more particular embodiments, suitable injection needles may be similar in cross-sectional diameter and / or tip design to acupuncture needles and may have a cross-sectional diameter of approximately 250 microns. However, it will be recognized that suitable injection needles may have larger or smaller cross-sectional diameters as required for a particular application.

[0146] In still further embodiments, the tip of the needle (while present) can be angled downward from the end of the sensor 104 so that the needle first penetrates the tissue and opens an access path for the sensor 104. In other exemplary embodiments, the sensor 104 can be present within the lumen of the needle or at the bevel of the needle, which similarly opens an access path for the sensor 104. In either case, the needle is then withdrawn after facilitating insertion of the sensor.

[0147] A sensor can contain or include one or more (e.g., multiple) protein switches on the active area of ​​a single working electrode or on two or more separate working electrodes. Single working electrode configurations for sensors can use a two-electrode sensing motif or a three-electrode sensing motif. Hereinafter, sensor configurations featuring a single working electrode are described with reference to Figures 2A-2C. Hereinafter, sensor configurations featuring multiple working electrodes are separately described with reference to Figure 3. Multiple protein switches can be incorporated into any of the sensor configurations described hereinbelow, with specific configurations suitable for incorporating multiple protein switches described in further detail hereinbelow.

[0148] When a single working electrode is present in the sensor, a three-electrode detection motif can include a working electrode, a counter electrode, and a reference electrode. A related two-electrode detection motif can include a working electrode and a second electrode, where the second electrode functions as both the counter electrode and the reference electrode (i.e., the counter / reference electrode). In both two-electrode and three-electrode detection motifs, one or more active regions of the sensor can be in contact with the working electrode. The one or more active regions can include multiple protein switches, and one or more (e.g., multiple) protein switches can be present in a single active region and / or in multiple active regions. In some embodiments, the various electrodes can be at least partially stacked (layered) on top of each other, as described in more detail herein below. In some or other embodiments, the various electrodes can be laterally spaced apart from each other on the sensor tail. Similarly, the associated active regions on each electrode can be vertically stacked or laterally spaced apart from each other. In either case, the various electrodes may be electrically isolated from one another by a dielectric material or similar insulator.

[0149] FIG. 2A shows a schematic of an exemplary two-electrode sensor configuration having a single working electrode, which is compatible with use in some embodiments of the present disclosure herein. As shown, sensor 200 includes a substrate 212 disposed between a working electrode 214 and a counter / reference electrode 216. Alternatively, working electrode 214 and counter / reference electrode 216 can be disposed on the same side of substrate 212 with a dielectric material interposed therebetween (configuration not shown). Active region 218 is disposed as at least one layer over at least a portion of working electrode 214. In various embodiments, active region 218 can include multiple spots or a single spot configured for detection of one or more analytes of interest. One or more (e.g., multiple) protein switches can be present within active region 218 (i.e., within a single spot or within multiple spots).

[0150] 2A , membrane 220 overcoats at least active area 218 and, according to some embodiments, may optionally overcoat some or all of working electrode 214 and / or counter / reference electrode 216, or the entire sensor 200. One or both sides of sensor 200 may be overcoated with membrane 220. Membrane 220 may comprise one or more polymeric membrane materials capable of restricting analyte flux to active area 218. As described further herein, the composition of membrane 220 may vary depending on the identification of the analyte.

[0151] 2B and 2C show schematics of an exemplary three-electrode sensor configuration with a single working electrode. A three-electrode sensor configuration using a single working electrode can be similar to the configuration shown for sensor 200 in FIG. 2A, except for the incorporation of additional electrode 217 in sensors 201 and 202 (FIGS. 2B and 2C). Additional electrode 217 then allows counter / reference electrode 216 to function as a counter electrode or a reference electrode, and additional electrode 217 performs other electrode functions not otherwise performed. Working electrode 214 continues to perform its original function. Additional electrode 217 can be disposed on working electrode 214 or electrode 216, with a separating layer of dielectric material between them. For example, as depicted in FIG. 2B, dielectric layers 219a and 219b separate electrodes 214, 216, and 217 from one another, providing electrical insulation. Alternatively, at least one of electrodes 214, 216, and 217 may be disposed on opposite sides of substrate 212, as shown in FIG. 2C. Thus, in some embodiments, electrode 214 (working electrode) and electrode 216 (counter electrode) may be disposed on opposite sides of substrate 212, with electrode 217 (reference electrode) disposed on one of electrodes 214 or 216 and spaced therefrom by a dielectric material. Reference material layer 230 (e.g., Ag / AgCl) may be present on electrode 217, and the location of reference material layer 230 is not limited to the location depicted in FIGS. 2B and 2C. Similar to sensor 200 shown in FIG. 2A, active area 218 in sensors 201 and 202 may include multiple spots or a single spot configured for detection of one or more analytes of interest. One or more (e.g., multiple protein switches) may be present within active area 218 of sensors 201 and 202.

[0152] As with sensor 200, membrane 220 may also overcoat active area 218 as well as other sensor components within sensors 201 and 202. In some embodiments, additional electrode 217 may be overcoated with membrane 220. While FIGS. 2B and 2C depict electrodes 214, 216, and 217 as all being overcoated with membrane 220, it should be appreciated that in some embodiments, only working electrode 214 may be overcoated. Furthermore, the thickness of membrane 220 on each of electrodes 214, 216, and 217 may be the same or different. As with the two-electrode sensor configuration ( FIG. 2A ), in the sensor configuration of FIGS. 2B and 2C , one or both sides of sensors 201 and 202 may be overcoated with membrane 220, or the entirety of sensors 201 and 202 may be overcoated. Therefore, the three-electrode sensor configuration shown in Figures 2B and 2C should be understood to be a non-limiting example of the embodiments disclosed herein, and alternative electrode and / or layer configurations remain within the scope of the present disclosure.

[0153] Sensor configurations having multiple working electrodes will now be described in further detail. While the following description is primarily directed to sensor configurations having two working electrodes, it will be understood that more than two working electrodes can be successfully incorporated through extensions of the present disclosure herein. Additional working electrodes may allow additional active area and corresponding sensing capabilities to be imparted to sensors having such features.

[0154] FIG. 3 shows a schematic of an exemplary sensor configuration having two working electrodes, a reference electrode, and a counter electrode, which is compatible with use in some embodiments of the present disclosure. As shown in FIG. 3 , sensor 300 includes working electrodes 304 and 306 disposed on opposite sides of substrate 302. Active region 310 is disposed on the surface of working electrode 304, and active region 312 is disposed on the surface of working electrode 306. Collectively, one or more (e.g., multiple) protein switches can be present within active region 310 and active region 312, with each active region 310, 312 containing one or more (e.g., multiple) protein switches. For example, in certain embodiments, a first protein switch including an analyte-binding domain capable of binding to troponin and a glucose oxidase domain responsive to glucose can be present within active region 310, and a second protein switch including an analyte-binding domain capable of binding to BNP and a glucose oxidase domain responsive to glucose can be present within active region 312. Alternatively, in certain embodiments, a second protein switch comprising an analyte-binding domain capable of binding BNP and a lactate oxidase domain responsive to lactate can be present within active region 312. Counter electrode 320 is electrically insulated from working electrode 304 by dielectric layer 322, and reference electrode 321 is electrically insulated from working electrode 306 by dielectric layer 323. Outer dielectric layers 330 and 332 are disposed on reference electrode 321 and counter electrode 320, respectively. Membrane 340 can overcoat at least active regions 310 and 312 according to various embodiments. Other components of sensor 300 can also be overcoated with membrane 340, and similarly to the above, one or both surfaces or portions thereof of sensor 300 can also be overcoated with membrane 340.

[0155] Alternative sensor configurations having multiple working electrodes and differing from the sensor configuration shown in Figure 3 may feature counter / reference electrodes rather than separate counter and reference electrodes 320, 321, and / or may feature layer and / or film arrangements different from those explicitly depicted. For example, the arrangement of counter electrode 320 and reference electrode 321 may be reversed from that depicted in Figure 3. Additionally, working electrodes 304 and 306 are not necessarily on opposite sides of substrate 302 in the manner shown in Figure 3.

[0156] A sensor configuration featuring a working electrode with an active area remote from the working electrode is shown in Figures 5A and 5B and is discussed further below.

[0157] According to some embodiments, an electron transfer agent can be present in one or more of the sensing regions (e.g., active regions) according to any of the sensors or sensor configurations disclosed herein. A suitable electron transfer agent / mediator compound can facilitate the transfer of electrons to the working electrode when a reactant undergoes an oxidation-reduction reaction. The selection of the electron transfer agent within each active region can dictate the oxidation-reduction potential observed for each active region. When multiple active regions are present, the electron transfer agent within each active region can be the same or different.

[0158] Suitable electron transfer agents may include electroreducible ions, complexes, or molecules (e.g., quinones) and electrooxidizable ones having oxidation-reduction potentials several hundred millivolts above or below the oxidation-reduction potential of a standard calomel electrode (SCE). According to some embodiments, suitable electron transfer agents may include low-potential osmium complexes, such as those described in U.S. Pat. Nos. 6,134,461 and 6,605,200, which are incorporated herein by reference in their entireties. Further examples include those described in U.S. Pat. Nos. 6,736,957, 7,501,053, and 7,754,093, the disclosures of each of which are incorporated herein by reference in their entireties. Other suitable electron transfer agents may include metal compounds or complexes, including metallocene compounds thereof, such as those of ruthenium, osmium, iron (e.g., polyvinylferrocene or hexacyanoferrate), or cobalt. Suitable examples of electron transfer mediators and polymer-bound electron transfer mediators include those described in U.S. Pat. Nos. 8,444,834, 8,268,143, and 6,605,201, the disclosures of each of which are incorporated herein by reference in their entireties. Suitable ligands for metal complexes can also include, for example, bidentate or higher ligands, such as bipyridine, biimidazole, phenanthroline, or pyridyl (imidazole). Other suitable bidentate ligands can include, for example, amino acids, oxalic acid, acetylacetone, diaminoalkanes, or o-diaminoarenes. Any combination of monodentate, bidentate, tridentate, tetradentate, or higher ligands can be present in the metal complex to achieve a full coordination sphere.

[0159] In yet other embodiments, the active or sensing region may also include a cofactor capable of catalyzing a reaction of a reactant associated with at least one oxidase domain portion or dehydrogenase domain portion of the protein switch. In some embodiments, the cofactor is a non-protein organic molecule, such as pyroquinoline quinone (PQQ), flavin adenine dinucleotide (FAD), nicotinamide adenine dinucleotide (NAD), or flavin mononucleotide (FMN). In certain embodiments, the cofactor may be conjugated to a polymer that crosslinks the cofactor with the electron transfer agent. In certain embodiments, a second cofactor may also be used.

[0160] In other embodiments, a polymer may be present within each active region of any of the sensors or sensor configurations disclosed herein. Polymers suitable for incorporation within the active region may include, but are not limited to, polyvinylpyridine (e.g., poly(4-vinylpyridine)), polyvinylimidazole (e.g., poly(1-vinylimidazole)), or any copolymers thereof. Exemplary copolymers that may be suitable for incorporation within the active region include copolymers containing monomer units such as, for example, styrene, acrylamide, methacrylamide, or acrylonitrile. When multiple active regions are present, the polymer within each active region may be the same or different.

[0161] In some embodiments, the electron transfer agent can be covalently bonded to the polymer in each active region. The manner of covalent bonding is not considered to be particularly limiting. The covalent bonding of the electron transfer agent to the polymer can occur by polymerizing polymer monomer units bearing the covalently bonded electron transfer agent, or the electron transfer agent can be reacted separately with the polymer after the polymer has already been synthesized. In other embodiments, a bifunctional spacer can covalently bond the electron transfer agent to the polymer in the active region, with a first functional group reactive with the polymer (e.g., a functional group capable of quaternizing the nitrogen atom of pyridine or the nitrogen atom of imidazole) and a second functional group reactive with the electron transfer agent (e.g., a functional group reactive with a ligand that coordinates to a metal ion).

[0162] Similarly, in yet other embodiments, protein switches in one or more of the active regions can be covalently bound to the polymer. When multiple protein switches are present in a single active region, in some embodiments, all of the multiple protein switches can be covalently bound to the polymer, while in other embodiments, only some of the multiple protein switches can be covalently bound to the polymer. For example, a first protein switch can be covalently bound to the polymer, and a second protein switch can be non-covalently associated with the polymer. According to more specific embodiments, the covalent binding of the protein switch to the polymer can occur via a cross-linker introduced by an appropriate cross-linking agent. Cross-linkers suitable for reacting with free amino groups in the protein switch can include, for example, polyethylene glycol diglycosidyl ether (PEGDGE) or other polyepoxides, cyanuric chloride, N-hydroxysuccinimide, imidoesters, epichlorohydrin, or derivatized variants thereof. Cross-linkers suitable for reacting with free carboxylic acid groups in the protein switch can include, for example, carbodiimides. Crosslinking is generally intermolecular, but in some embodiments can be intramolecular.

[0163] The electron transfer agent and / or one or more (e.g., multiple) protein switches may be associated with the polymer in the active region through means other than covalent bonding. In some embodiments, the electron transfer agent and / or one or more (e.g., multiple) protein switches may be ionically or coordinatively associated with the polymer. For example, a charged polymer may be ionically associated with an oppositely charged electron transfer agent or protein switch. In yet other embodiments, the electron transfer agent and / or protein switch may be physically entrained within the polymer without being bound to the polymer.

[0164] As already mentioned, the present disclosure contemplates a variety of suitable configurations for arranging multiple protein switches within a sensor. Multiple protein switches can be arranged within one or more active regions of the sensor. The size of the active region can be approximately 0.01 mm. 2 ~about 1mm 2 , although larger or smaller active areas are also contemplated herein.

[0165] In other embodiments, sensors containing multiple protein switches, whether operating independently or cooperatively, can function with enhanced stability in the presence of an appropriate stabilizer. Stabilizers that can be used include, for example, catalase or albumin (e.g., bovine serum albumin or human serum albumin).

[0166] In still other embodiments, multiple protein switches can be arranged in separate active regions on a single working electrode. When multiple protein switches are arranged in this manner, each active region can facilitate the detection of a separate (e.g., two or more different) analyte, as described hereinbelow. At least one of the active regions can produce a signal independent of the other active regions.

[0167] According to some embodiments, a sensor having multiple active regions on a single working electrode can include at least one working electrode and a sensor tail including at least two active regions disposed on the surface of the working electrode. Each active region includes a protein switch and a polymer, and the protein switch in each active region is different. Each active region has an oxidation-reduction potential, and the oxidation-reduction potential of a first active region is sufficiently separated from the oxidation-reduction potential of a second active region to enable generation of a signal from the first active region independent of the signal from the second active region. In more specific embodiments, such a sensor can include a single working electrode having at least two active regions. An electron transfer agent can be incorporated into each active region to facilitate electron transfer.

[0168] An alternative sensor configuration can include a single active region containing both a first protein switch and a second protein switch along with an electron transfer agent. Each protein switch can be covalently bound to a separate polymer moiety within the single active region. The single active region can facilitate analyte detection in a manner similar to that described below for separate active regions, provided that the sensing chemical reactants for facilitating electron transfer for each analyte are not excessively diluted within the single active region. Such a sensor configuration can be feasible, particularly when the analytes assayed by the first protein switch and the second protein switch have comparable membrane permeability values.

[0169] In more specific embodiments, the sensor tail can be configured for insertion into tissue. Suitable tissues are not considered particularly limiting and are addressed in more detail above. Similarly, considerations for placing the sensor tail at a particular location within tissue are addressed above.

[0170] In other embodiments, the oxidation-reduction potential associated with the first active region can be separated from the oxidation-reduction potential of the second active region by at least about 100 mV, at least about 150 mV, or at least about 200 mV. The upper limit of the separation between the oxidation-reduction potentials is dictated by the in vivo electrochemical working window. By sufficiently separating the oxidation-reduction potentials of the active regions from each other, electrochemical reactions can occur in the first active region without substantially inducing electrochemical reactions in the second active region. Thus, a signal from the first active region can be independently generated above its corresponding oxidation-reduction potential. In contrast, above the oxidation-reduction potential of the second active region, electrochemical reactions can occur in both active regions. Thus, above the oxidation-reduction potential of the second active region, the resulting signal can include signal contributions from both the first active region and the second active region, and the signal is a composite signal. The signal contribution from the second active region above its corresponding redox potential can then be determined by subtracting from the composite signal the signal obtained from the first active region alone above its corresponding redox potential. Similar considerations apply to the analysis of signal contributions from a single active region containing two different protein switches that result in signals at different redox potentials.

[0171] In more specific embodiments, when the active regions are disposed on the same working electrode to provide oxidation-reduction potentials that are sufficiently separated in magnitude, a first active region and a second active region can contain different electron transfer agents. More specifically, the first active region can include a first electron transfer agent, and the second active region can include a second electron transfer agent, where the first and second electron transfer agents are different. According to various embodiments of the present disclosure, the metal center and / or ligands present in a given electron transfer agent can be varied to provide sufficient separation between the oxidation-reduction potentials of the first and second active regions. According to even more specific embodiments, the first electron transfer agent can be covalently bonded to a polymer in the first active region, and the second electron transfer agent can be covalently bonded to a polymer in the second active region. The covalent attachment scheme for the first electron transfer agent and the covalent attachment scheme for the second electron transfer agent can be the same or different, and similar considerations apply to selecting electron transfer agents suitable for use with a first protein switch and a second protein switch contained within a single active region in accordance with the present disclosure above.

[0172] In more specific embodiments of the present disclosure, the protein switch in each active region may be covalently bound (or otherwise immobilized) to a polymer in each active region. In even more specific embodiments, the protein switch and electron transfer agent in each active region may be covalently bound to a polymer in each active region. When contained within a single active region, a first protein switch and a first electron transfer agent may be covalently bound to a first portion of the polymer, and a second protein switch and a second electron transfer agent may be covalently bound to a second portion of the polymer. The polymer in the first portion and the polymer in the second portion may be the same or different.

[0173] In some embodiments, the first and second active regions disposed on a single working electrode can be configured to rapidly reach a steady-state current when the sensor is activated at a given potential. Rapid attainment of a steady-state current can be facilitated by selecting an electron transfer agent for each active region that rapidly changes its oxidation state when exposed to a potential equal to or greater than its redox potential. Making the active regions as thin as possible can also facilitate rapid attainment of a steady-state current. For example, a suitable thickness for the first and second active regions can range from about 0.1 microns to about 10 microns. In some or other embodiments, combining a conductive material, such as carbon nanotubes, graphene, or metal nanoparticles, in one or more of the active regions can facilitate rapid attainment of a steady-state current. The appropriate amount of conductive particles can range from about 0.1% to about 50% by weight, or from about 1% to about 50% by weight, or from about 0.1% to about 10% by weight, or from about 1% to about 10% by weight of the active region. Stabilizing agents may also be used to promote response stability.

[0174] It is also understood that the sensitivity (output current) of the sensor to each analyte can be varied by varying the coverage (area or size) of the active regions, the ratio of the areas of the active regions relative to one another, the identity and thickness of the mass transport limiting membrane overcoating the active regions, and any combination thereof. Variations in these parameters can be readily accomplished by one skilled in the art.

[0175] While the foregoing description is primarily directed to sensors configured to detect two different analytes, it is understood that the above concepts can be extended to detect more than two analytes using a corresponding number of active regions disposed on a single working electrode. Specifically, in further embodiments of the present disclosure, sensors employing more than two active regions and a corresponding number of different protein switches (and electron transfer agents) therein can be used to detect a similar number of different analytes. Provided that the oxidation-reduction potential of each active region is sufficiently separated from the oxidation-reduction potential of the other active regions, the signal contribution from each active region can be analyzed in a manner analogous to that described above to yield the concentration of each analyte.

[0176] For example, the first active region can include a first protein switch including an analyte-binding domain capable of binding to troponin and a glucose oxidase domain responsive to glucose, and the second active region can include a second protein switch including an analyte-binding domain capable of binding to BNP and a lactate oxidase domain responsive to lactate, in addition to a suitable electron transfer agent and polymer, as discussed in more detail above. For example, a sensor suitable for detecting troponin and BNP can include a working electrode having a first active region and a second active region disposed thereon, and a mass transport-limiting membrane overcoating the first active region and the second active region on the working electrode, where the second active region includes a polymer and an analyte-binding domain capable of binding to BNP and a lactate oxidase domain responsive to lactate, covalently bonded to the polymer, and the first active region includes an analyte-binding domain capable of binding to troponin and a glucose oxidase domain responsive to glucose, covalently bonded to the polymer. Alternatively, the lactate oxidase domain may be replaced with a glucose oxidase domain that is responsive to glucose. Within each active region, there may be a first electron transfer agent and a second electron transfer agent that are different from each other. In more specific embodiments, the mass transport limiting membrane may comprise at least a crosslinked homopolymer or copolymer of polyvinylpyridine. When a mass transport limiting membrane overcoats each active region, the compositions of the mass transport limiting membranes may be the same or different. In certain embodiments, the mass transport limiting membrane overcoating the first active region may be single-component (containing a single membrane polymer), and the mass transport limiting membrane overcoating the second active region may be multi-component (containing two or more different membrane polymers, one of which is a polyvinylpyridine homopolymer or copolymer), as a bilayer or homogeneous mixture.

[0177] It should also be understood that some sensors with two or more active regions disposed on a given working electrode may include two or more protein switches in at least one of the active regions. According to more specific embodiments, two or more protein switches in a given active region may interact cooperatively to generate a signal proportional to the concentration of a single analyte. Thus, the protein switches need not necessarily be present in a 1:1 ratio with a given analyte of choice. Sensors containing cooperatively interacting protein switches are described in further detail herein.

[0178] Thus, also described herein are methods for multiplexed analyte detection using a sensor featuring multiple protein switches disposed on a single working electrode. In various embodiments, such methods can include exposing the sensor to a fluid containing at least one analyte. The sensor includes at least one working electrode, particularly a single working electrode, and a sensor tail including at least two active regions disposed on the surface of the working electrode. Each active region includes a protein switch and a polymer, and the protein switches within each active region are different. Each active region has an oxidation-reduction potential, and the oxidation-reduction potential of a first active region is sufficiently separated from the oxidation-reduction potential of a second active region to allow generation of a signal from the first active region independent of generation of a signal from the second active region. The method additionally includes the steps of obtaining a first signal above the oxidation-reduction potential of the first active area, such that the first signal is proportional to the concentration of the first analyte; obtaining a second signal above the oxidation-reduction potential of the second active area, such that the second signal is a composite signal including a signal contribution from the first active area and a signal contribution from the second active area; and subtracting the first signal from the second signal to obtain a difference signal proportional to the concentration of the second analyte.

[0179] In more specific embodiments, the oxidation-reduction potential associated with the first active region can be separated from the oxidation-reduction potential of the second active region by at least about 100 mV, or at least about 150 mV, or at least about 200 mV to provide sufficient separation for independent generation of a signal from the first active region.

[0180] In some more specific or other more specific embodiments, the fluid is a biological fluid and the sensor is exposed to the biological fluid in vivo within an individual. Biological fluids suitable for analysis by a sensor having at least two distinct active areas disposed on a given working electrode can include any of the biological fluids previously discussed herein.

[0181] In some embodiments, the signal associated with each active area can be correlated with the corresponding analyte concentration by referencing a lookup table or calibration curve for each analyte. A lookup table for each analyte can be prepared by assaying multiple samples with known analyte concentrations and recording the sensor response at each concentration for each analyte. Similarly, a calibration curve for each analyte can be determined by plotting the sensor response for each analyte as a function of concentration. According to some embodiments, the calibration curve for the sensors of the present disclosure can be linear.

[0182] The processor may determine which sensor response value in the lookup table is closest to the measured sensor response value for the sample with the unknown analyte concentration and then report the analyte concentration accordingly. In some or other embodiments, if the sensor response value for the sample with the unknown analyte concentration is between the values ​​recorded in the lookup table, the processor may interpolate between the two lookup table values ​​to estimate the analyte concentration. Interpolation may assume a linear variation in concentration between the two values ​​reported in the lookup table. Interpolation may be used when the sensor response differs a sufficient amount from a given value in the lookup table, such as by a variation of about 10% or more.

[0183] Similarly, according to some or other various embodiments, the processor may input the sensor response values ​​for samples with unknown analyte concentrations into a corresponding calibration curve, which may then cause the sensor to report the analyte concentration.

[0184] Sensor embodiments having two distinct active regions disposed on a given working electrode are depicted in Figures 2A-2C and may use sensor configurations similar to those described above. However, it is understood that suitable sensors may also feature multiple working electrodes, such as the sensor configuration depicted in Figure 3, with at least one of the working electrodes having at least two active regions that are distinct from one another. It is also understood that other sensor configurations having two or more distinct active regions disposed on the surface of a given working electrode are also within the scope of the present disclosure. For example, the position, orientation, or functionality of the working electrode and counter and / or reference electrodes may differ from the position, orientation, or functionality depicted in the figures herein.

[0185] Figure 4 shows an exemplary sensor configuration compatible with use in some embodiments of the present disclosure, in which two distinct active regions are disposed on the surface of a single working electrode. The sensor configuration of Figure 4 bears the highest degree of similarity to the sensor configuration of Figure 2C. Where appropriate, for clarity, common reference features from Figure 2C are also used in Figure 4, and for brevity, features having common structure and / or function will not be described again in further detail. It should also be understood that other sensor configurations may incorporate the features described in Figure 4 below as well.

[0186] Referring to FIG. 4 , sensor 400 includes active regions 218a and 218b on the surface of working electrode 214. Active region 218a includes a first electron transfer agent and a first protein switch, which may be covalently bonded to a polymer comprising active region 218a. Active region 218b includes a second protein switch, which may be covalently bonded to a polymer comprising active region 218b. The first and second electron transfer agents may differ in composition to provide a separation between the oxidation-reduction potentials of first active region 218a and second active region 218b. In certain embodiments, active region 218b may include an analyte-binding domain capable of binding to BNP and a lactate oxidase domain responsive to lactate, and active region 218a may include an analyte-binding domain capable of binding to troponin and a glucose oxidase domain responsive to glucose. Alternatively, the lactate oxidase domain may be replaced by a glucose oxidase domain that is responsive to glucose.

[0187] The oxidation-reduction potentials of the first active region 218a and the second active region 218b are sufficiently separated from one another to allow signal generation from the first active region 218a independent of signal generation from the second active region 218b. In this manner, the sensor 400 can be operated at a first potential at which an oxidation-reduction reaction occurs in the first active region 218a but not in the second active region 218b. Thus, a first analyte (e.g., troponin) can be selectively detected at or above the oxidation-reduction potential of the first active region 218a, provided that the applied potential is not high enough to promote an oxidation-reduction reaction by the second active region 218b. The concentration of the first analyte can be determined from the sensor response of the first active region 218a by referencing a lookup table or calibration curve.

[0188] Above the oxidation-reduction potential of the second active region 218b, separate oxidation-reduction reactions can occur simultaneously or nearly simultaneously in both the first active region 218a and the second active region 218b. As a result, the signal produced above the oxidation-reduction potential of the second active region 218b can include a composite signal having signal contributions from both the first active region 218a and the second active region 218b. To determine the concentration of a second analyte (e.g., BNP) from the composite signal, the signal from the first active region 218a above its corresponding oxidation-reduction potential can be subtracted from the composite signal to produce a difference signal associated with the second active region 218b alone. Once the difference signal is determined, the concentration of the second analyte can be determined by reference to a lookup table or calibration curve.

[0189] As already mentioned, similar considerations also apply to the separation of a first signal and a second signal from a single active region containing two different protein switches in order to determine the concentrations of a first analyte and a second analyte that differ from each other.

[0190] As previously discussed herein, the sensor configurations shown in Figures 2A-4 all feature one or more working electrodes with one or more active areas disposed directly on the surface of each working electrode. In contrast, Figures 5A and 5B show schematics for working electrodes in which a first active area is disposed directly on the surface of the working electrode and a second active area is separated from (spaced apart from or remote from) the working electrode by a membrane. The working electrode configurations depicted in Figures 5A and 5B can replace any of the specific working electrode configurations depicted in Figures 2A-4. That is, the working electrode configurations depicted in Figures 5A and 5B can be combined in any suitable manner with counter and / or reference electrodes, membranes, substrates, and similar structures within a sensor.

[0191] As shown in FIG. 5A , working electrode 400 has active region 402 disposed directly on its surface. Active region 402 includes a first protein switch covalently bonded to a first polymer. Typically, an electron transfer agent is also present within active region 402, and the electron transfer agent is also covalently bonded to the polymer. Active region 402 is overcoated with membrane 404. Membrane 404 may also overcoat the surface of working electrode 400 as depicted, as well as other portions of the sensor where working electrode 400 resides. Membrane 404 insulates active region 406 from working electrode 400 so that electron exchange between the two is prevented. Active region 406 includes a second protein switch covalently bonded to a second polymer. Typically, an electron transfer agent is also present within active region 406, and the electron transfer agent is also covalently bonded to polymer 406. FIG. 5A shows active region 406 disposed directly on active region 402, although it should be understood that they can also be laterally spaced apart in alternative configurations compatible with the present disclosure. Membrane 408 overcoats active region 406 and optional other sensor components to provide mass transport limiting properties. Similarly, as shown in FIG. 5B , membrane 404 does not necessarily extend the same lateral distance as membrane 408 does over working electrode 400. In fact, membrane 404 in FIG. 5B overcoats active region 402, but only a portion of the surface of working electrode 400, with membrane 408 overcoating active region 406, the surface of membrane 404, and the remaining portions of the surface of working electrode 400 not overcoated by membrane 404. In some embodiments, active regions 402 and 406 can also be laterally offset from each other.

[0192] Membrane 408 is permeable to the analyte and any additional components required to facilitate the enzymatic reaction within active region 406. In contrast, membrane 404 is permeable to the product formed within active region 406. That is, the analyte reacts within active region 406 to form a first product, which then diffuses through membrane 404 and subsequently reacts further within active region 402 to form a second product. The second product is then detectable upon electron exchange with working electrode 400.

[0193] According to some embodiments, the first membrane polymer and the second membrane polymer can be different from each other. According to some embodiments, the first membrane polymer can be cross-linked polyvinylpyridine. In embodiments of the present disclosure, cross-linked polyvinylpyridine is readily permeable to acetaldehyde. The second membrane polymer can be a cross-linked polyvinylpyridine-co-styrene polymer in which some of the pyridine nitrogen atoms are functionalized with non-cross-linked poly(ethylene glycol) tails and some of the pyridine nitrogen atoms are functionalized with alkylsulfonic acid groups. Such second membrane polymers are readily permeable to both glucose and ethanol.

[0194] In still yet other embodiments, multiple protein switches can be positioned within the active regions of separate working electrodes. In this manner, signals associated with the enzymatic reactions occurring within each active region can be measured individually by interrogating each working electrode simultaneously or at different times. The signals associated with each active region can then be correlated with the concentration of a separate analyte.

[0195] As previously discussed herein, a membrane (i.e., a mass transport limiting membrane) may overcoat one or more of the active areas in a sensor to increase biocompatibility and modify analyte flux to the active area. Such a membrane may be present in any of the sensors disclosed herein. Because different analytes exhibit varying permeability values ​​within a given membrane, a sensor configured to analyze multiple analytes may exhibit different sensitivities for each analyte. One approach to handling different sensitivity values ​​may involve utilizing membranes of different thicknesses across each active area. While this approach is feasible, it may be difficult to achieve from a manufacturing standpoint. That is, it may be difficult to vary the membrane thickness at different locations using typical dip-coating methods used for membrane placement. Another possible approach is to use active areas of different sizes for each analyte.

[0196] In some embodiments, a sensor featuring two or more protein switches disposed on separate working electrodes may include a sensor tail including at least a first working electrode and a second working electrode, a first active region disposed on the surface of the first working electrode, a second active region disposed on the surface of the second working electrode, a multicomponent membrane overcoating the first active region, and a homogeneous membrane overcoating the second active region. The first active region includes a first protein switch reactive with a first polymer and a first analyte, and the second protein switch includes a second protein switch reactive with a second polymer and a second analyte. The first protein switch and the second protein switch are different and reactive with different analytes. The multicomponent membrane includes at least a first membrane polymer and a second membrane polymer that are different from each other. The homogeneous membrane includes one of the first membrane polymer and the second membrane polymer.

[0197] The particular configuration of multi-component membranes described above may comprise bilayer membranes in some embodiments or mixtures of membrane polymers in other embodiments. Surprisingly, bilayer and mixed membranes may function to equalize analyte permeability, as discussed further below.

[0198] Sensors of the present disclosure in which two distinct active regions are disposed on separate working electrodes may use a sensor configuration similar to or variations on the sensor configuration described above in FIG. 3. For example, in some embodiments, a counter / reference electrode may replace separate counter and reference electrodes in sensors having two or more working electrodes. Similarly, the configuration and arrangement of layers in sensors in which two distinct active regions are disposed on separate working electrodes may differ from the configuration and arrangement of layers depicted in FIG. 3.

[0199] For example, in some embodiments, a sensor having multiple working electrodes can include active regions in which an electron transfer agent is covalently bound to a polymer within each active region. In some or other embodiments, such sensors can feature a first protein switch covalently bound to a polymer within the first active region and a second protein switch covalently bound to a polymer within the second active region. Also, in certain embodiments, the first protein switch can include a first protein switch comprising an analyte-binding domain capable of binding to troponin and a glucose oxidase domain responsive to glucose, and the second protein switch can include a second protein switch comprising an analyte-binding domain capable of binding to BNP and a lactate oxidase domain responsive to lactate. Alternatively, the lactate oxidase domain can be replaced with a glucose oxidase domain responsive to glucose.

[0200] In still yet further embodiments, a sensor having multiple working electrodes can include a sensor tail configured for insertion into tissue.

[0201] In some embodiments, a bilayer membrane can overcoat a first active area of ​​one of the working electrodes. The bilayer membrane includes a first membrane polymer and a second membrane polymer layered on top of each other across the active area. In more specific embodiments, the first membrane polymer can be disposed directly on the active area of ​​the first working electrode, and the second membrane polymer can be disposed on the first membrane polymer to define a bilayer membrane. In such embodiments, the second membrane polymer is present in a homogeneous membrane disposed on the second working electrode. In some embodiments, such a bilayer configuration can be prepared by coating only the first working electrode with the first membrane polymer (e.g., by spray coating, painting, inkjet printing, roller coating, etc.) and then simultaneously coating both working electrodes with the second membrane polymer (e.g., by dip coating or a similar technique). In other embodiments, a bilayer membrane can be constructed as described above, with the first membrane polymer disposed on the second working electrode.

[0202] 6 shows an exemplary schematic diagram of a portion of a sensor having two working electrodes and featuring a bilayer membrane overcoating one of the two working electrodes, which is compatible for use in some embodiments of the present disclosure herein. As shown in FIG. 6, the sensor features a sensor tail 600 having working electrodes 614a and 614b disposed on opposite sides of a substrate 612. An active region 618a is disposed on working electrode 614a, and an active region 618b is disposed on working electrode 614b. Active regions 618a and 618b contain different protein switches and are configured to assay for different analytes in accordance with the disclosure herein. 6 shows active region 618a and active region 618b as generally disposed on opposite sides of substrate 612, it is understood that active region 618a and active region 618b may be laterally spaced (offset) from one another on opposite sides of substrate 612. A laterally spaced configuration for active regions 618a and 618b may be particularly advantageous for overcoating each active region 618a and 618b with a mass transport limiting film, as discussed hereinafter.

[0203] As further shown in Figure 6, active region 618a is overcoated with membrane layer 620. Membrane layer 620 is a homogeneous membrane comprising a single membrane polymer. Active region 618b is overcoated with bilayer membrane 621, comprising membrane layer 621a in direct contact with active region 618b and membrane layer 621b overlying membrane layer 621a. Membrane layer 621a and membrane layer 621b comprise different membrane polymers. In certain embodiments described above, membrane layer 620 and membrane layer 621b can comprise the same membrane polymer.

[0204] According to one or more embodiments, a sensor having multiple active regions on a separate working electrode, one of which is overcoated with a bilayer membrane, may exhibit equalized or independently variable analyte permeability. That is, the sensor may have sensitivity to two different analytes that are closer to each other than they would be if the bilayer membrane were not present. In such a sensor configuration, an active region overcoated with a homogeneous membrane (e.g., membrane layer 620 in FIG. 6 ) may exhibit an analyte permeability to a first analyte that is characteristic of that particular membrane polymer. Surprisingly, a bilayer membrane (e.g., bilayer membrane 621 in FIG. 6 ) may contain a membrane polymer that does not negatively affect the permeability of a second analyte (i.e., a membrane polymer with a neutral permeability impact), thereby allowing other membrane polymers, including the bilayer membrane, to exhibit their characteristic permeability to a second analyte equivalent to that in the absence of the first membrane polymer. Thus, according to various embodiments, the membrane polymer having a neutral permeability influence and the membrane polymer comprising the homogeneous membrane may comprise the same polymer.

[0205] In some specific embodiments, the membrane polymer having a neutral permeability influence can comprise the inner layer of a bilayer membrane. Thus, according to such embodiments, the inner layer of the bilayer membrane and the homogeneous membrane can comprise the same membrane polymer. In other specific embodiments, the outer layer of the bilayer membrane and the homogeneous membrane can comprise the same membrane polymer.

[0206] In still yet other embodiments, the multi-component membrane can include a mixture (homogeneous blend) of a first membrane polymer and a second membrane polymer. Such a sensor configuration can be similar in appearance to the sensor configuration shown in FIG. 6, except for the replacement of bilayer membrane 621 with a mixed membrane containing two different membrane polymers in a homogeneous blend. Similar to the sensor including a bilayer membrane disposed in one of the active regions, a homogeneous membrane including one of the first membrane polymer or the second membrane polymer of the mixed membrane can overcoat the other active region on the second working electrode.

[0207] Similar to bilayer membranes, a mixed membrane containing a membrane polymer that has a neutral effect on the permeability of a second analyte can allow the mixed membrane to exhibit permeability to the second analyte that is primarily characteristic of the other membrane polymer in the mixture. Thus, according to various embodiments of the present disclosure, one of the membrane polymers of the homogeneous membrane and the mixed membrane can be selected so that the permeability of the second analyte through the mixed membrane is not substantially altered by the membrane polymer. In certain embodiments, the first active region can include a first protein switch comprising an analyte-binding domain capable of binding to troponin and a glucose oxidase domain responsive to glucose, and the second active region can include a second protein switch comprising an analyte-binding domain capable of binding to BNP and a lactate oxidase domain responsive to lactate. Thus, according to such embodiments, the first active region containing the first protein switch can be overcoated with the mixed membrane, and the second active region containing the second protein switch can be overactivated by a homogeneous (single-component membrane polymer) membrane. In yet other embodiments, the second active region can comprise a polymer, albumin, and a second protein switch covalently bound to the polymer. In even more specific embodiments, the homogeneous membrane overcoating the second active region can comprise at least a cross-linked homopolymer or copolymer of polyvinylpyridine, and the mixed membrane overcoating the first active region can also comprise a homopolymer or copolymer of polyvinylpyridine.

[0208] As mentioned above, bilayer and mixed membranes can equalize analyte permeability within the sensors described herein, where two or more active regions can be spatially separated from one another and overcoated with different mass transport-limiting membranes. Specifically, bilayer and mixed membranes can equalize analyte permeability within sensors containing two or more active regions with separate working electrodes and different protein switches, with at least one active region disposed on each working electrode. Thus, such membranes can advantageously allow sensor sensitivity to be independently varied for each analyte. The membrane thickness and / or the relative ratio of the first membrane polymer to the second membrane polymer represent other parameters that can be varied to tailor the characteristic analyte permeability at each working electrode.

[0209] Thus, a method for using a sensor containing two working electrodes can include exposing the sensor to a fluid containing at least one analyte. The sensor includes a sensor tail including at least a first working electrode and a second working electrode. A first active region is disposed on the surface of the first working electrode, and a second active region is disposed on the surface of the second working electrode. The first active region includes a first protein switch reactive with a first polymer and a first analyte, and the second active region includes a second protein switch reactive with a second polymer and a second analyte. The first protein switch and the second protein switch are different. A multi-component membrane overcoats the first active region, and a homogeneous membrane overcoats the second active region. The multi-component membrane includes at least a first membrane polymer and a second membrane polymer that are different from each other, and the homogeneous membrane includes one of the first membrane polymer or the second membrane polymer. The method further includes obtaining a first signal at or above the oxidation-reduction potential of the first active area, obtaining a second signal at or above the oxidation-reduction potential of the second active area, and correlating the first signal with the concentration of the first analyte in the fluid and correlating the second signal with the concentration of the second analyte in the fluid, wherein the first signal is proportional to the concentration of the first analyte in the fluid and the second signal is proportional to the concentration of the second analyte in the fluid.

[0210] In other embodiments, the first signal and the second signal can be measured at different times. Thus, in such embodiments, a potential can be applied alternately to the first and second working electrodes. In other embodiments, the first and second signals can be measured simultaneously via the first and second channels, where a potential can be applied to both electrodes simultaneously.

[0211] In some embodiments, the sensor system may also contain a reactant storage device that can act as a reservoir holding a reactant for the enzyme portion of the protein switch, where the reactant is not available from the analyte-containing sample. Examples of reactants include glucose, lactate, and the like. The reactant storage device may release the reactant over an extended period of time. The storage device may be capable of delivering the reactant to the protein switch for at least 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, or 50 minutes, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours, or 1, 2, 3, 4, 5, 6, or 7 days, or 2, 3, or 4 weeks, or 2, 3, 4, 5, or 6 months. The enzyme storage device may include a mechanical or osmotic device for sustained delivery of the reactant from the enzyme storage device. The enzyme storage device may include a mechanical or osmotic device for sustained delivery of the reactant from the enzyme storage device. The enzyme storage device may also have combinations of the above to provide sustained release of the reactants. Other methods and techniques may also be used for sustained release of the reactants.

[0212] The storage device may have pores or holes through which the reactive agent is released. The pores or holes through which the reactive agent may be released to the protein switch may be small in diameter, allowing delivery of the reactive agent from the storage device over an extended period of time. Such holes or pores may be made by etching, laser machining, drilling, and / or conventional processes.

[0213] A membrane or coating can be placed over the pores or holes of the storage device to extend the time of delivery of the reactant from the storage device. The storage device can be constructed from a porous material, or a portion of the storage device can be constructed from a porous material, which can be coated with a polymer coating and / or polymer membrane that delays the delivery of the reactant from the storage device, extending the time of release of the reactant. The coating can be made from a polyurethane dispersion (such as BAYHDROL™), an acrylic latex dispersion, and a polymer dispersion such as EUDRAGIT®.

[0214] The reactant storage device may provide for delivery of the reactant via an osmotic delivery system. A "standard osmotic delivery system" that may be used is an elementary osmotic pump (EOP) system. EOPs are well known. This system may be used to deliver reactants, particularly those that provide "erratic" or incomplete release profiles. See, for example, Felix Theeuwes, "Elementary Osmotic Pump," Journal of Pharmaceutical Sciences, Vol. 64, No. 12, pp. 1987-1991, December 1975. The reactant storage device may contain an osmotic inducer or osmogen, including water-soluble compounds suitable for inducing osmotic pressure, i.e., pharmaceutically acceptable, pharmacologically inert, water-soluble compounds as referred to in pharmacopeias such as the United States Pharmacopoeia, as well as Remington, "Science and Practice of Pharmacy," 19th Edition, Mack Publishing Company, Easton, Pa. (1995). The osmotic inducer may be selected from pharmaceutically acceptable water-soluble salts of inorganic or organic acids or non-ionic organic compounds, such as carbohydrates or amino acids, that are highly water-soluble. Osmotic inducers may also include inorganic salts such as magnesium chloride or magnesium sulfate, lithium chloride, sodium chloride or potassium chloride, lithium hydrogen phosphate, sodium hydrogen phosphate or potassium hydrogen phosphate, lithium dihydrogen phosphate, sodium dihydrogen phosphate or potassium dihydrogen phosphate, salts of organic acids such as sodium acetate or potassium acetate, magnesium succinate, sodium benzoate, sodium citrate or sodium ascorbate; carbohydrates such as mannitol, sorbitol, arabinose, ribose, xylose, glucose, fructose, mannose, galactose, sucrose, maltose, lactose, raffinose; water-soluble amino acids such as glycine, leucine, alanine, or methionine; urea, etc., and mixtures thereof. The amount of osmogen used will depend on the particular osmogen used and may range from about 1% to about 60% by weight of the reactant mixture.The osmotic delivery system may also include a polymer, such as those described above as drug-eluting polymers. The osmotic delivery system may also include a coating and / or membrane that acts as a semi-permeable barrier between the enzyme and the reactant. Other suitable membranes and / or coatings are known and may be used.

[0215] The reactant storage device can include or be an element of a microfluidic system for delivering reactants to a protein switch. Microfluidic systems for sustained delivery of reactants to enzymes are well known to those skilled in the art. See, for example, U.S. Patent Nos. 9,194,859 and 8,460,607; Madou, "Fundamentals of Microfabrication: Science of Miniaturization," 2nd Edition (Hardcover), CRC, 2002; and microfluidic devices disclosed in Nguyen et al., "Fundamentals and Applications of Microfluidics," Artech House Publishers (2002).

[0216] E. Methods of Using Protein Switch-Containing Devices and Sensor Systems Devices containing one or more protein switches may detect, identify, and / or monitor any of the analytes described in Section B below. For example, devices containing protein switches may be used to monitor patients at risk for an adverse cardiac event. For example, a device may be capable of detecting cardiac troponin, and the device may be used to monitor patients at risk for myocardial infarction. The device may also be used to monitor cardiac patients after surgery or other procedures to monitor cardiac function and provide early warning of a potential adverse cardiac event.

[0217] In some embodiments, devices containing protein switches can be used to monitor a subject for exposure to an infectious agent. For example, the devices can be used to monitor a patient after acute exposure to an infectious agent and / or after the progression of an infection. In other embodiments, the devices can be used to monitor a subject's response to anti-infective drugs and / or anti-infective procedures administered to the subject to treat an infectious disease. In still further embodiments, the devices can also be used to personalize treatment so that subjects receive sufficient treatment to treat their sepsis or infection.

[0218] In still yet further embodiments, the devices may also be used to monitor drug treatment in subjects being treated with one or more drugs, hi yet other embodiments, devices containing protein switches may be used to detect the presence of one or more drugs and / or drug metabolites.

[0219] Devices containing protein switches can be used to automatically detect analytes. The devices can detect changes in the amount or level of the analyte in a bodily fluid. The devices can detect the rate of change of the analyte in a bodily fluid. The devices can detect when the analyte reaches a threshold level or amount. Additionally, the devices can automatically monitor multiple analytes in a subject's bodily fluid.

[0220] The present disclosure herein will be better understood from the following experimental details. However, those skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative, as more fully described in the claims that follow hereinafter. Unless otherwise indicated, the present disclosure is not limited to specific procedures, materials, etc., which may vary as such. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. [Example]

[0221] Example 1: Creation and identification of protein switches A glucose dehydrogenase (GDH) mutant library was prepared, including GDH polypeptides developed using GDH-105 (Codexis, Redwood, CA) as a scaffold. Approximately 2,800 GDH mutants were prepared. The mutants were screened for glucose activity against one or more of the analytes listed in Table 4 below. Glucose activity was determined by an NADH kinetic assay using a spectrometer set at UV 340 or UV 360 at various time intervals or by endpoint measurement of gluconate using a RapidFire mass spectrometer.

[0222] [Table 6]

[0223] Table 5 shows the protein switches identified as binding to one or more of the test analytes and whether binding of the analyte to the protein switch inhibited or activated glucose activity.

[0224] [Table 7]

[0225] Example 2: Evolution of a protein switch for warfarin GDH-2016 (also known as Rd2bb), a protein switch having the amino acid sequence of SEQ ID NO: 11, was evolved using routine techniques known in the art to create a library of mutants. Approximately 3024 GDH mutants were prepared.

[0226] Each mutant was screened for glucose activity and warfarin inhibition (% inhibition) by combining it with a reaction mixture containing 1.25 g / L glucose, 5 g / L NAD, 20% HTP GDH-NAD lysate with or without 0.1 g / L warfarin in 50 mM NaPO at pH 7.0. Glucose activity was assessed by NADH kinetic assay using a spectrometer (Molecular Device SpectraMax M5) set at UV 360 and read at 1-minute intervals over 8 minutes. The top mutants that showed improved inhibition (compared to Rd2bb) were grown at shake flask scale, and each mutant was further examined for warfarin inhibition (Ki) by combining it with a reaction mixture containing shake flask powder of GDH-NAD loaded at 0-10 g / L glucose, 25 g / L NAD+, 0-0.5 g / L warfarin, and a range of 0.5-2.5 g / L depending on the individual mutant, in 50 mM NaPO4 at pH 7.0. As shown in Table 6 below, protein switches exhibiting low Ki (high glucose inhibition) and high glucose activity (e.g., catalytic efficiency) were identified. These protein switches were determined to have the amino acid sequences shown in SEQ ID NOs: 1-10. The protein switch having the sequence of SEQ ID NO: 1 (GDH-2025; also known as Rd3bb) was selected for further evolution.

[0227] [Table 8]

[0228] A similar technique to that described above was used to evolve the protein switch, GDH-2025. Approximately 1680 mutants were prepared. Each mutant was screened for glucose activity and glucose inhibition (Ki) by combining it with a reaction mixture containing 3.5 g / L glucose, 12.5 NAD+, 10% HTP GDH-NAD lysate, and 0.003 g / L warfarin sodium in 50 mM NaPO4 at pH 7.0. Glucose activity was assessed using a spectrometer (Molecular Device SpectraMax M5) set at UV 360 and read at 1-minute intervals over an 8-minute period. The top mutants that showed improved inhibition (compared to Rd3bb) were grown at shake flask scale, and each mutant was further examined for warfarin inhibition (Ki) by combining it with a reaction mixture containing shake flask powder of 0-10 g / L glucose, 25 g / L NAD+, 0-0.5 g / L warfarin, and 0.5 g / L GDH-NAD in 50 mM NaPO4 at pH 7.0. As shown in Table 7 below, protein switches exhibiting low Ki (high glucose inhibition) and high glucose activity (e.g., catalytic efficiency) were identified. These protein switches were determined to have the amino acid sequences shown in SEQ ID NOs: 12-19. The protein switch (GDH-2028; also known as Rd4bb) having the sequence of SEQ ID NO: 15 was selected for further evolution.

[0229] [Table 9]

[0230] [Example 3] Protein switch sensor Sensor Preparation: Using the monolayer active region system, glucose-responsive active regions for experiments were prepared for GDH-105 (control), GDH-2004 (methotrexate (MTX)), GDH-2007 (MTX), GDH-2008 (MTX), GDH-2009 (MTX), GDH-2010 (MTX), GDH-2011 (MTX), GDH-2015 (MTX), GDH-2018 (T3), GDH-2019 (T3), GDH-2004 (cortisol (CT)), GDH-2005 (CT), GDH-2004 (warfarin (WF)), GDH-2009 (WF), GDH-2013 (WF), GDH-2016 (WF), and GDH-2024 (WF). The GDH was obtained from Codexis, Redwood City, CA. The carbon working electrode was coated with an active area of ​​monolayer composition (Table 8 below). The active area (0.11 mm 2 After placement of the PVP film, the active area was allowed to cure for 3 days at room temperature. A PVP film was then applied to the working electrode using a film-coating solution containing 4 mL of 100 mg / mL polyvinylpyridine and 200 μL of 100 mg / mL PEG-DGE 400. The film was placed over the active area (3 × 1 mm dipping per second) and allowed to cure for 2 days at 25°C and 60% relative humidity.

[0231] [Table 10]

[0232] In-beaker calibration: Glucose sensing analysis was performed on sensors containing GDH-105 and the above-mentioned protein switches, GDH-2004, GDH-2007, GDH-2008, GDH-2009, GDH-2010, GDH-2011, GDH-2015, GDH-2018, GDH-2019, GDH-2005, GDH-2013, GDH-2016, and GDH-2024, by immersing the electrodes in 100 mM PBS buffer containing varying concentrations of glucose (1, 2, 3, 5, 7, 10, 15, 20, 25, and 30 mM glucose) at room temperature. Figure 7 shows the response of each of the sensors from GDH-105, GDH-2004, GDH-2007, GDH-2008, GDH-2009, GDH-2010, GDH-2011, GDH-2015, GDH-2018, GDH-2019, GDH-2005, GDH-2013, GDH-2016, and GDH-2024. As shown in Table 9 below, each of the GDHs exhibits a measurable response to increasing glucose concentrations, with sensitivity varying from 0.09 to 0.73 nA per mM glucose. Figure 7 shows the linear sensitivity response of the GDH-105, GDH-2004, GDH-2007, GDH-2008, GDH-2009, GDH-2010, GDH-2011, GDH-2015, GDH-2018, GDH-2019, GDH-2005, GDH-2013, GDH-2016, and GDH-2024 sensors based on in-beaker calibration, confirming the positive driving force.

[0233] [Table 11]

[0234] In-beaker stability: The in-beaker stability (long-term stability) of sensors based on GDH-105 and the above-mentioned protein switches, GDH-2004, GDH-2007, GDH-2008, GDH-2009, GDH-2010, GDH-2011, GDH-2015, GDH-2018, GDH-2019, GDH-2005, GDH-2013, GDH-2016, and GDH-2024, was assessed in 100 mM PBS with 30 mM glucose at 33°C, as shown in Figure 8. As shown in Table 10, after 4 days, each signal underwent a decline in sensitivity (a decrease in stability for detecting glucose) at different rates. The signal declines for the GDH-105 and GDH-2018 sensors were substantially smaller than those for the other tested sensors.

[0235] [Table 12]

[0236] [Example 4] Warfarin sensor Warfarin Sensor: A single-layer active region system was used to prepare glucose-responsive active regions of GDH-105 and GDH-2016 for the experiments. GDH was obtained from Codexis, Redwood City, CA. A carbon working electrode was coated with an active region of single-layer composition (Table 8). After active region placement, the active region was allowed to cure at room temperature for 3 days.

[0237] Experimental procedure: The electrode was placed in a test beaker containing PBS buffer. +GDH-105 and GDH-2016 sensors were tested by immersion in 100 mM PBS buffer at 33°C supplemented with glucose (5 mM), followed by the addition of glucose (1 mM). Both GDH-105 and GDH-2016 sensors showed a response to the addition of glucose. Warfarin was then added and tested at different concentrations (10, 30, 80, 180, and 300 μM warfarin) in the test beaker. Figures 9A and 9B show the inhibition of the GDH-2016 sensor with increasing warfarin concentrations (approximately 55% inhibition at 300 μM warfarin), compared with no effect on the GDH-105 sensor.

[0238] Evolutionary Warfarin Sensor: The single-layer active region system was used to prepare the glucose-responsive active regions of GDH-105 (control), GDH-2025(WF), GDH-2026(WF), GDH-2027(WF), and GDH-2028(WF) for the experiments. GDH was obtained from Codexis, Redwood City, CA. A carbon working electrode was coated with an active region of single-layer composition (Table 8). The active region (0.33 mm) was 0.01 mm thick. 2 After placement of the PVP film, the active area was allowed to cure for 3 days at room temperature. A PVP film was then applied to the working electrode using a film-coating solution containing 4 mL of 100 mg / mL polyvinylpyridine and 200 μL of 100 mg / mL PEG-DGE 400. The film was placed over the active area (3 × 1 mm dipping per second) and allowed to cure for 2 days at 25°C and 60% relative humidity.

[0239] In-beaker calibration: Glucose sensing analysis was performed on the GDH-105, GDH-2025, GDH-2026, GDH-2027, and GDH-2028 sensors prepared as described above by immersing the electrodes in 100 mM PBS buffer containing varying concentrations of glucose (1, 2, 3, 5, 7, 10, 15, 20, 25, and 30 mM glucose) at room temperature. Figure 10 shows the response of each of the GDH-105, GDH-2025, GDH-2026, GDH-2027, and GDH-2028 sensors. As shown in Table 11, each of the GDHs exhibited a measurable response to increasing glucose concentrations, varying in sensitivity from 0.09 to 1.53 nA per mM glucose. FIG. 10 shows the linear sensitivity response of sensors with GDH-105, GDH-2025, GDH-2026, GDH-2027 and GDH-2028 based on in-beaker calibration, confirming the positive driving force.

[0240] [Table 13]

[0241] In-beaker stability: The in-beaker stability (long-term stability) of sensors based on GDH-105, GDH-2025, GDH-2026, GDH-2027, and GDH-2028 was assessed in 100 mM PBS with 30 mM glucose at 33°C, as shown in Figure 11. As shown in Table 12, after one day, each of the signals underwent a decline in sensitivity (a decrease in stability for detecting glucose) at different rates. The decline in signal for the GDH-105 sensor was substantially smaller than for the other sensors tested.

[0242] [Table 14]

[0243] Evolved warfarin sensors: Glucose-responsive active regions GDH-105, GDH-2016, GDH-2025, GDH-2026, GDH-2027, and GDH-2028 were prepared using a single-layer active region system. GDH was obtained from Codexis, Redwood City, CA. A carbon working electrode was attached to the active region (0.33 mm) of single-layer composition. 2 ) (Table 8). After placement of the active area, the active area was allowed to cure at room temperature for 3 days.

[0244] Experimental procedure: The electrode was placed in a test beaker containing PBS buffer. + Testing of sensors based on GDH-105, GDH-2016, GDH-2025, GDH-2026, GDH-2027, and GDH-2028 was performed by immersion in 100 mM PBS buffer at 33°C supplemented with glucose (10 mM), followed by the addition of glucose (5 mM). Sensors based on GDH-105, GDH-2016, GDH-2025, GDH-2026, GDH-2027, and GDH-2028 showed a response to the addition of glucose. Warfarin was then added and examined at different concentrations (10, 30, 80, 180, and 300 μM warfarin) in test beakers. Figures 12A and 12B and Figure 13 show the inhibition of GDH-105, GDH-2016, GDH-2025, GDH-2026, GDH-2027, and GDH-2028 sensors with increasing warfarin concentrations, compared to GDH-105 sensors, which remained unaffected. As shown in Table 13, the percentage inhibition of warfarin varied for GDH-105 sensors, GDH-2025, GDH-2026, GDH-2027, and GDH-2028.

[0245] [Table 15]

[0246] All publications, patents, and patent applications discussed and cited herein are incorporated herein by reference in their entirety. It is understood that the embodiments disclosed herein are not limited to the particular methodology, protocols, and materials described, as these may vary. It is also understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present disclosure, which is limited only by the appended claims.

[0247] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein which equivalents are intended to be encompassed by the following claims.

[0248] For reasons of completeness, various aspects of the disclosure are set out in the following numbered sections: Item 1. A protein switch comprising at least one non-naturally occurring polypeptide having: (a) at least one analyte-binding domain capable of binding to at least one analyte; and (b) at least one oxidase domain or dehydrogenase domain having oxidase activity or dehydrogenase activity and capable of binding to or reacting with at least one reactive agent, wherein (i) the analyte that binds to the analyte-binding domain is different from the reactive agent that binds to or reacts with the oxidase domain or the dehydrogenase domain; and (ii) when the analyte binds to the analyte-binding domain, the oxidase activity or the dehydrogenase activity is altered.

[0249] Item 2. The protein switch according to Item 1, wherein the oxidase is glucose oxidase or lactate oxidase.

[0250] Item 3. The protein switch according to Item 1, wherein the dehydrogenase is glucose dehydrogenase or lactate dehydrogenase.

[0251] Item 4. The protein switch according to any one of Items 1 to 3, wherein the activity of the oxidase or the dehydrogenase is reduced when the analyte-binding domain binds to an analyte.

[0252] Item 5. The protein switch according to any one of Items 1 to 3, wherein the activity of the oxidase or the dehydrogenase increases when the analyte-binding domain binds to an analyte.

[0253] Item 6. The protein switch according to any one of Items 4 to 5, wherein when the analyte-binding domain binds to an analyte, the activity of the oxidase or the dehydrogenase increases or decreases as a result of competitive inhibition, uncompetitive inhibition, or non-competitive inhibition.

[0254] Item 7. The protein switch according to any one of Items 4 to 5, wherein when the analyte-binding domain binds to an analyte, the activity of the oxidase or the dehydrogenase is reduced as a result of competitive inhibition.

[0255] Item 8. The protein switch according to any one of Items 1 to 7, wherein the analyte is warfarin, cortisol, methotrexate, or triiodothyronine.

[0256] Item 9. The protein switch according to any one of Items 1 to 8, wherein the reactant is glucose or lactic acid.

[0257] Item 10. A protein switch comprising at least seven mutations at amino acid positions 96, 155, 156, 159, 170, 198, and 252 of SEQ ID NO:20.

[0258] Item 11. Cysteine, phenylalanine, methionine, tryptophan, or tyrosine at amino acid position 96 of SEQ ID NO: 20; alanine, glycine, isoleucine, leucine, or valine at amino acid position 155 of SEQ ID NO: 20; threonine or serine at amino acid position 156 of SEQ ID NO: 20; threonine or serine at amino acid position 159 of SEQ ID NO: 20; a lysine, arginine, or histidine at amino acid position 170 of SEQ ID NO: 20; Glutamic acid or aspartic acid at amino acid position 198 of SEQ ID NO:20; and Alanine, glycine, isoleucine, leucine, or valine at amino acid position 252 of SEQ ID NO: 20 Item 11. The protein switch according to Item 10, comprising:

[0259] Item 12. Glycine, isoleucine, leucine, or valine at amino acid position 11 of SEQ ID NO: 20; Glycine, isoleucine, leucine, or valine at amino acid position 22 of SEQ ID NO: 20; Asparagine or glutamine at amino acid position 45 of SEQ ID NO: 20; an alanine, glycine, isoleucine, or leucine at amino acid position 48 of SEQ ID NO: 20; an aspartic acid or glutamic acid at amino acid position 55 of SEQ ID NO: 20; alanine, glycine, isoleucine, leucine, or valine at amino acid position 98 of SEQ ID NO: 20; Phenylanine, tryptophan, or tyrosine at amino acid position 137 of SEQ ID NO: 20; an alanine, glycine, leucine, or valine at amino acid position 141 of SEQ ID NO: 20; an alanine, glycine, isoleucine, or leucine at amino acid position 149 of SEQ ID NO: 20; an alanine, glycine, isoleucine, or valine at amino acid position 154 of SEQ ID NO: 20; threonine or serine at amino acid position 166 of SEQ ID NO: 20; Glycine, isoleucine, leucine, or valine at amino acid position 173 of SEQ ID NO: 20; threonine or serine at amino acid position 184 of SEQ ID NO: 20; histidine, leucine, or arginine at amino acid position 195 of SEQ ID NO: 20; threonine or serine at amino acid position 219 of SEQ ID NO: 20; an asparagine or glutamine at amino acid position 240 of SEQ ID NO: 20; and / or Alanine, glycine, isoleucine, leucine, or valine at amino acid position 251 of SEQ ID NO: 20 Item 12. The protein switch of item 11, further comprising one or more of:

[0260] Item 13. A protein switch comprising an amino acid sequence having at least 80% identity to SEQ ID NO:11.

[0261] Clause 14. The protein switch of Clause 13, comprising an amino acid sequence having at least 85% identity to SEQ ID NO:11.

[0262] Item 15. The protein switch according to any one of Items 13 to 14, comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 11.

[0263] Item 16. The protein switch according to any one of Items 13 to 15, comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 11.

[0264] Item 17. The protein switch according to any one of Items 13 to 16, comprising an amino acid sequence having at least 96% identity to SEQ ID NO: 11.

[0265] Item 18. The protein switch according to any one of Items 13 to 17, comprising an amino acid sequence having at least 97% identity to SEQ ID NO: 11.

[0266] Item 19. The protein switch according to any one of Items 13 to 18, comprising an amino acid sequence having at least 98% identity to SEQ ID NO: 11.

[0267] Item 20. The protein switch according to any one of Items 13 to 19, comprising an amino acid sequence having at least 99% identity to SEQ ID NO: 11.

[0268] Item 21. The protein switch according to any one of Items 13 to 20, comprising an amino acid sequence having at least 100% identity to SEQ ID NO: 11.

[0269] Item 22. The protein switch according to any one of Items 13 to 21, comprising any one of the amino acid sequences of SEQ ID NOs: 1 to 10 or 12 to 19.

[0270] Item 23. A composition or kit comprising at least one protein switch according to Item 1, 10, 11 or 13 and at least one reactive agent.

[0271] Item 24. The composition according to Item 23, wherein the reactant is glucose or lactic acid.

[0272] Item 25. A method for detecting an analyte, comprising: providing a protein switch comprising at least one polypeptide having (a) at least one analyte-binding domain capable of binding to at least one analyte; and (b) at least one oxidase or dehydrogenase domain having oxidase or dehydrogenase activity and capable of binding to or reacting with at least one reactive agent, wherein (i) the analyte that binds to the analyte-binding domain is different from the reactive agent that binds to or reacts with the oxidase or dehydrogenase domain; and (ii) when the analyte binds to the analyte-binding domain, the oxidase activity or the dehydrogenase activity is altered; contacting the protein switch with a fluid containing a reactive agent specific for the protein switch, wherein the analyte binding domain binds to the analyte in the fluid, thereby altering the oxidase activity or the dehydrogenase activity; and detecting a change in the rate of degradation of the reactive agent by the oxidase domain or the dehydrogenase domain of the protein switch. A method comprising:

[0273] Item 26. The method according to Item 25, wherein the oxidase is glucose oxidase or lactate oxidase.

[0274] Item 27. The method according to Item 25, wherein the dehydrogenase is glucose dehydrogenase or lactate dehydrogenase.

[0275] Item 28. The method according to any one of Items 26 to 27, wherein the reactant is glucose or lactic acid.

[0276] Item 29. A method for detecting an analyte, comprising: Providing a protein switch according to any one of items 10, 11 or 13; contacting the protein switch with a fluid containing at least one protein switch-specific reactive agent, wherein the analyte binding domain binds to an analyte in the fluid, thereby altering the oxidase or dehydrogenase activity; and detecting a change in the rate of degradation of the reactive agent by the oxidase domain or the dehydrogenase domain of the protein switch. A method comprising:

[0277] Clause 30. The method of clause 29, wherein the protein switch comprises an amino acid sequence having at least 85% identity to SEQ ID NO:11.

[0278] Clause 31. The method of clause 29 or 30, wherein the protein switch comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 11.

[0279] Item 32. The method of any one of Items 29 to 31, wherein the protein switch comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 11.

[0280] Item 33. The method of any of Items 29 to 32, wherein the protein switch comprises an amino acid sequence having at least 96% identity to SEQ ID NO: 11.

[0281] Item 34. The method of any of Items 29 to 33, wherein the protein switch comprises an amino acid sequence having at least 97% identity to SEQ ID NO: 11.

[0282] Item 35. The method of any of Items 29 to 34, wherein the protein switch comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 11.

[0283] Item 36. The method of any of Items 29 to 35, wherein the protein switch comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 11.

[0284] Item 37. The method of any of Items 29 to 36, wherein the protein switch comprises an amino acid sequence having at least 100% identity to SEQ ID NO: 11.

[0285] Item 38. The method according to any one of Items 29 to 37, wherein the protein switch comprises any one of the amino acid sequences of SEQ ID NOs: 1 to 10 or 12 to 19.

[0286] Item 39. The method according to any one of Items 29 to 38, wherein the oxidase is glucose oxidase or lactate oxidase.

[0287] Item 40. The method according to any one of Items 29 to 39, wherein the dehydrogenase is glucose dehydrogenase or lactate dehydrogenase.

[0288] Item 41. The method according to any one of Items 29 to 40, wherein the reactant is glucose or lactic acid.

[0289] Item 42. The method according to any one of Items 29 to 41, wherein the analyte is warfarin, cortisol, methotrexate, or triiodothyronine.

[0290] Item 43. A system for detecting or monitoring an analyte concentration, comprising a sensor control device and a signal detection device, wherein the sensor control device comprises at least one sensor comprising the protein switch according to Item 1, 10, 11 or 13.

[0291] Item 44. The system according to Item 43, wherein the oxidase is glucose oxidase or lactate oxidase.

[0292] Item 45. The system according to Item 43, wherein the dehydrogenase is glucose dehydrogenase or lactate dehydrogenase.

[0293] Item 46. The system according to any one of Items 43 to 45, wherein the protein switch comprises an amino acid sequence having at least 85% identity to SEQ ID NO: 11.

[0294] Item 47. The system according to any one of Items 43 to 46, wherein the protein switch comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 11.

[0295] Item 48. The system according to any one of Items 43 to 47, wherein the protein switch comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 11.

[0296] Item 49. The system according to any one of Items 43 to 48, wherein the protein switch comprises an amino acid sequence having at least 96% identity to SEQ ID NO: 11.

[0297] Item 50. The system according to any one of Items 43 to 49, wherein the protein switch comprises an amino acid sequence having at least 97% identity to SEQ ID NO: 11.

[0298] Item 51. The system according to any one of Items 43 to 50, wherein the protein switch comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 11.

[0299] Item 52. The system according to any one of Items 43 to 51, wherein the protein switch comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 11.

[0300] Item 53. The system according to any one of Items 43 to 52, wherein the protein switch comprises an amino acid sequence having at least 100% identity to SEQ ID NO: 11.

[0301] Item 54. The system according to any one of Items 43 to 53, wherein the protein switch comprises any one of the amino acid sequences of SEQ ID NOs: 1 to 10 or 12 to 19.

[0302] Clause 55. An analyte monitoring system comprising: a sensor comprising a substrate, a working electrode, and the protein switch of clause 1, 10, 11, or 13, wherein at least a portion of the sensor is adapted for implantation and in intimate contact with a bodily fluid, the sensor constructed and arranged to provide a signal representative of a level of an analyte in the bodily fluid; and a signal detection device for receiving the signal, wherein the signal is generated by contact of the analyte with the protein switch.

[0303] Item 56. The analyte monitoring system of Item 55, wherein the oxidase is glucose oxidase or lactate oxidase.

[0304] Clause 57. The analyte monitoring system of clause 55, wherein the dehydrogenase is glucose dehydrogenase or lactate dehydrogenase.

[0305] Item 58. The analyte monitoring system of Items 55 to 57, wherein the protein switch comprises an amino acid sequence having at least 85% identity to SEQ ID NO:11.

[0306] Item 59. The analyte monitoring system of Items 55 to 58, wherein the protein switch comprises an amino acid sequence having at least 90% identity to SEQ ID NO:11.

[0307] Item 60. The analyte monitoring system of Items 55 to 59, wherein the protein switch comprises an amino acid sequence having at least 95% identity to SEQ ID NO:11.

[0308] Item 61. The analyte monitoring system of Items 55 to 60, wherein the protein switch comprises an amino acid sequence having at least 96% identity to SEQ ID NO:11.

[0309] Item 62. The analyte monitoring system of Items 55 to 61, wherein the protein switch comprises an amino acid sequence having at least 97% identity to SEQ ID NO:11.

[0310] Item 63. The analyte monitoring system of Items 55 to 62, wherein the protein switch comprises an amino acid sequence having at least 98% identity to SEQ ID NO:11.

[0311] Item 64. The analyte monitoring system of Items 55 to 63, wherein the protein switch comprises an amino acid sequence having at least 99% identity to SEQ ID NO:11.

[0312] Item 65. The analyte monitoring system of Items 55 to 64, wherein the protein switch comprises an amino acid sequence having at least 100% identity to SEQ ID NO:11.

[0313] Item 66. The analyte monitoring system according to Items 55 to 65, wherein the protein switch comprises any one of the amino acid sequences of SEQ ID NOs: 1 to 10 or 12 to 19.

[0314] Item 67. The analyte monitoring system of Items 55 to 66, wherein the analyte is warfarin, cortisol, methotrexate, or triiodothyronine.

Claims

1. 1. A protein switch comprising at least one non-naturally occurring polypeptide having: (a) at least one analyte binding domain capable of binding to at least one analyte; and (b) at least one oxidase or dehydrogenase domain having oxidase or dehydrogenase activity and capable of binding to or reacting with at least one reactive agent, wherein (i) the analyte that binds to the analyte binding domain is different from the reactive agent that binds to or reacts with the oxidase or dehydrogenase domain; and (ii) when the analyte binds to the analyte binding domain, the oxidase activity or the dehydrogenase activity is altered.

2. The protein switch according to claim 1 , wherein the oxidase is glucose oxidase or lactate oxidase.

3. The protein switch according to claim 1 , wherein the dehydrogenase is glucose dehydrogenase or lactate dehydrogenase.

4. The protein switch according to any one of claims 1 to 3, wherein when the analyte-binding domain binds to an analyte, the activity of the oxidase or the dehydrogenase is reduced.

5. The protein switch according to any one of claims 1 to 3, wherein the activity of the oxidase or the dehydrogenase increases when the analyte-binding domain binds to an analyte.

6. The protein switch according to any one of claims 4 to 5, wherein when the analyte-binding domain binds to an analyte, the activity of the oxidase or the dehydrogenase is increased or decreased as a result of competitive inhibition, uncompetitive or non-competitive inhibition.

7. The protein switch according to any one of claims 4 to 5, wherein when the analyte-binding domain binds to an analyte, the activity of the oxidase or the dehydrogenase is reduced as a result of competitive inhibition.

8. The protein switch according to any one of claims 1 to 7, wherein the analyte is warfarin, cortisol, methotrexate or triiodothyronine.

9. The protein switch according to any one of claims 1 to 8, wherein the reactant is glucose or lactate.

10. A protein switch comprising at least seven mutations at amino acid positions 96, 155, 156, 159, 170, 198 and 252 of SEQ ID NO:

20.

11. A cysteine, phenylalanine, methionine, tryptophan, or tyrosine at amino acid position 96 of SEQ ID NO:20; an alanine, glycine, isoleucine, leucine, or valine at amino acid position 155 of SEQ ID NO: 20; Threonine or serine at amino acid position 156 of SEQ ID NO: 20; Threonine or serine at amino acid position 159 of SEQ ID NO: 20; a lysine, arginine, or histidine at amino acid position 170 of SEQ ID NO: 20; Glutamic acid or aspartic acid at amino acid position 198 of SEQ ID NO:20; and Alanine, glycine, isoleucine, leucine, or valine at amino acid position 252 of SEQ ID NO:20 The protein switch of claim 10, comprising:

12. Glycine, isoleucine, leucine, or valine at amino acid position 11 of SEQ ID NO:20; Glycine, isoleucine, leucine, or valine at amino acid position 22 of SEQ ID NO:20; an asparagine or glutamine at amino acid position 45 of SEQ ID NO:20; an alanine, glycine, isoleucine, or leucine at amino acid position 48 of SEQ ID NO:20; an aspartic acid or glutamic acid at amino acid position 55 of SEQ ID NO:20; an alanine, glycine, isoleucine, leucine, or valine at amino acid position 98 of SEQ ID NO:20; A phenylalanine, tryptophan, or tyrosine at amino acid position 137 of SEQ ID NO:20; an alanine, glycine, leucine, or valine at amino acid position 141 of SEQ ID NO: 20; an alanine, glycine, isoleucine, or leucine at amino acid position 149 of SEQ ID NO: 20; an alanine, glycine, isoleucine, or valine at amino acid position 154 of SEQ ID NO: 20; Threonine or serine at amino acid position 166 of SEQ ID NO: 20; Glycine, isoleucine, leucine, or valine at amino acid position 173 of SEQ ID NO: 20; Threonine or serine at amino acid position 184 of SEQ ID NO: 20; histidine, leucine, or arginine at amino acid position 195 of SEQ ID NO: 20; Threonine or serine at amino acid position 219 of SEQ ID NO: 20; Asparagine or glutamine at amino acid position 240 of SEQ ID NO: 20; and / or Alanine, glycine, isoleucine, leucine, or valine at amino acid position 251 of SEQ ID NO:20 12. The protein switch of claim 11, further comprising one or more of:

13. A protein switch comprising an amino acid sequence having at least 80% identity to SEQ ID NO:

11.

14. 14. The protein switch of claim 13, comprising an amino acid sequence having at least 85% identity to SEQ ID NO:

11.

15. The protein switch according to any one of claims 13 to 14, comprising an amino acid sequence having at least 90% identity to SEQ ID NO:

11.

16. The protein switch according to any one of claims 13 to 15, comprising an amino acid sequence having at least 95% identity to SEQ ID NO:

11.

17. The protein switch according to any one of claims 13 to 16, comprising an amino acid sequence having at least 96% identity to SEQ ID NO:

11.

18. The protein switch according to any one of claims 13 to 17, comprising an amino acid sequence having at least 97% identity to SEQ ID NO:

11.

19. The protein switch according to any one of claims 13 to 18, comprising an amino acid sequence having at least 98% identity to SEQ ID NO:

11.

20. The protein switch according to any one of claims 13 to 19, comprising an amino acid sequence having at least 99% identity to SEQ ID NO:

11.

21. The protein switch according to any one of claims 13 to 20, comprising an amino acid sequence having at least 100% identity to SEQ ID NO:

11.

22. The protein switch according to any one of claims 13 to 21, comprising an amino acid sequence of any one of SEQ ID NOs: 1 to 10 or 12 to 19.

23. 19. A composition or kit comprising at least one protein switch according to claim 1, 10, 11 or 13 and at least one reactive agent.

24. 24. The composition of claim 23, wherein the reactant is glucose or lactic acid.

25. 1. A method for detecting an analyte, comprising: providing a protein switch comprising at least one polypeptide having (a) at least one analyte-binding domain capable of binding to at least one analyte; and (b) at least one oxidase or dehydrogenase domain having oxidase or dehydrogenase activity and capable of binding to or reacting with at least one reactive agent, wherein (i) the analyte that binds to the analyte-binding domain is different from the reactive agent that binds to or reacts with the oxidase or dehydrogenase domain; and (ii) the oxidase activity or the dehydrogenase activity is altered when the analyte binds to the analyte-binding domain; contacting the protein switch with a fluid containing a reactive agent specific for the protein switch, wherein the analyte binding domain binds to the analyte in the fluid, thereby altering the oxidase activity or the dehydrogenase activity; and detecting a change in the rate of degradation of the reactive agent by the oxidase domain or the dehydrogenase domain of the protein switch. A method comprising:

26. 26. The method of claim 25, wherein the oxidase is glucose oxidase or lactate oxidase.

27. 26. The method of claim 25, wherein the dehydrogenase is glucose dehydrogenase or lactate dehydrogenase.

28. The method according to any one of claims 26 to 27, wherein the reactant is glucose or lactic acid.

29. 1. A method for detecting an analyte, comprising: providing a protein switch according to any one of claims 10, 11 or 13; contacting the protein switch with a fluid containing a reactive agent specific for the protein switch, wherein the analyte binding domain binds to the analyte in the fluid, thereby altering the oxidase activity or the dehydrogenase activity; and detecting a change in the rate of degradation of the reactive agent by the oxidase domain or the dehydrogenase domain of the protein switch. A method comprising:

30. 30. The method of claim 29, wherein the protein switch comprises an amino acid sequence having at least 85% identity to SEQ ID NO:

11.

31. 31. The method of claim 29 or 30, wherein the protein switch comprises an amino acid sequence having at least 90% identity to SEQ ID NO:

11.

32. The method of any of claims 29 to 31, wherein the protein switch comprises an amino acid sequence having at least 95% identity to SEQ ID NO:

11.

33. The method of any of claims 29 to 32, wherein the protein switch comprises an amino acid sequence having at least 96% identity to SEQ ID NO:

11.

34. The method of any of claims 29 to 33, wherein the protein switch comprises an amino acid sequence having at least 97% identity to SEQ ID NO:

11.

35. The method of any of claims 29 to 34, wherein the protein switch comprises an amino acid sequence having at least 98% identity to SEQ ID NO:

11.

36. The method of any of claims 29 to 35, wherein the protein switch comprises an amino acid sequence having at least 99% identity to SEQ ID NO:

11.

37. The method of any of claims 29 to 36, wherein the protein switch comprises an amino acid sequence having at least 100% identity to SEQ ID NO:

11.

38. The method of any of claims 29 to 37, wherein the protein switch comprises an amino acid sequence of any one of SEQ ID NOs: 1 to 10 or 12 to 19.

39. 39. The method of any one of claims 29 to 38, wherein the oxidase is glucose oxidase or lactate oxidase.

40. 40. The method of any one of claims 29 to 39, wherein the dehydrogenase is glucose dehydrogenase or lactate dehydrogenase.

41. The method of any one of claims 29 to 40, wherein the reactant is glucose or lactic acid.

42. The method of any one of claims 29 to 41, wherein the analyte is warfarin, cortisol, methotrexate or triiodothyronine.

43. 14. A system for detecting or monitoring an analyte concentration, comprising a sensor control device and a signal detection device, wherein the sensor control device comprises at least one sensor comprising the protein switch of claim 1, 10, 11 or 13.

44. 44. The system of claim 43, wherein the oxidase is glucose oxidase or lactate oxidase.

45. 44. The system of claim 43, wherein the dehydrogenase is glucose dehydrogenase or lactate dehydrogenase.

46. The system of any of claims 43 to 45, wherein the protein switch comprises an amino acid sequence having at least 85% identity to SEQ ID NO:

11.

47. The system of any of claims 43 to 46, wherein the protein switch comprises an amino acid sequence having at least 90% identity to SEQ ID NO:

11.

48. The system of any of claims 43 to 47, wherein the protein switch comprises an amino acid sequence having at least 95% identity to SEQ ID NO:

11.

49. The system of any of claims 43 to 48, wherein the protein switch comprises an amino acid sequence having at least 96% identity to SEQ ID NO:

11.

50. The system of any of claims 43 to 49, wherein the protein switch comprises an amino acid sequence having at least 97% identity to SEQ ID NO:

11.

51. The system of any of claims 43 to 50, wherein the protein switch comprises an amino acid sequence having at least 98% identity to SEQ ID NO:

11.

52. The system of any of claims 43 to 51, wherein the protein switch comprises an amino acid sequence having at least 99% identity to SEQ ID NO:

11.

53. The system of any of claims 43 to 52, wherein the protein switch comprises an amino acid sequence having at least 100% identity to SEQ ID NO:

11.

54. The system of any one of claims 43 to 53, wherein the protein switch comprises an amino acid sequence of any one of SEQ ID NOs: 1 to 10 or 12 to 19.

55. 14. A sensor comprising a substrate, a working electrode, and the protein switch of claim 1, 10, 11, or 13, at least a portion of the sensor adapted for implantation and in intimate contact with a bodily fluid, the sensor constructed and arranged to provide a signal representative of a level of an analyte in the bodily fluid; and a signal detection device for receiving a signal, the signal being generated by contact of the analyte with the protein switch.

56. 56. The analyte monitoring system of claim 55, wherein the oxidase is glucose oxidase or lactate oxidase.

57. 56. The analyte monitoring system of claim 55, wherein the dehydrogenase is glucose dehydrogenase or lactate dehydrogenase.

58. 58. The analyte monitoring system of claims 55-57, wherein the protein switch comprises an amino acid sequence having at least 85% identity to SEQ ID NO:

11.

59. 59. The analyte monitoring system of claims 55-58, wherein the protein switch comprises an amino acid sequence having at least 90% identity to SEQ ID NO:

11.

60. 60. The analyte monitoring system of claims 55-59, wherein the protein switch comprises an amino acid sequence having at least 95% identity to SEQ ID NO:

11.

61. 61. The analyte monitoring system of claims 55-60, wherein the protein switch comprises an amino acid sequence having at least 96% identity to SEQ ID NO:

11.

62. 62. The analyte monitoring system of claims 55-61, wherein the protein switch comprises an amino acid sequence having at least 97% identity to SEQ ID NO:

11.

63. 63. The analyte monitoring system of claims 55-62, wherein the protein switch comprises an amino acid sequence having at least 98% identity to SEQ ID NO:

11.

64. 64. The analyte monitoring system of claims 55-63, wherein the protein switch comprises an amino acid sequence having at least 99% identity to SEQ ID NO:

11.

65. 65. The analyte monitoring system of claims 55-64, wherein the protein switch comprises an amino acid sequence having at least 100% identity to SEQ ID NO:

11.

66. 66. The analyte monitoring system of claims 55-65, wherein the protein switch comprises the amino acid sequence of any one of SEQ ID NOs: 1-10 or 12-19.

67. 67. The analyte monitoring system of claims 55-66, wherein the analyte is warfarin, cortisol, methotrexate, or triiodothyronine.

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