Detection of analytes by protein switches

Abstract

Substances, compositions, methods, devices, systems, and apparatuses for detecting an analyte are disclosed, including, for example, protein switches and their use in in vivo sensors. The protein switches can be used to determine analyte levels that are diagnostic of a subject's health and / or well-being.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of priority of U.S. Application No. 62 / 909,411, filed on October 2, 2019, the content of which is incorporated herein by reference.

[0003] Incorporation by Reference of Electronically Submitted Materials

[0004] This application contains a sequence listing that has been submitted electronically in ASCII format and is hereby incorporated 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. Technical Field

[0005] The present disclosure relates to material compositions, methods, devices, systems, and apparatuses for detecting and / or monitoring analytes, such as, for example, protein switches and their use in in - vivo sensors. The protein switches can be used to determine the levels of one or more analytes that are diagnostic and / or prognostic of the health and / or well - being of a subject. Background Art

[0006] The detection or monitoring of target analytes is beneficial for the health and / or well - being of certain individuals. Many disease conditions involve analytes that can be measured to diagnose and / or monitor the disease state of 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.

[0007] Key requirements for analyte detection and monitoring are the specificity and sensitivity of the assay. These requirements are particularly important when the target analyte is present in small or limited amounts or concentrations in a fluid or biological sample. Generally, the specificity and / or sensitivity of the assay are provided by capture and / or detection antibodies used to detect one or more analytes of interest. Diagnostic assays using this method are well - known and are commonly widely used in enzyme - linked immunosorbent sandwich assays (ELISA) or immunoassays. Generating antibodies that provide sufficient specificity and / or sensitivity against certain analytes can be challenging (and expensive).

[0008] Current assays for detecting target analytes for prognostic, diagnostic, and / or monitoring purposes have several limitations that restrict their widespread use in clinical, non - clinical (e.g., wearable), and point - of - care settings. Additionally, many of these assays require a significant amount of technical expertise and a set of expensive and specific reagents (such as antibodies) as well as sophisticated biomedical infrastructure, which is typically located in a professional laboratory environment.

[0009] Accordingly, there is a need for compositions, devices, systems, and methods that can detect a target analyte in a subject for prognostic, diagnostic, and / or monitoring purposes, which are produced relatively inexpensively, capable of measuring small or limited amounts or concentrations of an analyte in a fluid or biological sample, and suitable for widespread use in clinical, non-clinical, and point-of-care settings. SUMMARY OF THE INVENTION

[0010] 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 reactant, wherein (i) the analyte binding to the analyte-binding domain is different from the reactant binding to or reacting with the oxidase or dehydrogenase domain; and (ii) when the analyte binds to the analyte-binding domain, the oxidase or dehydrogenase activity is altered. In a further aspect, the oxidase is glucose oxidase or lactate oxidase. In another aspect, the dehydrogenase is glucose dehydrogenase or lactate dehydrogenase.

[0011] In a further aspect, in the above protein switch, when the analyte-binding domain binds the analyte, the activity of the oxidase or dehydrogenase is decreased. In yet a further aspect, in the above protein switch, when the analyte-binding domain binds the analyte, the activity of the oxidase or dehydrogenase is increased. In still yet a further aspect, when the analyte-binding domain binds the analyte, the activity of the oxidase or dehydrogenase is increased or decreased due to competitive inhibition, uncompetitive inhibition, or non-competitive inhibition. In still yet another aspect, when the analyte-binding domain binds the analyte, the activity of the oxidase or dehydrogenase is decreased due to competitive inhibition.

[0012] In yet another aspect, in any of the above protein switches, the analyte is warfarin, cortisol, methotrexate, or triiodothyronine.

[0013] In still yet a further aspect, in any of the above protein switches, the reactant is glucose or lactate.

[0014] In another embodiment, the present disclosure relates to a protein switch comprising at least 7 mutations at amino acid positions 96, 155, 156, 159, 170, 198, and 252 of SEQ ID NO:20.

[0015] In some aspects, the protein switch comprises at least the following mutations: (a) cysteine, phenylalanine, methionine, tryptophan, or tyrosine at amino acid position 96 of SEQ ID NO:20; (b) alanine, glycine, isoleucine, leucine, or valine at amino acid position 155 of SEQ ID NO:20; (c) threonine or serine at amino acid position 156 of SEQ ID NO:20; (d) threonine or serine at amino acid position 159 of SEQ ID NO:20; (e) lysine, arginine, or histidine at amino acid position 170 of SEQ ID NO:20; (f) glutamate or aspartate at amino acid position 198 of SEQ ID NO:20; and (g) alanine, glycine, isoleucine, leucine, or valine at amino acid position 252 of SEQ ID NO:20.In other aspects, in addition to the above mutations (a)-(g), the protein switch further comprises one or more 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.

[0016] In yet another aspect, the protein switch comprises 7 mutations at amino acid positions 96, 155, 156, 159, 170, 198, and 252 of SEQ ID NO:20. In these aspects, the protein switch comprises cysteine or phenylalanine at amino acid position 96 of SEQ ID NO:20, alanine at amino acid position 155, serine at amino acid position 156, tyrosine at amino acid position 159, lysine at amino acid position 170, glutamic acid at amino acid position 198, and lysine or valine at amino acid position 252 of SEQ ID NO:20.

[0017] In another aspect, 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 aspects, the protein switch comprises cysteine or phenylalanine at amino acid position 96 of SEQ ID NO:20, leucine at amino acid position 98, alanine at amino acid position 155, serine at amino acid position 156, tyrosine at amino acid position 159, lysine at amino acid position 170, glutamate at amino acid position 198, and lysine or valine at amino acid position 252.

[0018] In another aspect, 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 aspects, the protein switch comprises cysteine or phenylalanine at amino acid position 96 of SEQ ID NO:20, alanine at amino acid position 155, serine at amino acid position 156, tyrosine at amino acid position 159, lysine at amino acid position 170, glutamate at amino acid position 198, threonine at amino acid position 219, and lysine or valine at amino acid position 252.

[0019] In another aspect, 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 aspects, the protein switch comprises cysteine or phenylalanine at amino acid position 96 of SEQ ID NO:20, alanine at amino acid position 155, serine at amino acid position 156, tyrosine at amino acid position 159, lysine at amino acid position 170, arginine at amino acid position 195, glutamate at amino acid position 198, and lysine or valine at amino acid position 252.

[0020] In another aspect, 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 aspects, the protein switch comprises cysteine or phenylalanine at amino acid position 96 of SEQ ID NO:20, valine at amino acid position 141, alanine at amino acid position 155, serine at amino acid position 156, tyrosine at amino acid position 159, lysine at amino acid position 170, glutamate at amino acid position 198, and lysine or valine at amino acid position 252.

[0021] In another aspect, 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 aspects, the protein switch comprises cysteine or phenylalanine at amino acid position 96 of SEQ ID NO:20, alanine at amino acid position 155, serine at amino acid position 156, tyrosine at amino acid position 159, lysine at amino acid position 170, glycine at amino acid position 173, glutamate at amino acid position 198, and lysine or valine at amino acid position 252.

[0022] In another aspect, 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 aspects, the protein switch comprises glutamate at amino acid position 55 of SEQ ID NO:20, cysteine or phenylalanine at amino acid position 96, alanine at amino acid position 155, serine at amino acid position 156, tyrosine at amino acid position 159, lysine at amino acid position 170, glutamate at amino acid position 198, and lysine or valine at amino acid position 252.

[0023] In another aspect, 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 aspects, the protein switch comprises cysteine or phenylalanine at amino acid position 96 of SEQ ID NO:20, tyrosine at amino acid position 137, alanine at amino acid position 155, serine at amino acid position 156, tyrosine at amino acid position 159, lysine at amino acid position 170, glutamate at amino acid position 198, and lysine or valine at amino acid position 252.

[0024] In another aspect, 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 aspects, the protein switch comprises alanine at amino acid position 48 of SEQ ID NO:20, cysteine or phenylalanine at amino acid position 96, alanine at amino acid position 155, serine at amino acid position 156, tyrosine at amino acid position 159, lysine at amino acid position 170, glutamate at amino acid position 198, and lysine or valine at amino acid position 252.

[0025] In another aspect, 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 aspects, the protein switch comprises cysteine or phenylalanine at amino acid position 96 of SEQ ID NO:20, alanine at amino acid position 155, serine at amino acid position 156, tyrosine at amino acid position 159, lysine at amino acid position 170, serine at amino acid position 184, glutamate at amino acid position 198, and lysine or valine at amino acid position 252.

[0026] In another aspect, 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 aspects, the protein switch comprises cysteine or phenylalanine at amino acid position 96 of SEQ ID NO:20, alanine at amino acid position 155, serine at amino acid position 156, tyrosine at amino acid position 159, serine at amino acid position 166, lysine at amino acid position 170, glutamate at amino acid position 198, and lysine or valine at amino acid position 252.

[0027] In another aspect, 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 aspects, the protein switch comprises cysteine or phenylalanine at amino acid position 96 of SEQ ID NO:20, alanine at amino acid position 155, serine at amino acid position 156, tyrosine at amino acid position 159, lysine at amino acid position 170, glutamate at amino acid position 198, glutamine at amino acid position 240, and lysine or valine at amino acid position 252.

[0028] In yet another aspect, 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 aspects, the protein switch comprises asparagine at amino acid position 45 of SEQ ID NO:20, cysteine or phenylalanine at amino acid position 96, alanine at amino acid position 149, alanine at amino acid position 155, serine at amino acid position 156, tyrosine at amino acid position 159, lysine at amino acid position 170, glutamate at amino acid position 198, and lysine or valine at amino acid position 252.

[0029] In yet another aspect, 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 aspects, the protein switch comprises glycine at amino acid position 22 of SEQ ID NO:20, cysteine or phenylalanine at amino acid position 96, glycine at amino acid position 154, alanine at amino acid position 155, serine at amino acid position 156, tyrosine at amino acid position 159, lysine at amino acid position 170, glutamate at amino acid position 198, and lysine or valine at amino acid position 252.

[0030] In yet another aspect, 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 aspects, the protein switch comprises cysteine or phenylalanine at amino acid position 96 of SEQ ID NO:20, valine at amino acid position 141, glycine at amino acid position 154, alanine at amino acid position 155, serine at amino acid position 156, tyrosine at amino acid position 159, lysine at amino acid position 170, glutamate at amino acid position 198, and lysine or valine at amino acid position 252.

[0031] In yet a further aspect, 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 aspects, the protein switch comprises lysine at amino acid position 12 of SEQ ID NO:20, cysteine or phenylalanine at amino acid position 96, alanine at amino acid position 155, serine at amino acid position 156, tyrosine at amino acid position 159, lysine at amino acid position 170, arginine at amino acid position 195, glutamate at amino acid position 198, lysine at amino acid position 251, and lysine or valine at amino acid position 252.

[0032] 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.

[0033] In one aspect, the protein switch comprises an amino acid sequence having at least 85% identity to SEQ ID NO:11.

[0034] In yet another aspect, the protein switch comprises an amino acid sequence having at least 90% identity with SEQ ID NO:11.

[0035] In still yet another aspect, the protein switch comprises an amino acid sequence having at least 95% identity with SEQ ID NO:11.

[0036] In still yet another aspect, the protein switch comprises an amino acid sequence having at least 96% identity with SEQ ID NO:11.

[0037] In still yet another aspect, the protein switch comprises an amino acid sequence having at least 97% identity with SEQ ID NO:11.

[0038] In still yet another aspect, the protein switch comprises an amino acid sequence having at least 98% identity with SEQ ID NO:11.

[0039] In still yet another aspect, the protein switch comprises an amino acid sequence having at least 99% identity with SEQ ID NO:11.

[0040] In still yet another aspect, the protein switch comprises an amino acid sequence having at least 100% identity with SEQ ID NO:11.

[0041] In still yet another aspect, the protein switch comprises the amino acid sequence of any one of SEQ ID NO.1 - 10 or 12 - 19.

[0042] In yet another embodiment, the present disclosure relates to a composition or a kit, the composition or the kit comprising at least one of the above protein switches and at least one reactant. In one aspect, the reactant in the composition or the kit is glucose or lactate.

[0043] In yet another embodiment, the present disclosure relates to a method for detecting an analyte. The method comprises the following steps:

[0044] a. Providing a protein switch, which comprises at least one polypeptide, the 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 reactant, wherein (i) the analyte binding to the analyte - binding domain is different from the reactant binding to or reacting with the oxidase or dehydrogenase domain; and (ii) when the analyte binds to the analyte - binding domain, the oxidase or dehydrogenase activity changes;

[0045] b. Contacting the protein switch with a fluid comprising a reactant specific for the protein switch, wherein the analyte binding domain binds the analyte in the fluid, whereby the oxidase or dehydrogenase activity is altered; and

[0046] c. Detecting a change in the rate of breakdown of the reactant by the oxidase or dehydrogenase domain of the protein switch.

[0047] In one aspect of the above method, the oxidase is glucose oxidase or lactate oxidase.

[0048] In another aspect of the above method, the dehydrogenase is glucose dehydrogenase or lactate dehydrogenase.

[0049] In yet another aspect of the above method, the reactant is glucose or lactate.

[0050] In still yet another aspect of the above method, the analyte is warfarin, cortisol, methotrexate or triiodothyronine.

[0051] In yet another embodiment, the present disclosure relates to a method for detecting an analyte. The method comprises the following steps:

[0052] a. Providing a protein switch as described above;

[0053] b. Contacting the protein switch with a fluid comprising a reactant specific for the protein switch, wherein the analyte binding domain binds the analyte in the fluid, whereby the oxidase or dehydrogenase activity is altered; and

[0054] c. Detecting a change in the rate of breakdown of the reactant by the oxidase or dehydrogenase domain of the protein switch.

[0055] In one aspect of the above method, the protein switch comprises an amino acid sequence having at least 80% identity to SEQ ID NO:11.

[0056] In one aspect of the above method, the protein switch comprises an amino acid sequence having at least 85% identity to SEQ ID NO:11.

[0057] In another aspect of the above method, the protein switch comprises an amino acid sequence having at least 90% identity to SEQ ID NO:11.

[0058] In yet another aspect of the above method, the protein switch comprises an amino acid sequence having at least 95% identity to SEQ ID NO:11.

[0059] In yet another aspect of the above method, the protein switch comprises an amino acid sequence having at least 96% identity with SEQ ID NO:11.

[0060] In yet another aspect of the above method, the protein switch comprises an amino acid sequence having at least 97% identity with SEQ ID NO:11.

[0061] In yet another aspect of the above method, the protein switch comprises an amino acid sequence having at least 98% identity with SEQ ID NO:11.

[0062] In yet another aspect of the above method, the protein switch comprises an amino acid sequence having at least 99% identity with SEQ ID NO:11.

[0063] In yet another aspect of the above method, the protein switch comprises an amino acid sequence having at least 100% identity with SEQ ID NO:11.

[0064] In yet another aspect of the above method, the protein switch comprises the amino acid sequence of any one of SEQ ID NO.1-10 or 12-19.

[0065] In yet another aspect of the above method, the oxidase is glucose oxidase or lactate oxidase.

[0066] In yet another aspect of the above method, the dehydrogenase is glucose dehydrogenase or lactate dehydrogenase.

[0067] In yet another aspect of the above method, the reactant is glucose or lactate.

[0068] In still yet another aspect of the above method, the analyte is warfarin, cortisol, methotrexate or triiodothyronine.

[0069] In yet another embodiment, the present disclosure relates to a system for detecting or monitoring the concentration of an analyte. The system includes a sensor control device and a signal detection device, wherein the sensor control device includes at least one sensor, and the at least one sensor includes one of the above protein switches.

[0070] In one aspect, the oxidase in the protein switch used in the sensor is glucose oxidase or lactate oxidase.

[0071] In another aspect, the dehydrogenase in the protein switch used in the sensor is glucose dehydrogenase or lactate dehydrogenase.

[0072] In yet another embodiment, the present disclosure relates to an analyte monitoring system. The analyte monitoring system includes: a sensor including a substrate, one or more working electrodes, and one of the above-described protein switches, at least a portion of the sensor being adapted for implantation and in intimate contact with a body fluid, the sensor being configured and arranged to generate a signal representative of the level of analyte in the body fluid; and signal detection means for receiving the signal, wherein the signal is generated by contact of the analyte with the protein switch.

[0073] In one aspect, the oxidase in the protein switch used in the analyte monitoring system is glucose oxidase or lactate oxidase.

[0074] In another aspect, the dehydrogenase in the protein switch used in the analyte monitoring system is glucose dehydrogenase or lactate dehydrogenase.

[0075] In yet another aspect, the analyte detected in the analyte monitoring system is warfarin, cortisol, methotrexate, or triiodothyronine. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1 A diagram showing an illustrative sensing system incorporating a sensor including at least one protein switch of the present disclosure.

[0077] Figure 2A A diagram showing an illustrative two-electrode sensor configuration having a single working electrode. Figure 2B and 2C A diagram showing an illustrative three-electrode sensor configuration having a single working electrode.

[0078] Figure 3 A diagram showing an illustrative sensor configuration having two working electrodes, a reference electrode, and a counter electrode.

[0079] Figure 4 A diagram showing an illustrative sensor configuration in which two different active regions are provided on the surface of a single working electrode.

[0080] Figure 5A and 5B A diagram showing an illustrative working electrode in which a first active region is provided directly on the surface of the working electrode and a second active region is separated from the working electrode by a membrane.

[0081] Figure 6 A diagram showing an illustrative schematic of a portion of a sensor having two working electrodes and characterized by a bilayer membrane coating one of the two working electrodes.

[0082] Figure 7Shows the linear sensitivity response of sensors containing the following protein switches to increasing glucose concentration: 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 the control.

[0083] Figure 8 Shows the beaker (long-term) stability of sensors containing the following 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 the control.

[0084] Figure 9A and 9B Shows the inhibition of sensors containing the protein switch GDH-2016 to increasing warfarin concentration, while the sensor containing GDH-105 (control) is not affected.

[0085] Figure 10 Shows the linear sensitivity response based on beaker calibration of sensors containing GDH-105 (control) and sensors containing the protein switches GDH-2025, GDH-2026, GDH-2027, and GDH-2028.

[0086] Figure 11 Shows the beaker (long-term) stability of sensors containing the protein switches GDH-2025, GDH-2026, GDH-2027, and GDH-2028. The sensor containing GDH-105 is the control.

[0087] Figure 12A and 12B Shows the inhibition of sensors containing the following protein switches to increasing warfarin concentration: GDH-2016, GDH-2025, GDH-2026, GDH-2027, and GDH-20288. The sensor containing GDH-105 is the control.

[0088] Figure 13Shown is the inhibition of increased warfarin concentration by sensors comprising: GDH-2016, GDH-2025, GDH-2026, GDH-2027, and GDH-20288. The sensor comprising GDH-105 is a control.

[0089] Figure 14 Shown are the sequences of SEQ ID NO. 1-20. Detailed Description

[0090] Exemplary aspects of substance compositions, methods, devices (e.g., sensors), systems, and apparatuses for detecting and / or monitoring a target analyte in a body fluid or biological sample are provided herein. It should be understood that the teachings of the present disclosure are not limited to the specific aspects described and may, of course, vary. It should also be understood that the terms used herein are for the purpose of describing specific aspects only and are not intended to be limiting.

[0091] For purposes of illustration and not limitation, a protein switch can be designed to identify, detect, and / or quantify one or more target analytes in a body fluid or biological sample. The exact nature and configuration of the protein switch can vary. The protein switch can have an analyte binding portion for detecting the target analyte and an enzyme portion that generates a product (e.g., a signal) or a change in the generated product (e.g., a change in the signal) upon binding the analyte. The protein switch can be utilized in a variety of conditions and configurations, including in a sensor for measuring analyte levels in a subject. The configuration of such a sensor can depend on the analyte being measured and the body fluid in which the device measures its analyte. The sensor can be configured to detect and / or measure an in vivo analyte in a subject. An in vivo sensor can include an insertion tip that can be positioned beneath the skin surface, e.g., penetrate the skin and enter the dermal or subcutaneous region. The sensor can test for analytes in dermal fluid, interstitial fluid, subcutaneous fluid, or blood (e.g., capillary).

[0092] The sensor can include one or more protein switches that bind an analyte (in an analyte binding part or portion) and then generate a product (such as a signal from an enzyme part or portion) that can be detected by the sensor. The protein switches in the sensor can have one activity level (e.g., lower or higher) when the analyte is unbound and a different activity (e.g., higher or lower) when the analyte is bound. For example, analyte binding by the analyte binding part of the protein switch can decrease enzyme activity. Alternatively, analyte binding by the analyte binding part of the protein switch can increase enzyme activity. The change in enzyme activity can be detected (directly or indirectly) by the sensor as a change in the signal based on the amount of the product made by the enzyme. The signal detected by the sensor can be correlated with the amount of the analyte that can be prognostic or diagnostic of a patient's health and / or well-being. Alternatively, the signal detected at the sensor can be correlated with the amount of the analyte for monitoring a condition of a patient's health and / or well-being. The amount of the measured analyte can also be correlated with and / or converted to the amount in blood or other body fluids.

[0093] A. Definition

[0094] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosure, the preferred methods and materials are described below. 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 not intended to be limiting.

[0095] As used herein, the terms “comprise,” “include,” “having,” “has,” “can,” “contain,” and variations thereof are intended to be open-ended conjunctions, terms, or words that do not preclude the possibility of additional acts or structures. Unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” include plural referents. The present disclosure also encompasses other aspects that “comprise” the aspects or elements presented herein, “consisting of,” and “consisting essentially of,” whether explicitly stated or not.

[0096] For the recitation of numerical ranges herein, each intermediate number therebetween having the same degree of precision is explicitly covered. For example, for the range 6 - 9, the numbers 7 and 8 are covered in addition to 6 and 9, and for the range 6.0 - 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 explicitly covered.

[0097] As used herein, "Affimer" generally refers to a peptide that specifically or selectively binds to a target (e.g., an analyte, a target tissue, a target molecule, a target cell, etc.). Generally, an Affimer can be a small peptide or protein that typically has a molecular weight of less than 12 kDa. An Affimer can have the ability to recognize a specific epitope or antigen and can have a binding affinity that can be close to that of an antibody (e.g., in the low nanomolar to picomolar range); however, as used herein, the term "Affimer" does not cover antibodies, immunoglobulins, the Fab region of an antibody, or the Fc region of an antibody. An Affimer can have the same specificity advantages as an antibody, but can be smaller, can be chemically synthesized or chemically modified, and has the advantage of being free of cell culture contaminants.

[0098] As used interchangeably herein, "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 can be converted into electrical signals that can be correlated with the amount, concentration, or level of the analyte in the sample or the activity of an enzyme.

[0099] "Enzyme" refers to a protein or a fragment thereof that has activity (e.g., catalytic activity, enzymatic activity, or enzyme activity) towards one or more reactants (e.g., enzyme substrates). Examples of one or more reactants (e.g., enzyme substrates) are glucose, lactate, glutamate, ascorbic acid, cholesterol, choline acetylcholine, hypoxanthine, norepinephrine, serotonin, phenylethylamine, and e / e - methylhistamine, polyphenols, ethanol, aldehydes, or malic acid.

[0100] As used interchangeably herein, "fluid", "body fluid", "sample", or "biological sample" refers to dermal fluid, interstitial fluid, subcutaneous fluid, or blood (e.g., such as capillary blood) obtained from a subject or patient. In one aspect, the fluid or sample is dermal fluid. In one aspect, the fluid or sample is interstitial fluid. In yet another aspect, the fluid is subcutaneous fluid. In yet another aspect, the fluid or sample is blood (such as capillary blood).

[0101] As used herein, "identical," "identity," or "sequence identity" in the context of two or more polypeptide or polynucleotide sequences can mean that the sequences have a specified percentage of identical residues over a specified region as determined using standard algorithms, taking into account the degree of sequence identity over the comparison window. Identity or sequence identity can be determined using computer algorithms such as GAP, BESTFIT, FASTA, and the BLAST family of programs, as disclosed, for example, 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 (John Wiley & Sons Inc NY, 1995 - 1999), edited by Ausubel et al. Suitably, sequence identity is measured over the entire length of SEQ ID NOs. 1 - 20.

[0102] As used herein, "isolated polynucleotide" can mean a polynucleotide (e.g., a polynucleotide of genomic, cDNA, or synthetic origin or a combination thereof) that, as a result of its origin, is not associated with all or a portion of the polynucleotide with which it would be associated in nature where the "isolated polynucleotide" is found.

[0103] As used herein, "sensing layer" refers to a component of a sensor that includes components that facilitate the electro - oxidation or electro - reduction of a compound directly at the electrode or via a combination of one or more electron transfer agents, one or more cofactors, or one or more electron transfer agents and one or more cofactors. In some aspects of the sensor, the sensing layer is disposed near or on the working electrode.

[0104] As used herein, "sensing region" refers to the active chemical region of a sensor.

[0105] "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, etc.), and humans). In some aspects, the subject can be human or non - human. In some aspects, the subject is human. The subject or patient can undergo one or more forms of treatment.

[0106] As used interchangeably herein, the terms "variant protein", "protein variant", or "variant" mean a protein that differs from a parental protein by at least one amino acid modification. The term "protein variant" can refer to the protein itself, a composition containing the protein, or an amino acid sequence encoding it. In some aspects, a protein variant has at least one amino acid modification compared to the parental or reference protein, such as from about one to about fifty amino acid modifications compared to the parental protein. In some aspects, a protein variant has from about one to about forty amino acid modifications compared to the parental protein. In some aspects, a protein variant has from about one to about thirty amino acid modifications compared to the parental protein. In some aspects, a protein variant has from about one to about twenty amino acid modifications compared to the parental protein. In some aspects, a protein variant has from about one to about ten amino acid modifications compared to the parental protein. In some aspects, a protein variant has from about one to about five amino acid modifications compared to the parental protein. In some aspects, the protein variant sequences herein will have at least about 80% identity to the parental protein sequence. In other aspects, the protein variant sequences herein will have at least about 90% identity. In still other aspects, the protein variant sequences will have at least about 95%, 96%, 97%, 98%, or 99% identity.

[0107] In this disclosure, amino acids are mentioned. In addition to the names of the amino acids, three-letter and single-letter codes are also used herein. For clarity purposes, the amino acids mentioned in this disclosure are referenced as follows: alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, C), glutamine (Gln, 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).

[0108] Unless otherwise defined herein, scientific and technical terms used in conjunction with this disclosure will have the meanings commonly understood by one of ordinary skill in the art. For example, any nomenclature and techniques associated with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry, as well as hybridization described herein are those well known and commonly used in the art. The meaning and scope of the terms should be clear; however, if there is any implicit ambiguity, the definitions provided herein will prevail over any dictionary or extrinsic definition. In addition, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular.

[0109] B. Protein switch

[0110] In some aspects, the present disclosure relates to a substance composition, which may comprise one or more protein switches. Specifically, the protein switch may include, but is not limited to, at least two parts: at least one analyte-binding part (or portion) and at least one enzyme part (or portion). The analyte-binding part regulates the activity of the enzyme part of the protein switch. As will be discussed in further detail herein, when an analyte binds to the analyte-binding part, the activity of the enzyme part changes (e.g., increases or decreases) as a result of competitive inhibition, uncompetitive inhibition, or non-competitive inhibition. In some aspects, when an analyte binds to the analyte-binding part, the activity of the enzyme part increases (when compared to the absence of the analyte). In other aspects, when an analyte binds to the analyte-binding part, the activity of the enzyme part decreases (when compared to the absence of the analyte). This regulation of enzyme activity can provide for the detection of an analyte.

[0111] The protein switches of the present disclosure comprise at least one non-naturally occurring polypeptide having at least two different domains or parts. In some aspects, the at least two different domains or parts may overlap with each other in their sequences (e.g., containing one or more overlapping nucleic acid or amino acid sequences) or overlap with each other in their spatial orientations. The first domain or part is an analyte-binding part (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 target analytes. The second domain or part is an enzyme part comprising at least one oxidase or dehydrogenase domain having enzyme activity (e.g., catalytic activity). In other words, the second domain or part is catalytically active. Additionally, the oxidase or dehydrogenase domain binds to, reacts with, is capable of binding to and / or reacting with one or more reactants and optionally one or more cofactors to generate or produce a product (e.g., a signal) that can be detected, which will be discussed in more detail herein.

[0112] In one aspect, 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 reactant, and the activity of the enzyme (e.g., oxidase or dehydrogenase) increases. Specifically, the amount of the product (e.g., signal) produced increases. The activity of the enzyme can increase (when compared to the wild-type enzyme) 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%, 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%, 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%.

[0113] In yet another aspect, in the presence of an analyte (e.g., when an analyte binding region binds one or more analytes), the oxidase or dehydrogenase domain does not bind or react with one or more reactants or exhibits reduced (e.g., inhibited) binding and / or reaction with one or more reactants, and the activity of the enzyme (e.g., oxidase or dehydrogenase) may be reduced. Specifically, the amount of the product (e.g., signal) produced is reduced. The activity of the enzyme (e.g., oxidase or dehydrogenase) may be reduced (when compared to the wild-type enzyme) 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%, 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%, 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% (e.g., complete inhibition of the activity of the enzyme (e.g., oxidase or dehydrogenase)).

[0114] In one aspect, the analyte binding domain can be derived from any protein or polypeptide that binds or is capable of binding one or more analytes of interest. For example, in one aspect, the analyte binding domain can be derived from one or more affimers, antibodies, peptides, receptors (including full-length and single-chain receptors), small molecules, artificial binding proteins (such as those made using scaffolds, display technologies, etc.), or any functional fragment or variant thereof. In another aspect, 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 aspects, the binding affinity of the analyte binding domain for the analyte allows the protein switch to detect one or more analytes at physiological levels.

[0115] The length of the analyte binding domain is not critical, provided that the analyte binding domain binds or is capable of binding one or more analytes of interest. For example, the length of the analyte binding domain can be 3 contiguous amino acids, 4 contiguous amino acids, 5 contiguous amino acids, 6 contiguous amino acids, 7 contiguous amino acids, 8 contiguous amino acids, 9 contiguous amino acids, 10 contiguous amino acids, 11 contiguous amino acids, 12 contiguous amino acids, 13 contiguous amino acids, 14 contiguous amino acids, 15 contiguous amino acids, 16 contiguous amino acids, 17 contiguous amino acids, 18 contiguous amino acids, 19 contiguous amino acids, 20 contiguous amino acids, 21 contiguous amino acids, 22 contiguous amino acids, 23 contiguous amino acids, 24 contiguous amino acids, 25 contiguous amino acids, 26 contiguous amino acids, 27 contiguous amino acids, 28 contiguous amino acids, 29 contiguous amino acids, 30 contiguous amino acids, 31 contiguous amino acids, 32 contiguous amino acids, 33 contiguous amino acids, 34 contiguous amino acids, 35 contiguous amino acids, 36 contiguous amino acids, 37 contiguous amino acids, 38 contiguous amino acids, 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.

[0116] On the other hand, at least one oxidase or dehydrogenase domain or portion contains an amino acid sequence encoding at least one oxidase and / or at least one dehydrogenase having enzymatic activity (e.g., catalytic activity), and binds to, reacts with, is capable of binding to and / or reacting with at least one reactant and an optional cofactor to produce a product (e.g., a signal). The amino acid sequence encoding the oxidase or dehydrogenase can be a natural (wild-type) sequence derived from, obtained from, 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 natural (wild-type) sequence encoding an oxidase or dehydrogenase having enzymatic activity. As with the analyte-binding domain, the length of the oxidase or dehydrogenase domain is not critical, provided that it encodes an enzyme (e.g., an entire enzyme), a functional fragment thereof, or a variant having enzymatic (e.g., oxidase or dehydrogenase) activity.For example, the length of the oxidase or dehydrogenase domain can 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, 75 consecutive amino acids, 80 consecutive amino acids, 85 consecutive amino acids, 90 consecutive amino acids, 95 consecutive amino acids, 100 consecutive amino acids, 125 consecutive amino acids, 150 consecutive amino acids, 175 consecutive amino acids, 200 consecutive amino acids, 225 consecutive amino acids, 250 consecutive amino acids, 275 consecutive amino acids, 300 consecutive amino acids, 325 consecutive amino acids, 350 consecutive amino acids, 375 consecutive amino acids, 400 consecutive amino acids, 425 consecutive amino acids, 450 consecutive amino acids, 475 consecutive amino acids or 500 consecutive amino acids.

[0117] Enzymes that can be used in at least one oxidase 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 aspects, the oxidase is glucose oxidase. In other aspects, the oxidase is lactate oxidase. In some aspects, the oxidase is glutamate oxidase. In still other aspects, the dehydrogenase is glucose dehydrogenase. In yet other aspects, the dehydrogenase is lactate dehydrogenase. In yet other aspects, the dehydrogenase is glutamate dehydrogenase.

[0118] The oxidases and / or dehydrogenases used in the oxidase and / or dehydrogenase domains can be derived from or encoded by microorganisms such as bacteria, viruses, or fungi. Examples of the sources of the oxidase or dehydrogenase domains used in the protein switches described herein are provided in Tables 1 and 2 below.

[0119] Table 1: Oxidases

[0120] Table 1

[0121]

[0122] Table 2: Dehydrogenases

[0123] Table 2

[0124]

[0125]

[0126] 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, pyrroloquinoline quinone (PQQ), flavin adenine dinucleotide (FAD), nicotinamide adenine dinucleotide (NAD), flavin mononucleotide (FMN)) to produce or generate a detectable product (e.g., a signal) are well known in the art. By way of example, methods for generating a detectable product using glucose oxidase and glucose dehydrogenase will now be disclosed.

[0127] The enzyme glucose oxidase consists of two identical protein subunits and a cofactor (i.e., flavin adenine dinucleotide (FAD)) at its active site. Due to FAD, glucose oxidase catalyzes the oxidation of its reactant glucose at its first hydroxyl group using molecular oxygen as an electron acceptor to produce the products gluconolactone and hydrogen peroxide. The hydrogen peroxide product can be detected using conventional techniques known in the art (such as, for example, electrochemical oxidation at an electrode and the number of electrons transferred detected). The reaction can be outlined as shown below:

[0128] Glucose + Glucose oxidase (GOx)-FAD + → Gluconolactone + GOx-FADH2

[0129] GOx-FADH2 + O2 → GOx-FAD + H2O2

[0130] H2O2 → 2H + + O2 + 2e -

[0131] Alternatively, oxygen consumption can be measured.

[0132] Glucose dehydrogenase can utilize a variety 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. NADH can be electrochemically oxidized at the electrode and the number of electron transfers can be detected. The reaction can be outlined as follows:

[0133] Glucose + Glucose dehydrogenase (GDH)-NAD + → Gluconolactone + GDH-NADH

[0134] NADH → NAD + + H + + 2e -

[0135] The target analyte detected by the protein switch can be prognostic or diagnostic for the health and / or well-being of a patient. Alternatively, or in addition, the target analyte can be used to monitor the health and / or well-being of a patient. For example, the analyte can be procalcitonin (PCT), cardiac troponin (such as cardiac troponin I or cardiac troponin T), creatinine, urea, guanidinosuccinic acid, p-cresol sulfate, an indicator, dimethylamine, CMPF, pseudouridine, oxalic acid, glyoxal, 2-oxoglutaric acid, glucose, lactate, cerebrospinal fluid glucose, glutamate, malic acid, acetylcarnitine, hypoxanthine, sialic acid, creatinine, pseudouridine, hydroxyphenyl lactic 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), 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), triiodothryxin, human growth hormone, cytokines, chemokines, interleukins, procalcitonin, coagulation factors, C-reactive protein (CRP), procalcitonin, soluble triggering receptor expressed on myeloid cells-1 (sTREM-1), pancreatic stone protein (PSP), circulating complement (C3 and C4), ferritin, cholesterol, albumin, and neutrophil gelatinase-associated lipocalin. Analytes diagnostic for cardiac function or heart disease include, for example, cardiac troponin, 2-oxoglutaric acid, creatine kinase (CK-MB), glycogen phosphorylase isoenzyme BD, BNP, myoglobin, ischemia-modified albumin, cardionatrin, and / or lactate dehydrogenase isoenzymes. The analyte can also be a nucleic acid (e.g., microRNA, CpG islands, and other nucleic acid markers in plasma or serum), polypeptide, metabolite, lipid, carbohydrate, or other molecule present in a subject that can be diagnostic for the health and / or well-being of the subject. Analytes diagnostic for renal function or kidney disease include, for example, creatinine and urea. Analytes diagnostic for liver function or liver disease include, for example, glucose, urea, albumin, and creatinine. Analytes diagnostic for neurological function or neurological 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, sialic acid, creatinine, pseudouridine, hydroxyphenyl lactate, hexanoylcarnitine, C-reactive protein (CRP), procalcitonin, soluble triggering receptor expressed on myeloid cells-1 (sTREM-1), pancreatic stone protein (PSP), circulating complement (C3 and C4), ferritin, cholesterol, albumin, cortisol, and neutrophil gelatinase-associated lipocalin. Still other analytes include drugs or drug metabolites. For example, warfarin, methotrexate, cyclosporine A, methotrexate, and cyclosporine. In one aspect, the analyte is warfarin, cortisol, methotrexate, triiodothyronine, cyclosporine A, GFAP, UCH-L1, S100B, NF-L, or cardiac troponin. In yet another aspect, the analyte is warfarin. In still another aspect, the analyte is cortisol. In yet another aspect, the analyte is methotrexate. In still another aspect, the analyte is triiodothyronine. In still another aspect, the analyte is cyclosporine A. In still another aspect, the analyte is GFAP. In still another aspect, the analyte is UCH-L1. In still another aspect, the analyte is S100B. In yet still another aspect, the analyte is NF-L. In still another aspect, the analyte is cardiac troponin (e.g., troponin I or troponin T). In still another aspect, the analyte is troponin I.

[0136] The analyte that binds to the analyte-binding domain of the protein switch and the reactant that binds and / or reacts with the oxidase or dehydrogenase domain must be different (e.g., cannot be the same). For example, if the analyte that binds to the analyte-binding domain is glucose, the reactant that binds or reacts with the oxidase or dehydrogenase domain cannot be glucose and must be a different reactant such as lactate. Alternatively, if the analyte that binds to the analyte-binding domain of the protein switch is warfarin, the reactant that binds or reacts with the oxidase or dehydrogenase can be glucose or lactate.

[0137] As previously discussed herein, when an analyte binds to the analyte-binding domain, the oxidase or dehydrogenase activity changes. In some aspects, 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 aspects, when an analyte binds to the analyte-binding domain, the oxidase or dehydrogenase activity decreases (e.g., the amount of product produced decreases).

[0138] Any reactant known in the art that pairs with an enzyme is suitable for use with the protein switches described herein. Examples of reactants that can be used include glucose or lactate. In some aspects, the reactant is glucose. In other aspects, the reactant is lactate.

[0139] In another aspect, the protein switch of the present disclosure is a glucose dehydrogenase polypeptide. In yet another aspect, the protein switch is a glucose dehydrogenase polypeptide having at least 7 mutations in the following amino acid sequence: MYPDLKGKVVAITGAASGLGKAMAIRFGKEQAKVVINYYSNKQDPNEVKEEVIKAGGEAVVVQGDVTKEEDVKNIVQTAIKEFGTLDIMINNAGLENPVPSHEMPLKDWDKVIGTNLTGAFLGSREAIKYFVENDIKGNVINMSSVHEVIPWPLFVHYAASKGGMKLMTETLALEYAPKGIRVNNIGPGAINTTINAGKFADPKQKADVESMIPMGYIGEPEEIAAVAAWLASKEASYVTGITLFADGGMTQYPSFQAGRG (SEQ ID NO:20).

[0140] In one aspect, the protein switch comprises at least 7 mutations at amino acid positions 96, 155, 156, 159, 170, 198, and 252 of SEQ ID NO:20. The 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.

[0141] Table 3A

[0142] Amino acid position SEQ ID NO:20 Mutation 96 E C, F, M, W, Y 155 F A, G, I, L, V 156 V T, S 159 A T, S 170 E K, R, H 198 G E, D 252 Q A, G, I, L, V

[0143] In another aspect, the protein switch comprises the mutations at amino acid positions 96, 155, 156, 159, 170, 198, and 252 of SEQ ID NO:20 as shown in Table 3A above and 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.

[0144] Table 3B

[0145] Amino acid position SEQ ID NO:20 Mutation 11 A G, I, L, V 22 A G, I, L, V 45 P N, Q 48 V A, G, I, L 55 A D, E 98 P A, G, I, L, V 137 K F, W, Y 141 I A, G, L, V 149 V A, G, I, L 154 L A, G, I, V 166 K S, T 173 A G, I, L, V 184 N S, T 195 I H, L, R 219 G S, T 240 T N, Q 251 T A, G, I, L, V

[0146] In yet another aspect, the protein switch comprises 7 mutations at amino acid positions 96, 155, 156, 159, 170, 198, and 252 of SEQ ID NO:20.

[0147] In another aspect, 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 aspect, 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 aspect, 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 aspect, 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 aspect, 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 aspect, 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 aspect, 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 aspect, 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 aspect, 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 aspect, 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 aspect, the protein switch comprises eight mutations at amino acid positions 96, 155, 156, 159, 170, 198, 240, and 252 of SEQ ID NO:20.

[0148] In yet another aspect, 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 aspect, 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 another aspect, 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.

[0149] In yet a further aspect, 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.

[0150] In another aspect, 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 aspects, the protein switch comprises cysteine or phenylalanine at amino acid position 96 of SEQ ID NO:20, alanine at amino acid position 155, serine at amino acid position 156, tyrosine at amino acid position 159, lysine at amino acid position 170, glutamate at amino acid position 198, and lysine or valine at amino acid position 252.

[0151] In another aspect, 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 aspects, the protein switch comprises cysteine or phenylalanine at amino acid position 96 of SEQ ID NO:20, leucine at amino acid position 98, alanine at amino acid position 155, serine at amino acid position 156, tyrosine at amino acid position 159, lysine at amino acid position 170, glutamate at amino acid position 198, and lysine or valine at amino acid position 252.

[0152] In another aspect, 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 aspects, the protein switch comprises cysteine or phenylalanine at amino acid position 96 of SEQ ID NO:20, alanine at amino acid position 155, serine at amino acid position 156, tyrosine at amino acid position 159, lysine at amino acid position 170, glutamate at amino acid position 198, threonine at amino acid position 219, and lysine or valine at amino acid position 252.

[0153] In another aspect, 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 aspects, the protein switch comprises cysteine or phenylalanine at amino acid position 96 of SEQ ID NO:20, alanine at amino acid position 155, serine at amino acid position 156, tyrosine at amino acid position 159, lysine at amino acid position 170, arginine at amino acid position 195, glutamate at amino acid position 198, and lysine or valine at amino acid position 252.

[0154] In another aspect, 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 aspects, the protein switch comprises cysteine or phenylalanine at amino acid position 96 of SEQ ID NO:20, valine at amino acid position 141, alanine at amino acid position 155, serine at amino acid position 156, tyrosine at amino acid position 159, lysine at amino acid position 170, glutamate at amino acid position 198, and lysine or valine at amino acid position 252.

[0155] In another aspect, 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 aspects, the protein switch comprises cysteine or phenylalanine at amino acid position 96 of SEQ ID NO:20, alanine at amino acid position 155, serine at amino acid position 156, tyrosine at amino acid position 159, lysine at amino acid position 170, glycine at amino acid position 173, glutamate at amino acid position 198, and lysine or valine at amino acid position 252.

[0156] In another aspect, 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 aspects, the protein switch comprises glutamate at amino acid position 55 of SEQ ID NO:20, cysteine or phenylalanine at amino acid position 96, alanine at amino acid position 155, serine at amino acid position 156, tyrosine at amino acid position 159, lysine at amino acid position 170, glutamate at amino acid position 198, and lysine or valine at amino acid position 252.

[0157] In another aspect, 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 aspects, the protein switch comprises cysteine or phenylalanine at amino acid position 96 of SEQ ID NO:20, tyrosine at amino acid position 137, alanine at amino acid position 155, serine at amino acid position 156, tyrosine at amino acid position 159, lysine at amino acid position 170, glutamate at amino acid position 198, and lysine or valine at amino acid position 252.

[0158] In another aspect, 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 aspects, the protein switch comprises alanine at amino acid position 48 of SEQ ID NO:20, cysteine or phenylalanine at amino acid position 96, alanine at amino acid position 155, serine at amino acid position 156, tyrosine at amino acid position 159, lysine at amino acid position 170, glutamate at amino acid position 198, and lysine or valine at amino acid position 252.

[0159] In another aspect, 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 aspects, the protein switch comprises cysteine or phenylalanine at amino acid position 96 of SEQ ID NO:20, alanine at amino acid position 155, serine at amino acid position 156, tyrosine at amino acid position 159, lysine at amino acid position 170, serine at amino acid position 184, glutamate at amino acid position 198, and lysine or valine at amino acid position 252.

[0160] In another aspect, 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 aspects, the protein switch comprises cysteine or phenylalanine at amino acid position 96 of SEQ ID NO:20, alanine at amino acid position 155, serine at amino acid position 156, tyrosine at amino acid position 159, serine at amino acid position 166, lysine at amino acid position 170, glutamate at amino acid position 198, and lysine or valine at amino acid position 252.

[0161] In another aspect, 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 aspects, the protein switch comprises cysteine or phenylalanine at amino acid position 96 of SEQ ID NO:20, alanine at amino acid position 155, serine at amino acid position 156, tyrosine at amino acid position 159, lysine at amino acid position 170, glutamate at amino acid position 198, glutamine at amino acid position 240, and lysine or valine at amino acid position 252.

[0162] In yet another aspect, 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 aspects, the protein switch comprises asparagine at amino acid position 45 of SEQ ID NO:20, cysteine or phenylalanine at amino acid position 96, alanine at amino acid position 149, alanine at amino acid position 155, serine at amino acid position 156, tyrosine at amino acid position 159, lysine at amino acid position 170, glutamate at amino acid position 198, and lysine or valine at amino acid position 252.

[0163] In yet another aspect, 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 aspects, the protein switch comprises glycine at amino acid position 22 of SEQ ID NO:20, cysteine or phenylalanine at amino acid position 96, glycine at amino acid position 154, alanine at amino acid position 155, serine at amino acid position 156, tyrosine at amino acid position 159, lysine at amino acid position 170, glutamate at amino acid position 198, and lysine or valine at amino acid position 252.

[0164] In yet another aspect, 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 aspects, the protein switch comprises cysteine or phenylalanine at amino acid position 96 of SEQ ID NO:20, valine at amino acid position 141, glycine at amino acid position 154, alanine at amino acid position 155, serine at amino acid position 156, tyrosine at amino acid position 159, lysine at amino acid position 170, glutamate at amino acid position 198, and lysine or valine at amino acid position 252.

[0165] In still yet other aspects, 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 aspects, the protein switch comprises lysine at amino acid position 12 of SEQ ID NO:20, cysteine or phenylalanine at amino acid position 96, alanine at amino acid position 155, serine at amino acid position 156, tyrosine at amino acid position 159, lysine at amino acid position 170, arginine at amino acid position 195, glutamate at amino acid position 198, lysine at amino acid position 251, and lysine or valine at amino acid position 252.

[0166] In yet another aspect, 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 combination of mutations listed in Table 3C and Figure 14 as listed below.

[0167] Table 3C

[0168]

[0169]

[0170] In another aspect, the protein switch of the present disclosure has an amino acid sequence of any one of SEQ ID NOs. 1-19, an amino acid sequence having at least 60% sequence identity with any one of SEQ ID NOs. 1-19, an amino acid sequence having at least 65% sequence identity with any one of SEQ ID NOs. 1-19, an amino acid sequence having at least 70% sequence identity with any one of SEQ ID NOs. 1-10 and 12-19, an amino acid sequence having at least 75% sequence identity with any one of SEQ ID NOs. 1-19, an amino acid sequence having at least 80% sequence identity with any one of SEQ ID NOs. 1-19, an amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs. 1-19, an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NOs. 1-19, an amino acid sequence having at least 95% sequence identity with any one of SEQ ID NOs. 1-19, an amino acid sequence having at least 96% sequence identity with any one of SEQ ID NOs. 1-19, an amino acid sequence having at least 97% sequence identity with any one of SEQ ID NOs. 1-19, an amino acid sequence having at least 98% sequence identity with any one of SEQ ID NOs. 1-19, an amino acid sequence having at least 99% sequence identity with any one of SEQ ID NOs. 1-19 or an amino acid sequence having at least 100% sequence identity with any one of SEQ ID NOs. 1-19. The protein switch comprising the amino acid sequence of SEQ ID NOs. 1-19 binds to the analyte warfarin.

[0171] In another aspect, the protein switch of the present disclosure has an amino acid sequence of any one of SEQ ID NO.1, 7, 12 or 15, has at least 60% sequence identity with any one of SEQ ID NO.1, 7, 12 or 15, has at least 65% sequence identity with any one of SEQ ID NO.1, 7, 12 or 15, has at least 70% sequence identity with any one of SEQ ID NO.1, 7, 12 or 15, has at least 75% sequence identity with any one of SEQ ID NO.1, 7, 12 or 15, has at least 80% sequence identity with any one of SEQ ID NO.1, 7, 12 or 15, has at least 85% sequence identity with any one of SEQ ID NO.1, 7, 12 or 15, has at least 90% sequence identity with any one of SEQ ID NO.1, 7, 12 or 15, has at least 95% sequence identity with any one of SEQ ID NO.1, 7, 12 or 15, has at least 96% sequence identity with any one of SEQ ID NO.1, 7, 12 or 15, has at least 97% sequence identity with any one of SEQ ID NO.1, 7, 12 or 15, has at least 98% sequence identity with any one of SEQ ID NO.1, 7, 12 or 15, has at least 99% sequence identity with any one of SEQ ID NO.1, 7, 12 or 15 or has at least 100% sequence identity with any one of SEQ ID NO.SEQ ID NO.1, 7, 12 or 15.

[0172] In yet another aspect, the protein switch of the present disclosure comprises any one of the enzymes of Example 1 or 2.

[0173] As will be 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 to detect one or more analytes in a sample in a sensing system.

[0174] C. Method for fabricating a protein switch

[0175] In some aspects, the present disclosure relates to methods of identifying, generating, and / or preparing protein switches. In certain aspects, the protein switches of the present disclosure can be generated by screening libraries of non-naturally occurring variant polypeptides for one or more analytes of interest using biochemical techniques known in the art. For example, libraries of variant polypeptides encoding oxidases (e.g., glucose oxidase or lactate oxidase) or dehydrogenases (e.g., glucose dehydrogenase or lactate dehydrogenase) can be screened for one or more analytes of interest. In one aspect, 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 that exhibit such an increase or decrease in enzyme activity can be selected. In some aspects, polypeptides that exhibit a decrease in enzyme activity as a result of screening for an analyte of interest are selected. In other aspects, polypeptides that exhibit an increase in enzyme activity as a result of screening for an analyte of interest are selected.

[0176] Once oxidase or dehydrogenase variants that exhibit increased or decreased enzyme activity are identified, mutant and / or evolved protein switches (e.g., enzymes) with increased or decreased oxidase or dehydrogenase activity can be readily generated using conventional techniques known in the art. See, for example, 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 U.S.A., 94:45-4-4509; Crameri et al., Nature Biotechnology 14:315-319 (1996); Stemmer, Nature, 370:389-391 (1994); Stemmer, Proceedings of the National Academy of Sciences, U.S.A., 91:10747-10751 (1994); WO 95 / 22625; WO 97 / 0078; WO 97 / 35966; WO 98 / 27230; WO 00 / 42651; WO 01 / 75767 and U.S. Patent No. 6,537,746. To maximize any diversity, several of the above techniques can be used in sequence. Typically, a library of variant polynucleotides is generated by one mutagenesis or evolution technique, and the expression products are screened to find polypeptides with increased or decreased oxidase or dehydrogenase activity.Then, a second mutagenesis or evolution technique is applied to the polynucleotide encoding the most or least enzyme activity to generate a second library, which is then screened for oxidase or dehydrogenase activity using the same technique. The process of mutation and screening, including insertion point mutations, can be repeated as needed to obtain a polynucleotide encoding a protein switch having the desired activity, thermal stability, cofactor preference, or other characteristics.

[0177] D. Device and sensor system

[0178] In other aspects, the present disclosure relates to one or more sensors that employ and / or contain one or more (e.g., multiple) of the protein switches described herein for detecting and / or monitoring at least one analyte. Such sensors can be prepared using conventional techniques in the art, as will be discussed in more detail herein. Such sensors can then be used in one or more sensor systems. A general description of suitable sensor configurations and sensor systems that employ these sensors utilizing the protein switches of the present disclosure is provided. However, this description should be understood as non-limiting with respect to the aspects disclosed herein, and alternative sensors and systems are considered to remain within the scope of the present disclosure.

[0179] Figure 1FIG. depicting an illustrative sensing system incorporating a sensor incorporating 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 over a local communication path or link, which may be wired or wireless, one-way or two-way, and encrypted or unencrypted. Reader device 120 may constitute an output medium for viewing analyte concentrations and alerts or notifications determined by sensor / sensors 104 or associated processors and for allowing input from one or more users. Reader device 120 may be a multi-purpose smart phone or a dedicated electronic reading instrument. Although only one reader device 120 is shown, in some cases there may be multiple reader devices 120. 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 or wireless, one-way or two-way, and encrypted or unencrypted. Reader device 120 may also or alternatively communicate with a network 150 (e.g., a mobile phone network, the Internet, or a cloud server) via communication path / link 151. Network 150 may be further communicatively coupled to remote terminal 170 via communication path / link 152 and / or to 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 in the absence of an intervening reader device 120. For example, according to some aspects, sensor 104 may communicate with remote terminal 170 and / or trusted computer system 180 via a direct communication link with network 150, as described in U.S. Patent Application Publication 2011 / 0213225, which is incorporated herein by reference in its entirety. 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), or low energy protocols, Wi-Fi, etc. According to some aspects, remote terminal 170 and / or trusted computer system 180 may be accessible by individuals other than the primary user who are interested in the user's analyte levels. Reader device 120 may include a display 122 and an optional input component 121. According to some aspects, display 122 may include a touch screen.

[0180] The sensor control device 102 includes a sensor housing 103 that can accommodate 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, where the processor is physically located within the sensor housing 103 or the reader device 120. The sensor 104 projects from the lower side of the sensor housing 103 and extends through an adhesive layer 105 that, according to some aspects, is adapted to adhere the sensor housing 103 to a tissue surface, such as the skin.

[0181] The sensor 104 is adapted to be at least partially inserted into a target tissue, such as into the dermis or subcutaneous layer of the skin. The sensor 104 may include a sensor tail having a sufficient length to be inserted to a desired depth in a given tissue. The sensor tail may include at least one working electrode and one or more active regions (sensing layers or sensing zones / spots) that are located on the at least one working electrode and are effective for sensing one or more target analytes. Collectively, the one or more active regions may include one or more protein switches. The active regions may include a polymeric material to which at least some of the one or more protein switches are covalently bonded. In various aspects, the analyte may be monitored in any target biological fluid, such as dermal fluid, interstitial fluid, subcutaneous fluid, blood (e.g., intravenous or capillary). In certain aspects, the sensor may be adapted to assay dermal fluid or interstitial fluid.

[0182] In some aspects, the sensor 104 may automatically transfer data (e.g., such as by transmitting a signal) to the reader device 120. For example, analyte concentration data (e.g., a signal representative of the level of one or more analytes) may be automatically and periodically sent, such as when the data is acquired or at a certain frequency after a certain period of time, and the data is stored in a memory until transmission (e.g., every minute, every five minutes, or other predetermined period). In other aspects, the sensor 104 may communicate with the reader device 120 in a non-automatic manner rather than according to a set schedule. For example, when the sensor of the electronic device comes within the communication range of the reader device 120, data may be sent from the sensor 104 using RFID technology. The data may remain stored in the memory of the sensor 104 until it is sent to the reader device 120. Thus, the patient does not have to always remain in close proximity to the reader device 120 but can upload the data at a convenient time. In still other aspects, a combination of automatic and non-automatic data transfer may be implemented. For example, data transfer may continue on an automatic basis until the reader device 120 is no longer within the communication range of the sensor 104.

[0183] It may be temporarily present in the introducer to facilitate introduction of the sensor 104 into tissue. In some aspects, the introducer may include a needle or similar sharp object. It should be appreciated that in alternative aspects there may be other types of introducers, such as a sheath or blade. More specifically, prior to insertion into tissue, the needle or other introducer may be temporarily positioned adjacent to the sensor 104 and then withdrawn thereafter. When present, the needle or other introducer may facilitate insertion of the sensor 104 into tissue by opening an access path for the sensor 104 to follow. For example, the needle may facilitate penetration of the epidermis, which serves as an access path to the dermis, to permit implantation of the sensor 104. After opening the access path, the needle or other introducer may be withdrawn so that it does not pose a sharp object hazard. In other aspects, suitable needles may be solid or hollow in cross-section, beveled or non-beveled, and / or round or non-round. In more specific aspects, suitable needles may be comparable in cross-sectional diameter and / or tip design to acupuncture needles, which may have a cross-sectional diameter of about 250 microns. However, it should be appreciated that suitable needles may have a larger or smaller cross-sectional diameter if required for a particular application.

[0184] In yet additional aspects, the tip of the needle (when present) may be angled relative to the distal end of the sensor 104 such that the needle first penetrates the tissue and opens an access path for the sensor 104. In other illustrative aspects, the sensor 104 may reside within the lumen or groove of the needle, where the needle similarly opens an access path for the sensor 104. In either case, after facilitating sensor insertion, the needle is subsequently withdrawn.

[0185] The sensor may contain or include one or more (e.g., multiple) protein switches on the active region of a single working electrode or on two or more separate working electrodes. A single working electrode configuration for the sensor may employ a two-electrode or three-electrode detection motif. Sensor configurations featuring a single working electrode are described below with reference to Figures 2A - 2C described. Sensor configurations featuring multiple working electrodes are described subsequently with reference to Figure 3 separately described. Multiple protein switches may be incorporated into any of the sensor configurations described below, where the specific configurations suitable for incorporating multiple protein switches are described in further detail below.

[0186] When there is a single working electrode in the sensor, the three - electrode detection motif can include a working electrode, a counter electrode, and a reference electrode. The related two - electrode detection motif can include a working electrode and a second electrode, where the second electrode functions as both a counter electrode and a reference electrode (i.e., a counter / reference electrode). In both the 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, where one or more (e.g., multiple) protein switches are present in a single active region and / or in multiple active regions. In some aspects, the respective electrodes can be at least partially stacked (laminated) on top of each other, as described in further detail below. In some or other aspects, the respective electrodes can be laterally spaced apart from each other on the sensor tail. Similarly, the related active regions on each electrode can be vertically stacked on top of each other or laterally spaced apart. In either case, the respective electrodes can be electrically separated from each other by a dielectric material or a similar insulator.

[0187] Figure 2A FIG. shows an illustrative two - electrode sensor configuration with a single working electrode, which is suitable for use in some aspects of the present disclosure. As shown, sensor 200 includes a substrate 212 disposed between a working electrode 214 and a counter / reference electrode 216. Alternatively, the working electrode 214 and the counter / reference electrode 216 can be on the same side of the substrate 212, with a dielectric material (not shown in the configuration) interposed therebetween. An active region 218 is provided as at least one layer on at least a portion of the working electrode 214. In various aspects, the active region 218 can include a plurality of spots or a single spot configured to detect one or more target analytes. One or more (e.g., multiple) protein switches can be present in the active region 218 (i.e., in the single spot or the plurality of spots).

[0188] Still referring to Figure 2A , a membrane 220 at least overcoats the active region 218 and can optionally overcoat some or all of the working electrode 214 and / or the counter / reference electrode 216, or according to some aspects, the entire sensor 200. One or both faces of the sensor 200 can be overcoated with the membrane 220. The membrane 220 can comprise one or more polymer membrane materials having the ability to limit the analyte flux to the active region 218. Depending on the identity of the analyte, the composition of the membrane 220 can be varied, as further described herein.

[0189] Figure 2B and 2C FIG. shows an illustrative three - electrode sensor configuration with a single working electrode. The three - electrode sensor configuration with a single working electrode can be similar to that for Figure 2AThe configuration shown by sensor 200 in [reference], except that in sensors 201 and 202 ( Figure 2B and 2C ), additional electrodes 217 are included. In the case of having additional electrodes 217, the counter / reference electrode 216 can then act as a counter electrode or a reference electrode, and the additional electrode 217 performs other electrode functions not considered. The working electrode 214 continues to perform its original function. The additional electrode 217 can be disposed on the working electrode 214 or the electrode 216, with a separation layer of dielectric material therebetween. For example, as depicted in Figure 2B , the dielectric layers 219a and 219b separate the electrodes 214, 216, and 217 from each other and provide electrical isolation. Alternatively, at least one of the electrodes 214, 216, and 217 can be located on the opposite side of the substrate 212, as shown in Figure 2C . Thus, in some aspects, the electrode 214 (working electrode) and the electrode 216 (counter electrode) can be located on opposite sides of the substrate 212, where the electrode 217 (reference electrode) is located on one of the electrodes 214 or 216 and is spaced therefrom by a dielectric material. A reference material layer 230 (e.g., Ag / AgCl) can be present on the electrode 217, where the position of the reference material layer 230 is not limited to that depicted in Figure 2B and 2C . Similar to the sensor 200 shown in Figure 2A , the active regions 218 in sensors 201 and 202 can include multiple spots or a single spot configured to detect one or more target analytes. One or more (e.g., multiple protein switches) are present in the active regions 218 of sensors 201 and 202.

[0190] Similar to sensor 200, the membrane 220 can also encapsulate the active regions 218 in sensors 201 and 202, as well as other sensor components. In some aspects, the additional electrode 217 can be encapsulated with the membrane 220. Although Figure 2B and 2C depict all of the electrodes 214, 216, and 217 as being encapsulated with the membrane 220, it should be recognized that in some aspects, only the working electrode 214 can be encapsulated. Additionally, the thickness of the membrane 220 can be the same or different at each of the electrodes 214, 216, and 217. As in the two-electrode sensor configuration ( Figure 2A ), in the sensor configurations of Figure 2B and 2C , one or both faces of sensors 201 and 202 can be encapsulated with the membrane 220, or all of sensors 201 and 202 can be encapsulated. Thus, Figure 2B and 2CThe three - electrode sensor configuration shown should be understood as non - limiting for the aspects disclosed herein, and alternative electrode and / or layer configurations are still within the scope of the present disclosure.

[0191] Sensor configurations with multiple working electrodes will now be described in further detail. Although the following description is mainly directed to sensor configurations with two working electrodes, it should be understood that more than two working electrodes can be successfully incorporated by extending the disclosure herein. Additional working electrodes can allow additional active regions and corresponding sensing capabilities to be imparted to sensors with such features.

[0192] Figure 3 A diagram showing an illustrative sensor configuration with two working electrodes, one reference electrode, and one counter electrode, which is suitable for use in some aspects of the present disclosure. As Figure 3 shown, sensor 300 includes working electrodes 304 and 306 disposed on opposite faces 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 in active regions 310 and 312, where each active region 310, 312 contains one or more (e.g., control) protein switches. For example, in a particular aspect, a first protein switch comprising an analyte - binding domain capable of binding troponin and a glucose oxidase domain responsive to glucose can be present in active region 310, and a second protein switch comprising an analyte - binding domain capable of binding BNP and a glucose oxidase domain responsive to glucose can be present in active region 312. Alternatively, in a particular aspect, a second protein switch comprising an analyte - binding domain capable of binding BNP and a lactate oxidase domain responsive to lactate can be present in active region 312. Counter electrode 320 is electrically isolated from working electrode 304 by dielectric layer 322, and reference electrode 321 is electrically isolated from working electrode 306 by dielectric layer 323. Outer dielectric layers 330 and 332 are positioned on reference electrode 321 and counter electrode 320, respectively. According to various aspects, membrane 340 can at least over - coat active regions 310 and 312. Other components of sensor 300 can also be over - coated with membrane 340, and as above, one or both faces or portions thereof of sensor 300 can be over - coated with membrane 340.

[0193] Having multiple working electrodes and different from Figure 3 The alternative sensor configurations shown can be characterized by a counter / reference electrode instead of separate counter electrode 320 and reference electrode 321, and / or they can be characterized by layer and / or membrane arrangements different from those explicitly shown. For example, the positioning of counter electrode 320 and reference electrode 321 can be different fromFigure 3 The orientation depicted in Figure 3 is reversed. In addition, the working electrodes 304 and 306 do not necessarily need to reside on opposite sides of the substrate 302 in the

[0194] A sensor configuration featuring a working electrode with an active region remote from it is shown in Figure 5A and 5B and is discussed further below.

[0195] According to some aspects, an electron transfer agent can be present in one or more sensing zones (e.g., active regions) of any of the sensors or sensor configurations disclosed herein. Suitable electron transfer agent / mediator compounds can facilitate the transfer of electrons to the working electrode when the reactant undergoes an oxidation-reduction reaction. The choice of electron transfer agent within each active region can determine the oxidation-reduction potential observed for each. When multiple active regions are present, the electron transfer agent within each active region can be the same or different.

[0196] Suitable electron transfer agents can include electroreducible and electrooxidizable ions, complexes, or molecules (e.g., quinones) having an oxidation-reduction potential hundreds of millivolts above or below that of the standard calomel electrode (SCE). According to some aspects, suitable electron transfer agents can include low-potential osmium complexes such as those described in U.S. Patent Nos. 6,134,461 and 6,605,200, which are incorporated herein by reference in their entirety. Additional examples include those described in U.S. Patent Nos. 6,736,957, 7,501,053, and 7,754,093, the disclosures of each of which are incorporated herein by reference in their entirety. Other suitable electron transfer agents can include, for example, metal compounds or complexes of ruthenium, osmium, iron (e.g., polyvinylferrocene or ferricyanide), or cobalt, including their metallocene compounds. Suitable examples of electron transfer mediators and polymer-bound electron transfer mediators can include those described in U.S. Patent Nos. 8,444,834, 8,268,143, and 6,605,201, the disclosures of which are incorporated herein by reference in their entirety. Suitable ligands for metal complexes can also include, for example, bidentate or more dentate ligands such as, for example, 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 more dentate ligands can be present in the metal complex to achieve a complete coordination layer.

[0197] In still other aspects, the active region or sensing region can also include a cofactor that is capable of catalyzing a reaction of a reactant associated with at least one oxidase or dehydrogenase domain portion of the protein switch. In some aspects, the cofactor is a non-protein organic molecule such as pyrroloquinoline quinone (PQQ), flavin adenine dinucleotide (FAD), nicotinamide adenine dinucleotide (NAD), flavin mononucleotide (FMN), etc.). In certain aspects, the cofactor can be attached to a polymer such that the cofactor is cross-linked with an electron transfer agent. In certain aspects, a second cofactor can also be used.

[0198] In other aspects, the polymer can be present in each active region of any of the sensors or sensor configurations disclosed herein. Suitable polymers for inclusion in the active region can include, but are not limited to, polyvinylpyridine (e.g., poly(4-vinylpyridine)), polyvinylimidazole (e.g., poly(1-vinylimidazole)), or any copolymer thereof. Illustrative copolymers that can be suitable for inclusion in the active region include those containing monomer units such as styrene, acrylamide, methacrylamide, or acrylonitrile, for example. When there are multiple active regions, the polymers within each active region can be the same or different.

[0199] In some aspects, 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 limited. The covalent bonding of the electron transfer agent to the polymer can occur by polymerizing a monomer unit of the polymer carrying the covalently bound electron transfer agent, or the electron transfer agent can be reacted with the polymer separately after the polymer has been synthesized. In other aspects, a bifunctional spacer can covalently bond the electron transfer agent to the polymer within the active region, where the first functional group is reactive with the polymer (e.g., a functional group capable of quaternizing a pyridine nitrogen atom or an imidazole nitrogen atom) and the second functional group is reactive with the electron transfer agent (e.g., a functional group reactive with a ligand-coordinated metal ion).

[0200] Similarly, in yet other aspects, a protein switch having one or more active regions can be covalently bonded to a polymer. When multiple protein switches are present in a single active region, all of the multiple protein switches can, in some aspects, be covalently bonded to the polymer, and in other aspects, only a portion of the multiple protein switches can be covalently bonded to the polymer. For example, a first protein switch can be covalently bonded to the polymer while a second protein switch can be non-covalently associated with the polymer. According to more specific aspects, the covalent bonding of the protein switch to the polymer can occur via crosslinks introduced by a suitable crosslinking agent. Crosslinking agents suitable for reacting with free amino groups in the protein switch can include crosslinking agents such as, for example, polyethylene glycol diglycidyl ether (PEGDGE) or other polyepoxides, cyanuric chloride, N-hydroxysuccinimide, imido esters, epichlorohydrin, or derivatized variants thereof. Suitable crosslinking agents suitable for reacting with free carboxylic acid groups in the protein switch can include, for example, carbodiimides. Crosslinking is generally intermolecular, but in some aspects can be intramolecular.

[0201] The electron transfer agent and / or one or more (e.g., multiple) protein switches can also be associated with the polymer in the active region by means other than covalent bonds. In some aspects, the electron transfer agent and / or one or more (e.g., multiple) protein switches can be ionically or coordinatively associated with the polymer. For example, a charged polymer can be ionically associated with an electron transfer agent or protein switch having an opposite charge. In still other aspects, the electron transfer agent and / or protein switch can be physically entrapped within the polymer without being bonded thereto.

[0202] As previously mentioned, various configurations for arranging multiple protein switches in a sensor are contemplated within the present disclosure. Multiple protein switches can be disposed within one or more active regions of the sensor. The size of the active region can range from about 0.01 mm 2 to about 1 mm 2 , but larger or smaller active regions are also contemplated herein.

[0203] In other aspects, a sensor containing multiple protein switches (whether operating independently or in concert) can function with enhanced stability in the presence of a suitable stabilizer. Stabilizers that can be used include, for example, catalase or albumin (e.g., bovine serum albumin or human serum albumin).

[0204] In yet still other aspects, multiple protein switches can be disposed within separate active regions on a single working electrode. When multiple protein switches are disposed in this manner, each active region can facilitate the detection of a separate (e.g., two or more different) analyte, as described below. At least one active region can generate a signal independently of the other active regions.

[0205] According to some aspects, a sensor having multiple active regions on a single working electrode can include: a sensor tail including at least one working electrode and at least two active regions disposed on the surface of the working electrode. Each active region contains a protein switch and a polymer, wherein the protein switch in each active region is different. Each active region has a redox potential, and the redox potential of the first active region is sufficient to be separated from the redox potential of the second active region to allow generation of a signal from the first active region independent of a signal from the second active region. In more specific aspects, such a sensor can include a single working electrode having at least two active regions. An electron transfer agent can be incorporated within each active region to facilitate electron transfer.

[0206] An alternative sensor configuration can include a single active region containing a first protein switch and a second protein switch and an electron transfer agent. Each protein switch can be covalently bonded to a separate portion of the polymer in 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 chemistry for facilitating electron transfer of each analyte is not overly diluted in the single active region. Such a sensor configuration can be particularly feasible when the analytes to be assayed using the first and second protein switches have comparable membrane permeability values.

[0207] In more specific aspects, the sensor tail can be configured for insertion into tissue. Suitable tissues are not considered to be particularly limited and are presented in more detail above. Similarly, considerations for positioning the sensor tail at a particular location within the tissue are presented above.

[0208] In other aspects, 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. The upper limit of the separation between the oxidation-reduction potentials is determined by the in vivo electrochemical window. By separating the oxidation-reduction potentials of the active regions from each other in magnitude, an electrochemical reaction can occur within the first active region without substantially inducing an electrochemical reaction within the second active region. Thus, a signal from the first active region can be generated independently when at or above its corresponding oxidation-reduction potential. In contrast, when at or above the oxidation-reduction potential of the second active region, an electrochemical reaction can occur within both active regions. Thus, the signal generated when at or above the oxidation-reduction potential of the second active region can include signal contributions from both the first active region and the second active region, and the signal is a composite signal. Then the signal contribution from the second active region when at or above its oxidation-reduction potential can be determined by subtracting from the composite signal the signal obtained only from the first active region when at or above the corresponding oxidation-reduction potential of the first active region. Similar considerations apply to analyzing the signal contribution from a single active region containing two protein switches that generate signals at different oxidation-reduction potentials.

[0209] In a more specific aspect, when the active regions are located on the same working electrode, the first and second active regions can contain different electron transfer agents to provide oxidation-reduction potentials that are sufficiently separated in magnitude. More specifically, the first active region can contain a first electron transfer agent, and the second active region can contain a second electron transfer agent, where the first and second electron transfer agents are different. According to various aspects of the present disclosure, the metal center and / or ligand present in a given electron transfer agent can be varied to provide sufficient separation of the oxidation-reduction potentials of the first and second active regions. According to an even more specific aspect, 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 manner of covalent bonding for the first electron transfer agent and the second electron transfer agent can be the same or different. Similar considerations apply to selecting electron transfer agents suitable for use in combination with a first protein switch and a second protein switch contained within a single active region as disclosed above.

[0210] In more specific aspects of the present disclosure, the protein switches in each active region can be covalently bonded (or otherwise immobilized) to the polymer within each active region. In even more specific aspects, the protein switches and electron transfer agents in each active region can be covalently bonded to the polymer within each active region. When included in a single active region, the first protein switch and the first electron transfer agent can be covalently bonded to a first portion of the polymer, and the second protein switch and the second electron transfer agent can be covalently bonded to a second portion of the polymer. The polymers in the first and second portions can be the same or different.

[0211] In some aspects, the first and second active regions located on a single working electrode can be configured to rapidly reach a steady-state current when operating the sensor at a given potential. The rapid attainment of the steady-state current can be facilitated by selecting the electron transfer agent for each active region, which rapidly changes its oxidation state when exposed to a potential at or above the redox potential of the active region. Making the active regions as thin as possible can also facilitate the rapid attainment of the steady-state current. For example, the suitable thickness range of the first and second active regions can be from about 0.1 micrometer to about 10 micrometers. In some or other aspects, combining a conductive material such as, for example, carbon nanotubes, graphene, or metal nanoparticles within one or more active regions can facilitate the rapid attainment of the steady-state current. The suitable amount range of the conductive particles can be from about 0.1 wt% to about 50 wt% of the active region, or from about 1 wt% to about 50 wt%, or from about 0.1 wt% to about 10 wt% or from about 1 wt% to about 10 wt%. Stabilizers can also be employed to promote response stability.

[0212] It should also be understood that the sensitivity (output current) of the sensor to each analyte can be altered by changing the coverage (area or size) of the active regions, the area ratio of the active regions relative to each other, the properties and thickness of the mass transfer limiting membrane that envelopes the active regions, and any combination thereof. Alterations of these parameters can be readily made by those of ordinary skill in the art.

[0213] Although the foregoing description has been primarily directed to sensors configured to detect two different analytes, it should be understood that the above concepts can be extended to detect more than two analytes using a corresponding number of active regions located on a single working electrode. Specifically, in additional aspects of the present disclosure, sensors employing more than two active regions and a corresponding number of protein switches (and electron transfer agents) can be used to detect a similar number of different analytes. Provided that the redox potential of each active region is sufficiently separated from the redox potential of the other active regions, the signal contributions from each active region can be analyzed in a manner related to the manner described above to provide the concentration of each analyte.

[0214] For example, in addition to the suitable electron transfer agents discussed in more detail above, the first active region can include a first protein switch containing an analyte binding domain capable of binding troponin and a glucose oxidase domain responsive to glucose, and the second active region can include a second protein switch containing an analyte binding domain capable of binding BNP and a lactate oxidase domain responsive to lactate. 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 transfer limiting membrane that overcoats the first and second active regions on the working electrode, wherein the second active region contains a polymer and an analyte binding domain capable of binding BNP and a lactate oxidase domain responsive to lactate covalently bonded to the polymer, and the first active region contains an analyte binding domain capable of binding troponin and a glucose oxidase domain responsive to glucose covalently bonded to the polymer. Alternatively, the lactate oxidase domain can be replaced with a glucose oxidase domain responsive to glucose. First and second electron transfer agents that are different from each other can be present in each active region. In a more specific aspect, the mass transfer limiting membrane can include at least one crosslinked polyvinylpyridine homopolymer or copolymer. The composition of the mass transfer limiting membrane can be the same or different, wherein the mass transfer limiting membrane overcoats each active region. In a particular aspect, the mass transfer limiting membrane that overcoats the first active region can be a single component (containing a single membrane polymer) and the mass transfer limiting membrane that overcoats the second active region can be a multi-component (containing two or more different membrane polymers, wherein one is a polyvinylpyridine homopolymer or copolymer), as a bilayer or homogeneous mixture.

[0215] Similarly, it should also be understood that some sensors having two or more active regions located on a given working electrode can include two or more protein switches in at least one active region. According to a more specific aspect, two or more protein switches in a given active region can interact synergistically with each other to generate a signal proportional to the concentration of a single analyte. Thus, for a given selection of analyte, the protein switches do not need to be present in a 1:1 ratio. Sensors containing synergistically interacting protein switches are described in further detail herein.

[0216] Accordingly, the present disclosure also describes a multi - analyte detection method using a sensor featuring multiple protein switches disposed on a single working electrode. In various aspects, such methods can include: exposing the sensor to a fluid containing at least one analyte. The sensor includes a sensor tail, the sensor tail including at least one working electrode, particularly a single working electrode, and at least two active regions disposed on the surface of the working electrode. Each active region contains a protein switch and a polymer, and the protein switches in each active region are different. Each active region has a redox potential, and the redox potential of the first active region is sufficiently separated from the redox potential of the second active region to permit the generation of a signal from the first active region independently of the generation of a signal from the second active region. The method further includes: obtaining a first signal when at or above the redox potential of the first active region, such that the first signal is proportional to the concentration of the first analyte; obtaining a second signal when at or above the redox potential of the second active region, such that the second signal is a composite signal comprising a signal contribution from the first active region and a signal contribution from the second active region; and subtracting the first signal from the second signal to obtain a difference signal, the difference signal being proportional to the concentration of the second analyte.

[0217] In a more specific aspect, the redox potential associated with the first active region can be separated from the redox 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 to independently generate a signal from the first active region.

[0218] In some or other more specific aspects, the fluid is a biological fluid and the sensor is exposed to an in - vivo biological fluid within an individual. Biological fluids suitable for analysis with a sensor having at least two different active regions located on a given working electrode can include any of the biological fluids previously discussed herein.

[0219] In some aspects, the signals associated with each active region can be correlated with the corresponding analyte concentration by querying a look - up table or calibration curve for each analyte. The look - up table for each analyte can be populated by assaying a plurality of samples having known analyte concentrations and recording the sensor response at each concentration of each analyte. Similarly, the calibration curve for each analyte can be determined by plotting the sensor response for each analyte as a function of concentration. According to some aspects, the calibration curve of the sensors of the present disclosure can be linear.

[0220] The processor can determine which sensor response value in the lookup table is closest to the value measured for a sample with an unknown analyte concentration and then report the analyte concentration accordingly. In some or other aspects, if the sensor response value of a sample with an unknown analyte concentration is between the recorded values in the lookup table, the processor can interpolate between the two lookup table values to estimate the analyte concentration. The interpolation can assume a linear concentration change between the two values reported in the lookup table. Interpolation can be employed when the sensor response differs from a given value in the lookup table by a sufficient amount, such as a change of about 10% or greater.

[0221] Similarly, according to some or other various aspects, the processor can input the sensor response value of a sample with an unknown analyte concentration into a corresponding calibration curve. Then the sensor can report the analyte concentration accordingly.

[0222] Aspects of sensors having two different active regions disposed on a given working electrode can employ sensor configurations related to those depicted in Figures 2A - 2C and described above. However, it should be understood that suitable sensors can also be characterized by multiple working electrodes, such as the sensor configuration shown in Figure 3 wherein at least one working electrode has at least two active regions that are different from each other. It should also be understood that other sensor configurations having two or more different 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 the counter and / or reference electrodes can be different from those shown in the figures herein.

[0223] Figure 4 An illustrative sensor configuration suitable for use in some aspects of the present disclosure is shown, wherein two different active regions are disposed on the surface of a single working electrode. Figure 4 The sensor configuration of Figure 2C has the greatest similarity to the sensor configuration of Figure 2C For clarity, common reference characters from Figure 4 are used in Figure 4 and, for brevity, features having common structure and / or function are not described in further detail again. Similarly, it should be understood that other sensor configurations can be incorporated with features described below for

[0224] Refer to Figure 4, the sensor includes active regions 218a and 218b on the surface of the working electrode 214. Active region 218a contains a first electron transfer agent and a first protein switch that can be covalently bonded to the polymer constituting active region 218a. Active region 218b similarly contains a second electron transfer agent and a second protein switch that can be covalently bonded to the polymer constituting active region 218b. The first electron transfer agent and the second electron transfer agent can be compositionally different to provide separation of the oxidation-reduction potentials of the first active region 218a and the second active region 218b. In certain aspects, active region 218b can contain an analyte binding domain capable of binding BNP and a lactate oxidase domain responsive to lactate, and active region 218a contains an analyte binding domain capable of binding troponin and a glucose oxidase domain responsive to glucose. Alternatively, the lactate oxidase domain can be replaced by a glucose oxidase domain responsive to glucose.

[0225] The oxidation-reduction potentials of the first active region 218a and the second active region 218b can be sufficiently separated from each other to allow the generation of a signal from the first active region 218a independent of the signal generation from the second active region 218b. Thus, the sensor can be operated at a first potential at which an oxidation-reduction reaction occurs in the first active region 218a and no oxidation-reduction reaction occurs 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 with respect to 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 reference to a look-up table or calibration curve.

[0226] At or 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. Thus, the signal generated at or 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 the second analyte (e.g., BNP) from the composite signal, the signal from the first active region 218a at or above its corresponding oxidation-reduction potential can be subtracted from the composite signal to provide a difference signal associated with the second active region 218b alone. Once the difference signal has been determined, the concentration of the second analyte can be determined by reference to a look-up table or calibration curve.

[0227] As previously mentioned, similar considerations also apply to separating the first or second signal from a single active region containing two different protein switches in order to determine the concentrations of first and second analytes that are different from each other.

[0228] As previously discussed herein, Figures 2A - 4 All of the sensor configurations shown therein are characterized by one or more working electrodes having one or more active regions disposed directly on the surface of each working electrode. In contrast, Figure 5A and 5B show diagrams of working electrodes where a first active region is disposed directly on the surface of the working electrode and a second active region is separated (spaced apart or away) from the working electrode by a membrane. Figure 5A and 5B The working electrode configurations depicted in Figures 2A - 4 can replace any of the specific working electrode configurations depicted in Figure 5A and 5B That is, the working electrode configurations depicted in

[0229] As Figure 5A shown, the working electrode 400 has an active region 402 disposed directly on its surface. The active region 402 contains a first protein switch covalently bound to a first polymer. Typically, an electron transfer agent is also present in the active region 402, where the electron transfer agent is also covalently bound to the polymer. The active region 402 is coated with a membrane 404. As depicted, the membrane 404 can also coat the surface of the working electrode 400 and other parts of the sensor in which the working electrode 400 is present. The membrane 404 isolates the active region 406 from the working electrode 400 such that electron exchange between the two is blocked. The active region 406 contains a second protein switch covalently bound to a second polymer. Typically, an electron transfer agent is also present in the active region 406, where the electron transfer agent is also covalently bound to the second polymer. Although Figure 5A shows the active region 406 disposed directly on the active region 402, it should be understood that they can be laterally spaced apart from each other in alternative configurations that are also compatible with the present disclosure. The membrane 408 coats the active region 406 and optional other sensor components to provide mass transfer limiting characteristics. Similarly, as Figure 5B shown, the membrane 404 does not necessarily extend the same lateral distance on the working electrode 400 as the membrane 408. In fact, Figure 5BThe membrane 404 in [it] coats the active region 402, but only coats a part of the surface of the working electrode 400, wherein the membrane 408 coats the active region 406, the surface of the membrane 404, and the remaining part of the surface of the working electrode 400 that is not coated by the membrane 404. In some aspects, the active regions 402 and 406 can also be laterally offset from each other.

[0230] The membrane 408 is permeable to the analyte and any additional components required to facilitate the enzymatic reaction in the active region 406. In contrast, the membrane 404 is permeable to the product formed in the active region 406. That is, the analyte reacts in the active region 406 to form a first product, which then diffuses through the membrane 404 and subsequently further reacts in the active region 402 to form a second product. The second product is then detectable based on the electron exchange with the working electrode 400.

[0231] According to some aspects, the first membrane polymer and the second membrane polymer can be different from each other. According to some aspects, the first membrane polymer can be cross-linked polyvinylpyridine. In aspects 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, wherein a part of the pyridine nitrogen atoms are functionalized with non-cross-linked poly(ethylene glycol) tails and a part of the pyridine nitrogen atoms are functionalized with alkylsulfonic acid groups. Such a second membrane polymer is readily permeable to both glucose and ethanol.

[0232] In still other aspects, a plurality of protein switches can be arranged within the active regions of individual working electrodes. Because of this, the signals associated with the enzymatic reactions occurring within each active region can be individually measured by interrogating each working electrode simultaneously or at different times. Then the signals associated with each active region can be correlated with the concentration of the individual analyte.

[0233] As previously discussed herein, a membrane (i.e., a mass transfer limiting membrane) can coat one or more active regions in a sensor to increase biocompatibility and alter the analyte flux to the active regions. Such membranes can be present in any of the sensors disclosed herein. Since different analytes can exhibit altered permeability values in a given membrane, a sensor configured to analyze multiple analytes can exhibit different sensitivities for each analyte. One method for addressing different sensitivity values can involve using different membrane thicknesses over each active region. Although feasible, this method may be difficult to implement from a manufacturing perspective. That is, it may be difficult to vary the membrane thickness at different locations using typical dip coating techniques for membrane deposition. Other possible methods are to use active regions of different sizes for each analyte.

[0234] In some aspects, a sensor featuring two or more protein switches disposed on separate working electrodes may include: a sensor tail that at least includes a first working electrode and a second working electrode, a first active region located on the surface of the first working electrode, a second active region located on the surface of the second working electrode, a multi-component film coating the first active region, and a homogeneous film coating the second active region. The first active region contains a first polymer and a first protein switch that reacts with a first analyte, and the second protein switch contains a second polymer and a protein switch that reacts with a second analyte. The first protein switch and the second protein switch are different and reactive with different analytes. The multi-component film at least contains a first film polymer and a second film polymer that are different from each other. The homogeneous film contains one of the first film polymer and the second film polymer.

[0235] The specific configuration of the multi-component film described above may include a bilayer film in some aspects or may include a blend of film polymers in other aspects. Surprisingly, the bilayer film and the blend film can be used to equalize analyte permeability, as discussed further below.

[0236] The sensors of the present disclosure having two different active regions located on separate working electrodes may employ a sensor configuration similar to the sensor configuration described above in Figure 3 or a variant thereof. For example, in some aspects, the counter / reference electrode may replace the separate counter and reference electrodes in a sensor having two or more working electrodes. Similarly, the layer configuration and arrangement within a sensor having two different active regions located on separate working electrodes may be different from that depicted in Figure 3 as described above.

[0237] For example, in some aspects, a sensor having multiple working electrodes may include active regions where an electron transfer agent is covalently bonded to the polymer in each active region. In some or other aspects, such a sensor may be characterized by a first protein switch covalently bonded to the polymer in the first active region and a second protein switch covalently bonded to the polymer in the second active region. Also, in certain aspects, the first protein switch may include a first protein switch containing an analyte-binding domain capable of binding troponin and a glucose oxidase domain responsive to glucose, and the second protein switch may include a second protein switch containing an analyte-binding domain capable of binding BNP and a lactate oxidase domain responsive to lactate. Alternatively, the lactate oxidase domain may be replaced by a glucose oxidase domain responsive to glucose.

[0238] In still further aspects, a sensor having multiple working electrodes may include a sensor tail configured for insertion into tissue.

[0239] In some aspects, the bilayer membrane can overcoat a first active region on one of the working electrodes. The bilayer membrane comprises a first membrane polymer and a second membrane polymer laminated on top of each other above the active region. In more specific aspects, the first membrane polymer can be directly disposed on the active region of the first working electrode, and the second membrane polymer can be disposed on the first membrane polymer to define the bilayer membrane. In such aspects, the second membrane polymer is present in the homogeneous membrane located on the second working electrode. In some aspects, such a bilayer configuration can be prepared by coating the first membrane polymer only on the first working electrode (e.g., by spraying, painting, inkjet printing, roll coating, etc.), and then coating the second membrane polymer on both working electrodes simultaneously (e.g., by dip coating or a similar technique). In other aspects, the bilayer membrane can be configured as above, where the first membrane polymer is located on the second working electrode.

[0240] Figure 6 An illustrative schematic diagram showing a portion of a sensor is provided. The sensor has two working electrodes and is characterized by a bilayer membrane overcoating one of the two working electrodes, and is suitable for use in some aspects of the present disclosure. As Figure 6 shown, the sensor is characterized by a sensor tail 600 having working electrodes 614a and 614b disposed on opposite faces of a substrate 612. An active region 618a is disposed on the working electrode 614a, and an active region 618b is disposed on the working electrode 614b. According to the present disclosure, the active regions 618a and 618b contain different protein switches and are configured to assay different analytes. Although Figure 6 the active regions 618a and 618b have been shown to be disposed generally opposite to each other with respect to the substrate 612, it should be understood that the active regions 618a and 618b can be laterally spaced apart (offset) from each other on opposite faces of the substrate 612. The laterally spaced-apart configuration of the active regions 618a and 618b can be particularly advantageous for overcoating each of the active regions 618a and 618b with a mass transfer limiting membrane, as discussed below.

[0241] As Figure 6 further shown, the active region 618a is overcoated with a membrane layer 620. The membrane layer 620 is a homogeneous membrane comprising a single membrane polymer. The active region 618b is overcoated with a bilayer membrane 621, which includes a membrane layer 621a in direct contact with the active region 618b and a membrane layer 621b covering the membrane layer 621a. The membrane layers 621a and 621b comprise different membrane polymers. As described above, in certain aspects, the membrane layer 620 and the membrane layer 621b can comprise the same membrane polymer.

[0242] According to one or more aspects, a sensor having multiple active regions on separate working electrodes, where one of the active regions is coated with a bilayer membrane, can exhibit either homogenized or independently variable analyte permeability. That is, the sensor can have sensitivities to two different analytes that are closer to each other than when there is no bilayer membrane. In such a sensor configuration, an active region coated with a homogeneous membrane (e.g., Figure 6 the membrane layer 620 in Figure 6 ) can exhibit analyte permeability characteristic of its particular membrane polymer for a first analyte. Surprisingly, the bilayer membrane (e.g., the bilayer membrane 621 in

[0243] ) can contain a membrane polymer that does not negatively impact the permeability of a second analyte (i.e., a membrane polymer with a neutral permeability effect), such that the other membrane polymers making up the bilayer membrane can exhibit their characteristic permeability to the second analyte as if the first membrane polymer were not present. Thus, according to various aspects, a membrane polymer with a neutral permeability effect and the membrane polymers making up the homogeneous membrane can be considered the same polymer.

[0244] In some other specific aspects, a membrane polymer with a neutral permeability effect can include the inner layer of the bilayer membrane. Thus, according to such aspects, the inner layer of the bilayer membrane and the homogeneous membrane can be considered the same membrane polymer. In other specific aspects, the outer layer of the bilayer membrane and the homogeneous membrane can be considered the same membrane polymer. Figure 6 shown in

[0245] , except that the bilayer membrane 621 is replaced with a mixed membrane containing a homogeneous blend of two different membrane polymers. Similar to a sensor including a bilayer membrane disposed on one of the active regions, a homogeneous membrane containing one of the first or second membrane polymers of the mixed membrane can coat the other active region on the second working electrode.Similar to the bilayer membrane, a mixed membrane containing a membrane polymer that neutrally affects the permeability of a second analyte can allow the mixed membrane to exhibit a permeability characteristic mainly of another membrane polymer in the mixture for the second analyte. Thus, in accordance with various aspects of the present disclosure, one of the membrane polymers of the homogeneous membrane and the membrane polymer of the mixed membrane can be selected such that the permeability of the second analyte through the mixed membrane is substantially unaltered by the membrane polymer. In a particular aspect, the first active region can comprise a first protein switch containing an analyte-binding domain capable of binding troponin and a glucose oxidase domain responsive to glucose, and the second active region can comprise a second protein switch containing an analyte-binding domain capable of binding BNP and a lactate oxidase domain responsive to lactate. Thus, in accordance with such aspects, the first active region containing the first protein switch can be overcoated with a mixed membrane, and the second active region containing the second protein switch can be overcoated with a homogeneous (single-component membrane polymer) membrane. In yet other aspects, the second active region can comprise a polymer, albumin, and a second protein switch of a covalently bonded polymer. In yet still more specific aspects, the homogeneous membrane overcoating the second active region can comprise at least a crosslinked polyvinylpyridine homopolymer or copolymer, and the mixed membrane overcoating the first active region can also comprise a polyvinylpyridine homopolymer or copolymer.

[0246] As mentioned above, bilayer membranes and mixed membranes can equalize analyte permeability in the sensors described herein, where two or more active regions are spatially separated from each other and can be overcoated with different mass transfer-limiting membranes. Specifically, bilayer membranes and mixed membranes can equalize analyte permeability in sensors having separate working electrodes and comprising two or more active regions with different protein switches (where at least one active region is located at each working electrode). Thus, such membranes can advantageously allow the sensor sensitivity to be changed independently for each analyte. 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 adjust the characteristic permeability of the analyte at each working electrode.

[0247] Accordingly, a method for using a sensor having two working electrodes may include exposing the sensor to a fluid containing at least one analyte. The sensor includes a sensor tail that includes 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 contains a first polymer and a first protein switch that reacts with a first analyte, and the second active region contains a second polymer and a protein switch that reacts with a second analyte. The first protein switch and the second protein switch are different. A multi-component film coats the first active region, and a homogeneous film coats the second active region. The multi-component film contains at least a first film polymer and a second film polymer that are different from each other, and the homogeneous film contains one of the first film polymer or the second film polymer. The method further includes obtaining a first signal when at or above the redox potential of the first active region, obtaining a second signal when at or above the redox potential of the second active region, 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. 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.

[0248] In other aspects, the first signal and the second signal may be measured at different times. Accordingly, in such aspects, a potential may be applied alternately to the first working electrode and the second working electrode. In other aspects, the first signal and the second signal may be measured simultaneously via a first channel and a second channel, in which case a potential may be applied to both electrodes simultaneously.

[0249] In some aspects, the sensor system may further include a reactant storage device that may act as a reservoir for holding reactants for the enzyme portion of the protein switch, where the reactants are not obtained from the analyte-containing sample. Examples of reactants are glucose, lactate, etc. The reactant storage device may release the reactants over an extended period of time. The storage device may be capable of delivering the reactants 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 device or an osmotic device for extended delivery of the reactants from the enzyme storage device. The enzyme storage device may include a mechanical device or an osmotic device for extended delivery of the reactants from the enzyme storage device. The enzyme storage device may also have the above combinations for providing an extended release of the reactants. Other methods and techniques may also be used for extended release of the reactants.

[0250] The storage device can have pores or holes through which the reactant is released. The pores or holes through which the reactant is released to the protein switch can be very small, and the delivery of the reactant from the storage device can occur over an extended period of time. Such pores or holes can be made by etching, laser machining, machining, drilling, and / or conventional processes.

[0251] A membrane or coating can be placed over the pores or holes of the storage device to extend the delivery time of the reactant from the storage device. The storage device can be composed of a porous material or a portion of the storage device can be composed of a porous material, and the porous material can be coated with a polymer coating and / or membrane that delays the release of the reactant from the storage device, thereby delaying the release time of the reactant. The coating can be made from a polymer dispersion such as a polyurethane dispersion (BAYHDROL TM etc.), an acrylic latex dispersion, and made.

[0252] The reactant storage device can provide the delivery of reactants through an osmotic delivery system. A “standard osmotic delivery system” that can be used is the elementary osmotic pump (EOP) system. The EOP is well-known. This system can be used to deliver reactants and is particularly suitable for reactants that give an “unstable” release profile or provide an incomplete release profile. 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 can include a water-soluble compound suitable for inducing osmosis, i.e., an osmotic agent or osmogent, including pharmaceutically acceptable and pharmacologically inert water-soluble compounds mentioned in pharmacopoeias such as the United States Pharmacopia and in Remington: The Science and Practice of Pharmacy; 19th Edition; Mack Publishing Company, Easton, Pa. (1995). The osmotic agent can be selected from pharmaceutically acceptable water-soluble salts of inorganic or organic acids or nonionic organic compounds with high water solubility, such as carbohydrates (such as sugars) or amino acids. The osmotic agent can 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 osmotic agent that can be used depends on the specific osmotic agent used and can range from about 1 wt% to about 60 wt% of the reactant mixture. The osmotic delivery system can also include polymers, such as those described above as drug-eluting polymers. The osmotic delivery system can also include a coating and / or membrane that acts as a semipermeable barrier between the enzyme and the reactants. Other suitable membranes and / or coatings are known and can be used.

[0253] The reactant storage device can include or can be part of a microfluidic system for delivering reactants to the protein switch. Microfluidic systems for delivering reactants to an enzyme over an extended period of time are well known to those of ordinary skill in the art. See, for example, U.S. Patent Nos. 9,194,859 and 8,460,607; Madou, Fundamentals of Microfabrication: The Science of Miniaturization, 2nd Edition (Hardcover), CRC Press, 2002; and microfluidics disclosed in Nguyen et al., Fundamentals and Applications of Microfluidics, Artech House Publishers, (2002).

[0254] E. Method of using a device and sensor system comprising a protein switch

[0255] Devices containing one or more protein switches can detect, identify, and / or monitor any of the analytes described in Part B below. For example, a device containing a protein switch can be used to monitor a patient at risk of a cardiac adverse event. For example, the device can detect cardiac troponin, and the device can be used to monitor a patient at risk of myocardial infarction. The device can also be used to monitor cardiac patients after surgery or other treatment to monitor cardiac function and provide early warning of potential adverse cardiac events.

[0256] In some aspects, a device containing a protein switch can be used to monitor a subject's exposure to an infectious agent. For example, the device can be used to monitor a patient after acute exposure to an infectious agent and / or to track the progression of an infection. In other aspects, the device can be used to monitor a subject's response to an anti-infective drug and / or procedure administered to the subject to treat an infectious disease. In yet other aspects, the device can also be used for personalized treatment such that the subject receives sufficient treatment to treat their sepsis or infection.

[0257] In still yet other aspects, the device can also be used to monitor drug therapy in a subject being treated with one or more drugs. In still other aspects, a device containing a protein switch can be used to detect the presence of one or more drugs and / or drug metabolites.

[0258] A device containing a protein switch can be used to automatically detect an analyte. The device can detect a change in the amount or level of an analyte in a body fluid. The device can detect the rate of change of an analyte in a body fluid. The device can detect the time at which an analyte reaches a threshold level or amount. Additionally, the device can automatically monitor multiple analytes in a subject's body fluid.

[0259] The present disclosure will be better understood in light of 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. Unless otherwise specified, the present disclosure is not limited to specific procedures, materials, etc., and may thus vary. It should also be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting.

[0260] Examples

[0261] Example 1 - Generation and Identification of Protein Switches

[0262] A library of glucose dehydrogenase (GDH) variants containing GDH polypeptides developed using GDH-105 (Codexis, Redwood, CA) as a scaffold was prepared. Approximately 2,800 GDH variants were prepared. The glucose activity of the variants was screened against one or more of the analytes listed in Table 4 below. Glucose activity was determined by NADH kinetics at different time intervals using a spectrophotometer set at UV 340 or UV 360 or by endpoint measurement of gluconic acid using a RapidFire mass spectrometer.

[0263] Table 4: Amounts of Reaction Mixtures and Analytes

[0264] Table 4

[0265]

[0266] Table 5 shows the protein switches identified as binding to one or more of the test analytes and whether the binding of the analyte to the protein switch inhibits or activates glucose activity.

[0267] Table 5

[0268]

[0269]

[0270] Example 2 - Evolution of a Protein Switch Against Warfarin

[0271] The protein switch GDH-2016 (also known as Rd2bb) having the amino acid sequence of SEQ ID NO:11 was evolved using conventional techniques known in the art to generate a library of variants. Approximately 3,024 variants were prepared.

[0272] The glucose activity and warfarin inhibition (inhibition %) of each variant were screened by combining each variant with a reaction mixture containing 1.25 g / L glucose, 5 g / L NAD+, 20% HTP GDH-NAD lysate in 50 mM NaPO4 (pH 7.0) with or without 0.1 g / L warfarin. Glucose activity was evaluated by NADH kinetics assay using a spectrophotometer (Molecular Devices SpectraMax M5) set at UV 360, and readings were taken at 1-minute intervals for 8 minutes. The top variants showing improved inhibition (relative to Rd2bb) were grown at shake flask scale and further tested for warfarin inhibition (Ki) by combining each variant with a reaction mixture containing 0 - 10 g / L glucose, 25 g / L NAD+, 0 - 0.5 g / L warfarin, GDH-NAD shake flask powder (loaded in the range of 0.5 to 2.5 g / L depending on the individual variant) in 50 mM NaPO4 (pH 7.0). Protein switches were identified that showed low Ki (high glucose inhibition) and high glucose activity (e.g., catalytic efficiency) as shown in Table 6 below. These protein switches were determined to have the amino acid sequences shown in SEQ ID NO.: 1 - 10. The protein switch with the sequence of SEQ ID NO.1 (GDH-2025; also known as Rd3bb) was selected for further evolution.

[0273] Table 6

[0274]

[0275] Evolve the protein switch GDH-2025 using a similar technique as described above. Approximately 1680 variants were prepared. Screen the glucose activity and inhibition (Ki) of each variant by combining each variant with a reaction mixture containing 3.5 g / L glucose, 12.5 NAD+, 10% HTP GDH-NAD lysis solution, and 0.003 g / L sodium warfarin in 50 mM NaPO4 (pH 7.0). Evaluate the glucose activity using a spectrophotometer (Molecular Devices SpectraMax M5) set at UV 360 and read for 8 minutes at 1-minute intervals. Grow the top variants showing improved inhibition (relative to Rd3bb) at shake flask scale and further test the warfarin inhibition (Ki) by combining each variant with a reaction mixture containing 0 - 10 g / L glucose, 25 g / L NAD+, 0 - 0.5 g / L warfarin, and 0.5 g / L GDH-NAD shake flask powder in 50 mM NaPO4 (pH 7.0). Identify protein switches that exhibit low Ki (high glucose inhibition) and high glucose activity (e.g., catalytic efficiency) as shown in Table 7 below. Determine that these protein switches have the amino acid sequences shown in SEQ ID NO.: 12 - 19. Select the protein switch with the sequence of SEQ ID NO. 15 (GDH-2028; also known as Rd4bb) for further evolution.

[0276] Table 7

[0277]

[0278] Example 3 - Protein Switch Sensor

[0279] Skin preparation . Prepare experimental 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) glucose-responsive active regions using a single-layer active area system. GDH was obtained from Codexis, Redwood City, CA. Coat the active regions on the carbon working electrode as a single-layer composition (Table 8 below). After depositing the active regions (0.11 mm2 ) After that, the active area is cured at room temperature for three days. Then, a PVP membrane is applied to the working electrode using a membrane coating solution containing 4 mL of 100 mg / mL polyvinylpyridine and 200 μL of 100 mg / mL PEGDGE 400. The membrane is deposited on the active area (dipping at 3 x 1 mm / second) and cured at 25 °C and 60% relative humidity for two days.

[0280] Table 8

[0281]

[0282] Beaker calibration. Glucose sensing analysis of sensors including GDH-105 and sensors including 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 as described above is carried out as follows: The electrodes are immersed in 100 mM PBS buffer solution at room temperature and different glucose concentrations (1, 2, 3, 5, 7, 10, 15, 20, 25, and 30 mM glucose). Figure 7 Shows the response of each 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. As shown in Table 9 below, each GDH shows a measurable response to increasing glucose concentration, where the sensitivity varies from 0.09 to 0.73 nA / mM glucose. Figure 7 Shows the linear sensitivity response based on beaker calibration 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, which shows a positive driving force.

[0283] Table 9

[0284] Project ID Sensitivity (nA / mM glucose) GDH - 105 (control) 0.55 GDH - 2004 (MTX) 0.30 GDH - 2007 (MTX 0.40 GDH - 2008 (MTX) 0.49 GDH - 2009 (MTX) 0.62 GDH - 2010 (MTX) 0.30 GDH - 2011 (MTX) 0.34 GDH - 2015 (MTX) 0.13 GDH - 2018 (T3) 0.63 GDH - 2019 (T3) 0.67 GDH - 2004 (CT) 0.23 GDH - 2005 (CT) 0.73 GDH - 2004 (WF) 0.28 GDH - 2009 (WF) 0.55 GDH - 2013 (WF) 0.46 GDH - 2016 (WF) 0.56 GDH - 2024 (WF) 0.09

[0285] Beaker stability.The beaker stability (long-term stability) of the sensor GDH-105 and sensors including 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 described above was evaluated in 30 mM glucose in 100 mM PBS at 33 °C, as Figure 8 shown. After 4 days, each signal experienced sensor decline (decline in stability for glucose detection) at different rates, as shown in Table 10. The signal decline of the GDH-105 and GDH-2018 sensors was significantly less than that of the other tested sensors.

[0286] Table 10

[0287] Project ID Signal decline (4 days) GDH - 105 (control) -2％ GDH - 2004 (MTX) -96％ GDH - 2007 (MTX) -100％ GDH - 2008 (MTX) -77％ GDH - 2009 (MTX) -45％ GDH - 2010 (MTX) -98％ GDH - 2011 (MTX) -85％ GDH - 2015 (MTX) -76％ GDH - 2018 (T3) -8％ GDH - 2019 (T3) -46％ GDH - 2004 (CT) -56％ GDH - 2005 (CT) -29％ GDH - 2004 (WF) -89％ GDH - 2009 (WF) -56％ GDH - 2013 (WF) -76％ GDH - 2016 (WF) -87％ GDH - 2024 (WF) -100％

[0288] Example 4 - Warfarin Sensor

[0289] Warfarin sensor. Experimental GDH-105 and GDH-2016 glucose-responsive active regions were prepared using a single-layer active region system. GDH was obtained from Codexis, Redwood City, CA. The active regions were coated on carbon working electrodes as a single-layer composition (Table 8). After depositing the active regions, the active regions were cured at RT for three days.

[0290] Experimental procedure. The testing of the GDH-105 and GDH-2016 sensors was carried out as follows: The electrodes were immersed in a 100 mM PBS buffer solution at 33 °C with NAD + (5 mM), where NAD was provided to the test beaker containing the PBS buffer, and then glucose (1 mM) was added. Both the GDH-105 and GDH-2016 sensors responded to the addition of glucose. Then warfarin was added to the test beaker at different concentrations (10, 30, 80, 180, and 300 μM warfarin). Figure 9A And B shows the inhibition of the GDH-2016 sensor by increasing warfarin concentrations (about 55% inhibition at 300 μM warfarin), while the GDH-105 sensor was not affected.

[0291] Evolved warfarin sensor。The experimental GDH-105 (control), GDH-2025 (WF), GDH-2026 (WF), GDH-2027 (WF), and GDH-2028 (WF) glucose-responsive active regions were prepared using a single-layer active region system. The GDH was obtained from Codexis, Redwood City, CA. The active regions were coated on the carbon working electrode as a single-layer composition (Table 8). After depositing the active regions (0.33 mm 2 ), the active regions were cured at room temperature for three days. Then, a PVP membrane was applied to the working electrode using a membrane coating solution containing 4 mL of 100 mg / mL polyvinylpyridine and 200 μL of 100 mg / mL PEGDGE 400. The membrane was deposited on the active region (dipping at 3x1 mm / second) and cured at 25 °C and 60% relative humidity for two days.

[0292] Beaker calibration. The glucose sensing analysis of the GDH-105, GDH-2025, GDH-2026, GDH-2027, and GDH-2028 sensors prepared as described above was performed by immersing the electrodes in 100 mM PBS buffer solution at room temperature and different glucose concentrations (1, 2, 3, 5, 7, 10, 15, 20, 25, and 30 mM glucose). Figure 10 The response of each of the GDH-105, GDH-2025, GDH-2026, GDH-2027, and GDH-2028 sensors is shown. As shown in Table 11, each GDH exhibited a measurable response to increasing glucose concentration, with the sensitivity varying from 0.09 to 1.53 nA / mM glucose. Figure 10 The linear sensitivity response based on beaker calibration of the GDH-105, GDH-2025, GDH-2026, GDH-2027, and GDH-2028 sensors is shown, which exhibited a positive driving force.

[0293] Table 11

[0294] Enzyme ID Sensitivity (nA / mM) GDH - 105 (control) 1.53 GDH - 2025 (WF) 0.32 GDH - 2026 (WF) 0.22 GDH - 2027 (WF) 0.29 GDH - 2028 (WF) 0.09

[0295] Beaker stability. The beaker stability (long-term stability) of the GDH-105, GDH-2025, GDH-2026, GDH-2027, and GDH-2028 sensors was evaluated in 30 mM glucose in 100 mM PBS at 33 °C, as Figure 11 shown. After 1 day, each signal experienced a different rate of sensor decline (decline in stability for glucose detection), as provided in Table 12. The signal decline of the GDH-105 sensor was significantly less than that of the other tested sensors.

[0296] Table 12

[0297] Enzyme ID Signal decline (1 day) GDH-105 (Control) -0％ GDH-2025 (WF) -58％ GDH-2026 (WF) -69％ GDH-2027 (WF) -64％ GDH-2028 (WF) -68％

[0298] Evolved Warfarin Sensor 。Using a single-layer active region system to prepare GDH-105, GDH-2016, GDH-2025, GDH-2026, GDH-2027, and GDH-2028 glucose-responsive active regions. GDH was obtained from Codexis, Redwood City, CA. The active regions (0.33 mm 2 ) were coated onto the carbon working electrode as a single-layer composition (Table 8). After depositing the active regions, the active regions were cured at room temperature for three days.

[0299] Experimental Procedure. The testing of GDH-105, GDH-2016, GDH-2025, GDH-2026, GDH-2027, and GDH-2028 sensors was conducted as follows: The electrode was immersed in a 100 mM PBS buffer solution at 33 °C with NAD + (10 mM), where NAD was provided to the test beaker containing the PBS buffer, and then glucose (5 mM) was added. The GDH-105, GDH-2016, GDH-2025, GDH-2026, GDH-2027, and GDH-2028 sensors showed a response to the addition of glucose. Then warfarin was added to the test beaker at different concentrations (10, 30, 80, 180, and 300 μM warfarin). Figure 12A and 12B as well as Figure 13 showed inhibition of the GDH-105, GDH-2016, GDH-2025, GDH-2026, GDH-2027, and GDH-2028 sensors for increasing warfarin concentrations, while the GDH-105 sensor remained unaffected. The warfarin inhibition percentages changed for sensors GDH-105, GDH-2025, GDH-2026, GDH-2027, and GDH-2028, as shown in Table 13.

[0300] Table 13

[0301] Enzyme ID GDH Inhibition at 0.3 mM WF GDH-105 (Control) 5％ GDH-2025 (WF) 79％ GDH-2026 (WF) 78％ GDH-2027 (WF) 79％ GDH-2028 (WF) 84％

[0302] All publications, patents, and patent applications discussed and cited herein are incorporated herein by reference in their entirety. It should be understood that the aspects disclosed herein are not limited to the specific methods, protocols, and materials described, and thus these methods, protocols, and materials may vary. It should also be understood that the terms used herein are for the purpose of describing particular aspects only and are not intended to limit the scope of the present disclosure, the scope of the invention being defined only by the appended claims.

[0303] Those skilled in the art will recognize or be able to ascertain many equivalents of the specific aspects of the present disclosure described herein using only routine experimentation. Such equivalents are intended to be encompassed by the following claims.

[0304] For completeness, the various aspects of the present disclosure are set forth in the following numbered clauses:

[0305] Clause 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 reactant, wherein (i) the analyte that binds to the analyte-binding domain is different from the reactant that binds to or reacts with the oxidase or dehydrogenase domain; and (ii) when the analyte binds to the analyte-binding domain, the oxidase or dehydrogenase activity is altered.

[0306] Clause 2. The protein switch according to Clause 1, wherein the oxidase is glucose oxidase or lactate oxidase.

[0307] Clause 3. The protein switch according to Clause 1, wherein the dehydrogenase is glucose dehydrogenase or lactate dehydrogenase.

[0308] Clause 4. The protein switch according to any one of Clauses 1-3, wherein when the analyte-binding domain binds to the analyte, the activity of the oxidase or dehydrogenase is reduced.

[0309] Clause 5. The protein switch according to any one of Clauses 1-3, wherein when the analyte-binding domain binds to the analyte, the activity of the oxidase or dehydrogenase is increased.

[0310] Clause 6. The protein switch according to any one of Clauses 4-5, wherein when the analyte-binding domain binds to the analyte, the activity of the oxidase or dehydrogenase is increased or reduced due to competitive inhibition, uncompetitive inhibition, or non-competitive inhibition.

[0311] Clause 7. The protein switch according to any one of Clauses 4-5, wherein when the analyte-binding domain binds to the analyte, the activity of the oxidase or dehydrogenase is reduced due to competitive inhibition.

[0312] Clause 8. The protein switch according to any one of Clauses 1-7, wherein the analyte is warfarin, cortisol, methotrexate, or triiodothyronine.

[0313] Clause 9. A protein switch as described in any one of Clauses 1-8, wherein the reactant is glucose or lactate.

[0314] Clause 10. A protein switch comprising at least 7 mutations at amino acid positions 96, 155, 156, 159, 170, 198, and 252 of SEQ ID NO:20.

[0315] Clause 11. The protein switch as described in Clause 10, wherein the protein switch comprises:

[0316] cysteine, phenylalanine, methionine, tryptophan, or tyrosine at amino acid position 96 of SEQ ID NO:20;

[0317] alanine, glycine, isoleucine, leucine, or valine at amino acid position 155 of SEQ ID NO:20;

[0318] threonine or serine at amino acid position 156 of SEQ ID NO:20;

[0319] threonine or serine at amino acid position 159 of SEQ ID NO:20;

[0320] lysine, arginine, or histidine at amino acid position 170 of SEQ ID NO:20;

[0321] glutamic acid or aspartic acid at amino acid position 198 of SEQ ID NO:20; and

[0322] alanine, glycine, isoleucine, leucine, or valine at amino acid position 252 of SEQ ID NO:20.

[0323] Clause 12. The protein switch as described in Clause 11, further comprising one or more of the following:

[0324] glycine, isoleucine, leucine, or valine at amino acid position 11 of SEQ ID NO:20;

[0325] glycine, isoleucine, leucine, or valine at amino acid position 22 of SEQ ID NO:20;

[0326] asparagine or glutamine at amino acid position 45 of SEQ ID NO:20;

[0327] alanine, glycine, isoleucine, or leucine at amino acid position 48 of SEQ ID NO:20;

[0328] Aspartic acid or glutamic acid at amino acid position 55 of SEQ ID NO:20;

[0329] Alanine, glycine, isoleucine, leucine or valine at amino acid position 98 of SEQ ID NO:20;

[0330] Phenylalanine, tryptophan or tyrosine at amino acid position 137 of SEQ ID NO:20;

[0331] Alanine, glycine, leucine or valine at amino acid position 141 of SEQ ID NO:20;

[0332] Alanine, glycine, isoleucine or leucine at amino acid position 149 of SEQ ID NO:20;

[0333] Alanine, glycine, isoleucine or valine at amino acid position 154 of SEQ ID NO:20;

[0334] Threonine or serine at amino acid position 166 of SEQ ID NO:20;

[0335] Glycine, isoleucine, leucine or valine at amino acid position 173 of SEQ ID NO:20;

[0336] Threonine or serine at amino acid position 184 of SEQ ID NO:20;

[0337] Histidine, leucine or arginine at amino acid position 195 of SEQ ID NO:20;

[0338] Threonine or serine at amino acid position 219 of SEQ ID NO:20;

[0339] Asparagine or glutamine at amino acid position 240 of SEQ ID NO:20; and / or

[0340] Alanine, glycine, isoleucine, leucine or valine at amino acid position 251 of SEQ ID NO:20.

[0341] Clause 13. A protein switch comprising an amino acid sequence having at least 80% identity to SEQ ID NO.:11.

[0342] Clause 14. The protein switch according to Clause 13, comprising an amino acid sequence having at least 85% identity to SEQ ID NO.:11.

[0343] Clause 15. The protein switch as described in any one of Clauses 13 - 14 contains an amino acid sequence having at least 90% identity with SEQ ID NO.: 11.

[0344] Clause 16. The protein switch as described in any one of Clauses 13 - 15 contains an amino acid sequence having at least 95% identity with SEQ ID NO.: 11.

[0345] Clause 17. The protein switch as described in any one of Clauses 13 - 16 contains an amino acid sequence having at least 96% identity with SEQ ID NO.: 11.

[0346] Clause 18. The protein switch as described in any one of Clauses 13 - 17 contains an amino acid sequence having at least 97% identity with SEQ ID NO.: 11.

[0347] Clause 19. The protein switch as described in any one of Clauses 13 - 18 contains an amino acid sequence having at least 98% identity with SEQ ID NO.: 11.

[0348] Clause 20. The protein switch as described in any one of Clauses 13 - 19 contains an amino acid sequence having at least 99% identity with SEQ ID NO.: 11.

[0349] Clause 21. The protein switch as described in any one of Clauses 13 - 20 contains an amino acid sequence having at least 100% identity with SEQ ID NO.: 11.

[0350] Clause 22. The protein switch as described in any one of Clauses 13 - 21, wherein the protein switch contains the amino acid sequence of any one of SEQ ID NO. 1 - 10 or 12 - 19.

[0351] Clause 23. A composition or kit, which contains at least one protein switch as described in Clause 1, 10, 11 or 13 and at least one reactant.

[0352] Clause 24. The composition as described in Clause 23, wherein the reactant is glucose or lactic acid.

[0353] Clause 25. A method for detecting an analyte, the method comprising:

[0354] Provided is a protein switch, the 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 reactant, wherein (i) the analyte binding to the analyte-binding domain is different from the reactant binding to or reacting with the oxidase or dehydrogenase domain; and (ii) when the analyte binds to the analyte-binding domain, the oxidase or dehydrogenase activity changes;

[0355] Contacting the protein switch with a fluid comprising a reactant specific for the protein switch, wherein the analyte-binding domain binds the analyte in the fluid, whereby the oxidase or dehydrogenase activity changes; and

[0356] Detecting a change in the rate of decomposition of the reactant by the oxidase or dehydrogenase domain of the protein switch.

[0357] Clause 26. The method according to clause 25, wherein the oxidase is glucose oxidase or lactate oxidase.

[0358] Clause 27. The method according to clause 25, wherein the dehydrogenase is glucose dehydrogenase or lactate dehydrogenase.

[0359] Clause 28. The method according to any one of clauses 26-27, wherein the reactant is glucose or lactate.

[0360] Clause 29. A method for detecting an analyte, the method comprising:

[0361] Providing a protein switch according to any one of clauses 10, 11 and 13,

[0362] Contacting the protein switch with a fluid comprising at least one reactant specific for the protein switch, wherein the analyte-binding domain binds the analyte in the fluid, whereby the oxidase or dehydrogenase activity changes; and

[0363] Detecting a change in the rate of decomposition of the reactant by the oxidase or dehydrogenase domain of the protein switch.

[0364] Clause 30. The method according to clause 29, wherein the protein switch comprises an amino acid sequence having at least 85% identity to SEQ ID NO.:11.

[0365] Clause 31. The method as described in Clause 29 or 30, wherein the protein switch comprises an amino acid sequence having at least 90% identity with SEQ ID NO.: 11.

[0366] Clause 32. The method as described in any one of Clauses 29-31, wherein the protein switch comprises an amino acid sequence having at least 95% identity with SEQ ID NO.: 11.

[0367] Clause 33. The method as described in any one of Clauses 29-32, wherein the protein switch comprises an amino acid sequence having at least 96% identity with SEQ ID NO.: 11.

[0368] Clause 34. The method as described in any one of Clauses 29-33, wherein the protein switch comprises an amino acid sequence having at least 97% identity with SEQ ID NO.: 11.

[0369] Clause 35. The method as described in any one of Clauses 29-34, wherein the protein switch comprises an amino acid sequence having at least 98% identity with SEQ ID NO.: 11.

[0370] Clause 36. The method as described in any one of Clauses 29-35, wherein the protein switch comprises an amino acid sequence having at least 99% identity with SEQ ID NO.: 11.

[0371] Clause 37. The method as described in any one of Clauses 29-36, wherein the protein switch comprises an amino acid sequence having at least 100% identity with SEQ ID NO.: 11.

[0372] Clause 38. The method as described in any one of Clauses 29-37, wherein the protein switch comprises the amino acid sequence of any one of SEQ ID NO. 1-10 or 12-19.

[0373] Clause 39. The method as described in any one of Clauses 29-38, wherein the oxidase is glucose oxidase or lactate oxidase.

[0374] Clause 40. The method as described in any one of Clauses 29-39, wherein the dehydrogenase is glucose dehydrogenase or lactate dehydrogenase.

[0375] Clause 41. The method as described in any one of Clauses 29-40, wherein the reactant is glucose or lactate.

[0376] Clause 42. The method as described in any one of Clauses 29-41, wherein the analyte is warfarin, cortisol, methotrexate or triiodothyronine.

[0377] Clause 43. A system for detecting or monitoring the concentration of an analyte, comprising a sensor control device and a signal detection device, wherein the sensor control device comprises at least one sensor, and the at least one sensor comprises a protein switch as described in Clause 1, 10, 11 or 13.

[0378] Clause 44. The system according to Clause 43, wherein the oxidase is glucose oxidase or lactate oxidase.

[0379] Clause 45. The system according to Clause 43, wherein the dehydrogenase is glucose dehydrogenase or lactate dehydrogenase.

[0380] Clause 46. The system according to any one of Clauses 43-45, wherein the protein switch comprises an amino acid sequence having at least 85% identity with SEQ ID NO.: 11.

[0381] Clause 47. The system according to any one of Clauses 43-46, wherein the protein switch comprises an amino acid sequence having at least 90% identity with SEQ ID NO.: 11.

[0382] Clause 48. The system according to any one of Clauses 43-47, wherein the protein switch comprises an amino acid sequence having at least 95% identity with SEQ ID NO.: 11.

[0383] Clause 49. The system according to any one of Clauses 43-48, wherein the protein switch comprises an amino acid sequence having at least 96% identity with SEQ ID NO.: 11.

[0384] Clause 50. The system according to any one of Clauses 43-49, wherein the protein switch comprises an amino acid sequence having at least 97% identity with SEQ ID NO.: 11.

[0385] Clause 51. The system according to any one of Clauses 43-50, wherein the protein switch comprises an amino acid sequence having at least 98% identity with SEQ ID NO.: 11.

[0386] Clause 52. The system according to any one of Clauses 43-51, wherein the protein switch comprises an amino acid sequence having at least 99% identity with SEQ ID NO.: 11.

[0387] Clause 53. The system according to any one of Clauses 43-52, wherein the protein switch comprises an amino acid sequence having at least 100% identity with SEQ ID NO.: 11.

[0388] Clause 54. A system as described in any one of Clauses 43 - 53, wherein the protein switch comprises the amino acid sequence of any one of SEQ ID NOs. 1 - 10 or 12 - 19.

[0389] Clause 55. An analyte monitoring system, comprising: a sensor including a substrate, a working electrode, and a protein switch as described in Clause 1, 10, 11, or 13, at least a portion of the sensor being adapted for implantation and in intimate contact with a body fluid, the sensor being configured and arranged to generate a signal representative of the level of an analyte in the body fluid; and signal detection means for receiving the signal, wherein the signal is generated by contact of the analyte with the protein switch.

[0390] Clause 56. The analyte monitoring system as described in Clause 55, wherein the oxidase is glucose oxidase or lactate oxidase.

[0391] Clause 57. The analyte monitoring system as described in Clause 55, wherein the dehydrogenase is glucose dehydrogenase or lactate dehydrogenase.

[0392] Clause 58. The analyte monitoring system as described in any one of Clauses 55 - 57, wherein the protein switch comprises an amino acid sequence having at least 85% identity with SEQ ID NO.: 11.

[0393] Clause 59. The analyte monitoring system as described in any one of Clauses 55 - 58, wherein the protein switch comprises an amino acid sequence having at least 90% identity with SEQ ID NO.: 11.

[0394] Clause 60. The analyte monitoring system as described in any one of Clauses 55 - 59, wherein the protein switch comprises an amino acid sequence having at least 95% identity with SEQ ID NO.: 11.

[0395] Clause 61. The analyte monitoring system as described in any one of Clauses 55 - 60, wherein the protein switch comprises an amino acid sequence having at least 96% identity with SEQ ID NO.: 11.

[0396] Clause 62. The analyte monitoring system as described in any one of Clauses 55 - 61, wherein the protein switch comprises an amino acid sequence having at least 97% identity with SEQ ID NO.: 11.

[0397] Clause 63. The analyte monitoring system as described in any one of Clauses 55 - 62, wherein the protein switch comprises an amino acid sequence having at least 98% identity with SEQ ID NO.: 11.

[0398] Clause 64. An analyte monitoring system as described in any one of Clauses 55 - 63, wherein the protein switch comprises an amino acid sequence having at least 99% identity with SEQ ID NO.: 11.

[0399] Clause 65. An analyte monitoring system as described in any one of Clauses 55 - 64, wherein the protein switch comprises an amino acid sequence having at least 100% identity with SEQ ID NO.: 11.

[0400] Clause 66. An analyte monitoring system as described in any one of Clauses 55 - 65, wherein the protein switch comprises the amino acid sequence of any one of SEQ ID NOs. 1 - 10 or 12 - 19.

[0401] Clause 67. An analyte monitoring system as described in any one of Clauses 55 - 66, wherein the analyte is warfarin, cortisol, methotrexate, or triiodothyronine. Sequence Listing <110> Abbott Diabetes Care Inc., USA <120> Detection of Analytes by Protein Switches <130> ABBTD - 37810.601 <150> US 62 / 909,411 <151> 2019 - 10 - 02 <160> 20 <170> PatentIn version 3.5 <210> 1 <211> 262 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 1 Ala Met Tyr Pro Asp Leu Lys Gly Lys Val Val Ala Ile Thr Gly Ala 1 5 10 15 Ala Ser Gly Leu Gly Lys Ala Met Ala Ile Arg Phe Gly Lys Glu Gln 20 25 30 Ala Lys Val Val Ile Asn Tyr Tyr Ser Asn Lys Gln Asp Pro Asn Glu 35 40 45 Val Lys Glu Glu Val Ile Lys Ala Gly Gly Glu Ala Val Val Val Gln 50 55 60 Gly Asp Val Thr Lys Glu Glu Asp Val Lys Asn Ile Val Gln Thr Ala 65 70 75 80 Ile Lys Glu Phe Gly Thr Leu Asp Ile Met Ile Asn Asn Ala Gly Leu 85 90 95 Phe Asn Pro Val Pro Ser His Glu Met Pro Leu Lys Asp Trp Asp Lys 100 105 110 Val Ile Gly Thr Asn Leu Thr Gly Ala Phe Leu Gly Ser Arg Glu Ala 115 120 125 Ile Lys Tyr Phe Val Glu Asn Asp Ile Lys Gly Asn Val Ile Asn Met 130 135 140 Ser Ser Val His Glu Val Ile Pro Trp Pro Leu Ala Ser His Tyr Thr 145 150 155 160 Ala Ser Lys Gly Gly Met Lys Leu Met Thr Lys Thr Leu Ala Leu Glu 165 170 175 Tyr Ala Pro Lys Gly Ile Arg Val Asn Asn Ile Gly Pro Gly Ala Ile 180 185 190 Asn Thr Thr Ile Asn Ala Glu Lys Phe Ala Asp Pro Lys Gln Lys Ala 195 200 205 Asp Val Glu Ser Met Ile Pro Met Gly Tyr Ile Gly Glu Pro Glu Glu 210 215 220 Ile Ala Ala Val Ala Ala Trp Leu Ala Ser Lys Glu Ala Ser Tyr Val 225 230 235 240 Thr Gly Ile Thr Leu Phe Ala Asp Gly Gly Met Thr Leu Tyr Pro Ser 245 250 255 Phe Gln Ala Gly Arg Gly 260 <210> 2 <211> 261 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 2 Met Tyr Pro Asp Leu Lys Gly Lys Val Val Ala Ile Thr Gly Ala Ala 1 5 10 15 Ser Gly Leu Gly Lys Ala Met Ala Ile Arg Phe Gly Lys Glu Gln Ala 20 25 30 Lys Val Val Ile Asn Tyr Tyr Ser Asn Lys Gln Asp Pro Asn Glu Val 35 40 45 Lys Glu Glu Val Ile Lys Ala Gly Gly Glu Ala Val Val Val Gln Gly 50 55 60 Asp Val Thr Lys Glu Glu Asp Val Lys Asn Ile Val Gln Thr Ala Ile 65 70 75 80 Lys Glu Phe Gly Thr Leu Asp Ile Met Ile Asn Asn Ala Gly Leu Cys 85 90 95 Asn Leu Val Pro Ser His Glu Met Pro Leu Lys Asp Trp Asp Lys Val 100 105 110 Ile Gly Thr Asn Leu Thr Gly Ala Phe Leu Gly Ser Arg Glu Ala Ile 115 120 125 Lys Tyr Phe Val Glu Asn Asp Ile Lys Gly Asn Val Ile Asn Met Ser 130 135 140 Ser Val His Glu Val Ile Pro Trp Pro Leu Ala Ser His Tyr Thr Ala 145 150 155 160 Ser Lys Gly Gly Met Lys Leu Met Thr Lys Thr Leu Ala Leu Glu Tyr 165 170 175 Ala Pro Lys Gly Ile Arg Val Asn Asn Ile Gly Pro Gly Ala Ile Asn 180 185 190 Thr Thr Ile Asn Ala Glu Lys Phe Ala Asp Pro Lys Gln Lys Ala Asp 195 200 205 Val Glu Ser Met Ile Pro Met Gly Tyr Ile Gly Glu Pro Glu Glu Ile 210 215 220 Ala Ala Val Ala Ala Trp Leu Ala Ser Lys Glu Ala Ser Tyr Val Thr 225 230 235 240 Gly Ile Thr Leu Phe Ala Asp Gly Gly Met Thr Leu Tyr Pro Ser Phe 245 250 255 Gln Ala Gly Arg Gly 260 <210> 3 <211> 261 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 3 Met Tyr Pro Asp Leu Lys Gly Lys Val Val Ala Ile Thr Gly Ala Ala 1 5 10 15 Ser Gly Leu Gly Lys Ala Met Ala Ile Arg Phe Gly Lys Glu Gln Ala 20 25 30 Lys Val Val Ile Asn Tyr Tyr Ser Asn Lys Gln Asp Pro Asn Glu Val 35 40 45 Lys Glu Glu Val Ile Lys Ala Gly Gly Glu Ala Val Val Val Gln Gly 50 55 60 Asp Val Thr Lys Glu Glu Asp Val Lys Asn Ile Val Gln Thr Ala Ile 65 70 75 80 Lys Glu Phe Gly Thr Leu Asp Ile Met Ile Asn Asn Ala Gly Leu Cys 85 90 95 Asn Pro Val Pro Ser His Glu Met Pro Leu Lys Asp Trp Asp Lys Val 100 105 110 Ile Gly Thr Asn Leu Thr Gly Ala Phe Leu Gly Ser Arg Glu Ala Ile 115 120 125 Lys Tyr Phe Val Glu Asn Asp Ile Lys Gly Asn Val Ile Asn Met Ser 130 135 140 Ser Val His Glu Val Ile Pro Trp Pro Leu Ala Ser His Tyr Thr Ala 145 150 155 160 Ser Lys Gly Gly Met Lys Leu Met Thr Lys Thr Leu Ala Leu Glu Tyr 165 170 175 Ala Pro Lys Gly Ile Arg Val Asn Asn Ile Gly Pro Gly Ala Ile Asn 180 185 190 Thr Thr Ile Asn Ala Glu Lys Phe Ala Asp Pro Lys Gln Lys Ala Asp 195 200 205 Val Glu Ser Met Ile Pro Met Gly Tyr Ile Thr Glu Pro Glu Glu Ile 210 215 220 Ala Ala Val Ala Ala Trp Leu Ala Ser Lys Glu Ala Ser Tyr Val Thr 225 230 235 240 Gly Ile Thr Leu Phe Ala Asp Gly Gly Met Thr Leu Tyr Pro Ser Phe 245 250 255 Gln Ala Gly Arg Gly 260 <210> 4 <211> 261 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 4 Met Tyr Pro Asp Leu Lys Gly Lys Val Val Ala Ile Thr Gly Ala Ala 1 5 10 15 Ser Gly Leu Gly Lys Ala Met Ala Ile Arg Phe Gly Lys Glu Gln Ala 20 25 30 Lys Val Val Ile Asn Tyr Tyr Ser Asn Lys Gln Asp Pro Asn Glu Val 35 40 45 Lys Glu Glu Val Ile Lys Ala Gly Gly Glu Ala Val Val Val Gln Gly 50 55 60 Asp Val Thr Lys Glu Glu Asp Val Lys Asn Ile Val Gln Thr Ala Ile 65 70 75 80 Lys Glu Phe Gly Thr Leu Asp Ile Met Ile Asn Asn Ala Gly Leu Cys 85 90 95 Asn Pro Val Pro Ser His Glu Met Pro Leu Lys Asp Trp Asp Lys Val 100 105 110 Ile Gly Thr Asn Leu Thr Gly Ala Phe Leu Gly Ser Arg Glu Ala Ile 115 120 125 Lys Tyr Phe Val Glu Asn Asp Ile Lys Gly Asn Val Ile Asn Met Ser 130 135 140 Ser Val His Glu Val Ile Pro Trp Pro Leu Ala Ser His Tyr Thr Ala 145 150 155 160 Ser Lys Gly Gly Met Lys Leu Met Thr Lys Thr Leu Ala Leu Glu Tyr 165 170 175 Ala Pro Lys Gly Ile Arg Val Asn Asn Ile Gly Pro Gly Ala Ile Asn 180 185 190 Thr Thr Arg Asn Ala Glu Lys Phe Ala Asp Pro Lys Gln Lys Ala Asp 195 200 205 Val Glu Ser Met Ile Pro Met Gly Tyr Ile Gly Glu Pro Glu Glu Ile 210 215 220 Ala Ala Val Ala Ala Trp Leu Ala Ser Lys Glu Ala Ser Tyr Val Thr 225 230 235 240 Gly Ile Thr Leu Phe Ala Asp Gly Gly Met Thr Leu Tyr Pro Ser Phe 245 250 255 Gln Ala Gly Arg Gly 260 <210> 5 <211> 261 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 5 Met Tyr Pro Asp Leu Lys Gly Lys Val Val Ala Ile Thr Gly Ala Ala 1 5 10 15 Ser Gly Leu Gly Lys Ala Met Ala Ile Arg Phe Gly Lys Glu Gln Ala 20 25 30 Lys Val Val Ile Asn Tyr Tyr Ser Asn Lys Gln Asp Asn Asn Glu Val 35 40 45 Lys Glu Glu Val Ile Lys Ala Gly Gly Glu Ala Val Val Val Gln Gly 50 55 60 Asp Val Thr Lys Glu Glu Asp Val Lys Asn Ile Val Gln Thr Ala Ile 65 70 75 80 Lys Glu Phe Gly Thr Leu Asp Ile Met Ile Asn Asn Ala Gly Leu Phe 85 90 95 Asn Pro Val Pro Ser His Glu Met Pro Leu Lys Asp Trp Asp Lys Val 100 105 110 Ile Gly Thr Asn Leu Thr Gly Ala Phe Leu Gly Ser Arg Glu Ala Ile 115 120 125 Lys Tyr Phe Val Glu Asn Asp Ile Lys Gly Asn Val Ile Asn Met Ser 130 135 140 Ser Val His Glu Ala Ile Pro Trp Pro Leu Ala Ser His Tyr Thr Ala 145 150 155 160 Ser Lys Gly Gly Met Lys Leu Met Thr Lys Thr Leu Ala Leu Glu Tyr 165 170 175 Ala Pro Lys Gly Ile Arg Val Asn Asn Ile Gly Pro Gly Ala Ile Asn 180 185 190 Thr Thr Ile Asn Ala Glu Lys Phe Ala Asp Pro Lys Gln Lys Ala Asp 195 200 205 Val Glu Ser Met Ile Pro Met Gly Tyr Ile Gly Glu Pro Glu Glu Ile 210 215 220 Ala Ala Val Ala Ala Trp Leu Ala Ser Lys Glu Ala Ser Tyr Val Thr 225 230 235 240 Gly Ile Thr Leu Phe Ala Asp Gly Gly Met Thr Leu Tyr Pro Ser Phe 245 250 255 Gln Ala Gly Arg Gly 260 <210> 6 <211> 261 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 6 Met Tyr Pro Asp Leu Lys Gly Lys Val Val Ala Ile Thr Gly Ala Ala 1 5 10 15 Ser Gly Leu Gly Lys Gly Met Ala Ile Arg Phe Gly Lys Glu Gln Ala 20 25 30 Lys Val Val Ile Asn Tyr Tyr Ser Asn Lys Gln Asp Pro Asn Glu Val 35 40 45 Lys Glu Glu Val Ile Lys Ala Gly Gly Glu Ala Val Val Val Gln Gly 50 55 60 Asp Val Thr Lys Glu Glu Asp Val Lys Asn Ile Val Gln Thr Ala Ile 65 70 75 80 Lys Glu Phe Gly Thr Leu Asp Ile Met Ile Asn Asn Ala Gly Leu Phe 85 90 95 Asn Pro Val Pro Ser His Glu Met Pro Leu Lys Asp Trp Asp Lys Val 100 105 110 Ile Gly Thr Asn Leu Thr Gly Ala Phe Leu Gly Ser Arg Glu Ala Ile 115 120 125 Lys Tyr Phe Val Glu Asn Asp Ile Lys Gly Asn Val Ile Asn Met Ser 130 135 140 Ser Val His Glu Val Ile Pro Trp Pro Gly Ala Ser His Tyr Thr Ala 145 150 155 160 Ser Lys Gly Gly Met Lys Leu Met Thr Lys Thr Leu Ala Leu Glu Tyr 165 170 175 Ala Pro Lys Gly Ile Arg Val Asn Asn Ile Gly Pro Gly Ala Ile Asn 180 185 190 Thr Thr Ile Asn Ala Glu Lys Phe Ala Asp Pro Lys Gln Lys Ala Asp 195 200 205 Val Glu Ser Met Ile Pro Met Gly Tyr Ile Gly Glu Pro Glu Glu Ile 210 215 220 Ala Ala Val Ala Ala Trp Leu Ala Ser Lys Glu Ala Ser Tyr Val Thr 225 230 235 240 Gly Ile Thr Leu Phe Ala Asp Gly Gly Met Thr Leu Tyr Pro Ser Phe 245 250 255 Gln Ala Gly Arg Gly 260 <210> 7 <211> 261 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 7 Met Tyr Pro Asp Leu Lys Gly Lys Val Val Ala Ile Thr Gly Ala Ala 1 5 10 15 Ser Gly Leu Gly Lys Ala Met Ala Ile Arg Phe Gly Lys Glu Gln Ala 20 25 30 Lys Val Val Ile Asn Tyr Tyr Ser Asn Lys Gln Asp Pro Asn Glu Val 35 40 45 Lys Glu Glu Val Ile Lys Ala Gly Gly Glu Ala Val Val Val Gln Gly 50 55 60 Asp Val Thr Lys Glu Glu Asp Val Lys Asn Ile Val Gln Thr Ala Ile 65 70 75 80 Lys Glu Phe Gly Thr Leu Asp Ile Met Ile Asn Asn Ala Gly Leu Phe 85 90 95 Asn Pro Val Pro Ser His Glu Met Pro Leu Lys Asp Trp Asp Lys Val 100 105 110 Ile Gly Thr Asn Leu Thr Gly Ala Phe Leu Gly Ser Arg Glu Ala Ile 115 120 125 Lys Tyr Phe Val Glu Asn Asp Ile Lys Gly Asn Val Val Asn Met Ser 130 135 140 Ser Val His Glu Val Ile Pro Trp Pro Leu Ala Ser His Tyr Thr Ala 145 150 155 160 Ser Lys Gly Gly Met Lys Leu Met Thr Lys Thr Leu Ala Leu Glu Tyr 165 170 175 Ala Pro Lys Gly Ile Arg Val Asn Asn Ile Gly Pro Gly Ala Ile Asn 180 185 190 Thr Thr Ile Asn Ala Glu Lys Phe Ala Asp Pro Lys Gln Lys Ala Asp 195 200 205 Val Glu Ser Met Ile Pro Met Gly Tyr Ile Gly Glu Pro Glu Glu Ile 210 215 220 Ala Ala Val Ala Ala Trp Leu Ala Ser Lys Glu Ala Ser Tyr Val Thr 225 230 235 240 Gly Ile Thr Leu Phe Ala Asp Gly Gly Met Thr Leu Tyr Pro Ser Phe 245 250 255 Gln Ala Gly Arg Gly 260 <210> 8 <211> 261 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 8 Met Tyr Pro Asp Leu Lys Gly Lys Val Val Ala Ile Thr Gly Ala Ala 1 5 10 15 Ser Gly Leu Gly Lys Ala Met Ala Ile Arg Phe Gly Lys Glu Gln Ala 20 25 30 Lys Val Val Ile Asn Tyr Tyr Ser Asn Lys Gln Asp Pro Asn Glu Val 35 40 45 Lys Glu Glu Val Ile Lys Ala Gly Gly Glu Ala Val Val Val Gln Gly 50 55 60 Asp Val Thr Lys Glu Glu Asp Val Lys Asn Ile Val Gln Thr Ala Ile 65 70 75 80 Lys Glu Phe Gly Thr Leu Asp Ile Met Ile Asn Asn Ala Gly Leu Phe 85 90 95 Asn Pro Val Pro Ser His Glu Met Pro Leu Lys Asp Trp Asp Lys Val 100 105 110 Ile Gly Thr Asn Leu Thr Gly Ala Phe Leu Gly Ser Arg Glu Ala Ile 115 120 125 Lys Tyr Phe Val Glu Asn Asp Ile Lys Gly Asn Val Val Asn Met Ser 130 135 140 Ser Val His Glu Val Ile Pro Trp Pro Gly Ala Ser His Tyr Thr Ala 145 150 155 160 Ser Lys Gly Gly Met Lys Leu Met Thr Lys Thr Leu Ala Leu Glu Tyr 165 170 175 Ala Pro Lys Gly Ile Arg Val Asn Asn Ile Gly Pro Gly Ala Ile Asn 180 185 190 Thr Thr Ile Asn Ala Glu Lys Phe Ala Asp Pro Lys Gln Lys Ala Asp 195 200 205 Val Glu Ser Met Ile Pro Met Gly Tyr Ile Gly Glu Pro Glu Glu Ile 210 215 220 Ala Ala Val Ala Ala Trp Leu Ala Ser Lys Glu Ala Ser Tyr Val Thr 225 230 235 240 Gly Ile Thr Leu Phe Ala Asp Gly Gly Met Thr Leu Tyr Pro Ser Phe 245 250 255 Gln Ala Gly Arg Gly 260 <210> 9 <211> 261 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 9 Met Tyr Pro Asp Leu Lys Gly Lys Val Val Ala Leu Thr Gly Ala Ala 1 5 10 15 Ser Gly Leu Gly Lys Ala Met Ala Ile Arg Phe Gly Lys Glu Gln Ala 20 25 30 Lys Val Val Ile Asn Tyr Tyr Ser Asn Lys Gln Asp Pro Asn Glu Val 35 40 45 Lys Glu Glu Val Ile Lys Ala Gly Gly Glu Ala Val Val Val Gln Gly 50 55 60 Asp Val Thr Lys Glu Glu Asp Val Lys Asn Ile Val Gln Thr Ala Ile 65 70 75 80 Lys Glu Phe Gly Thr Leu Asp Ile Met Ile Asn Asn Ala Gly Leu Phe 85 90 95 Asn Pro Val Pro Ser His Glu Met Pro Leu Lys Asp Trp Asp Lys Val 100 105 110 Ile Gly Thr Asn Leu Thr Gly Ala Phe Leu Gly Ser Arg Glu Ala Ile 115 120 125 Lys Tyr Phe Val Glu Asn Asp Ile Lys Gly Asn Val Ile Asn Met Ser 130 135 140 Ser Val His Glu Val Ile Pro Trp Pro Leu Ala Ser His Tyr Thr Ala 145 150 155 160 Ser Lys Gly Gly Met Lys Leu Met Thr Lys Thr Leu Ala Leu Glu Tyr 165 170 175 Ala Pro Lys Gly Ile Arg Val Asn Asn Ile Gly Pro Gly Ala Ile Asn 180 185 190 Thr Thr Arg Asn Ala Glu Lys Phe Ala Asp Pro Lys Gln Lys Ala Asp 195 200 205 Val Glu Ser Met Ile Pro Met Gly Tyr Ile Gly Glu Pro Glu Glu Ile 210 215 220 Ala Ala Val Ala Ala Trp Leu Ala Ser Lys Glu Ala Ser Tyr Val Thr 225 230 235 240 Gly Ile Thr Leu Phe Ala Asp Gly Gly Met Leu Leu Tyr Pro Ser Phe 245 250 255 Gln Ala Gly Arg Gly 260 <210> 10 <211> 261 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 10 Met Tyr Pro Asp Leu Lys Gly Lys Val Val Ala Ile Thr Gly Ala Ala 1 5 10 15 Ser Gly Leu Gly Lys Ala Met Ala Ile Arg Phe Gly Lys Glu Gln Ala 20 25 30 Lys Val Val Ile Asn Tyr Tyr Ser Asn Lys Gln Asp Pro Asn Glu Val 35 40 45 Lys Glu Glu Val Ile Lys Ala Gly Gly Glu Ala Val Val Val Gln Gly 50 55 60 Asp Val Thr Lys Glu Glu Asp Val Lys Asn Ile Val Gln Thr Ala Ile 65 70 75 80 Lys Glu Phe Gly Thr Leu Asp Ile Met Ile Asn Asn Ala Gly Leu Cys 85 90 95 Asn Leu Val Pro Ser His Glu Met Pro Leu Lys Asp Trp Asp Lys Val 100 105 110 Ile Gly Thr Asn Leu Thr Gly Ala Phe Leu Gly Ser Arg Glu Ala Ile 115 120 125 Lys Tyr Phe Val Glu Asn Asp Ile Lys Gly Asn Val Ile Asn Met Ser 130 135 140 Ser Val His Glu Val Ile Pro Trp Pro Leu Ala Ser His Tyr Thr Ala 145 150 155 160 Ser Lys Gly Gly Met Lys Leu Met Thr Lys Thr Leu Ala Leu Glu Tyr 165 170 175 Ala Pro Lys Gly Ile Arg Val Asn Asn Ile Gly Pro Gly Ala Ile Asn 180 185 190 Thr Thr Ile Asn Ala Glu Lys Phe Ala Asp Pro Lys Gln Lys Ala Asp 195 200 205 Val Glu Ser Met Ile Pro Met Gly Tyr Ile Gly Glu Pro Glu Glu Ile 210 215 220 Ala Ala Val Ala Ala Trp Leu Ala Ser Lys Glu Ala Ser Tyr Val Thr 225 230 235 240 Gly Ile Thr Leu Phe Ala Asp Gly Gly Met Thr Leu Tyr Pro Ser Phe 245 250 255 Gln Ala Gly Arg Gly 260 <210> 11 <211> 261 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 11 Met Tyr Pro Asp Leu Lys Gly Lys Val Val Ala Ile Thr Gly Ala Ala 1 5 10 15 Ser Gly Leu Gly Lys Ala Met Ala Ile Arg Phe Gly Lys Glu Gln Ala 20 25 30 Lys Val Val Ile Asn Tyr Tyr Ser Asn Lys Gln Asp Pro Asn Glu Val 35 40 45 Lys Glu Glu Val Ile Lys Ala Gly Gly Glu Ala Val Val Val Gln Gly 50 55 60 Asp Val Thr Lys Glu Glu Asp Val Lys Asn Ile Val Gln Thr Ala Ile 65 70 75 80 Lys Glu Phe Gly Thr Leu Asp Ile Met Ile Asn Asn Ala Gly Leu Cys 85 90 95 Asn Pro Val Pro Ser His Glu Met Pro Leu Lys Asp Trp Asp Lys Val 100 105 110 Ile Gly Thr Asn Leu Thr Gly Ala Phe Leu Gly Ser Arg Glu Ala Ile 115 120 125 Lys Tyr Phe Val Glu Asn Asp Ile Lys Gly Asn Val Ile Asn Met Ser 130 135 140 Ser Val His Glu Val Ile Pro Trp Pro Leu Ala Ser His Tyr Thr Ala 145 150 155 160 Ser Lys Gly Gly Met Lys Leu Met Thr Lys Thr Leu Ala Leu Glu Tyr 165 170 175 Ala Pro Lys Gly Ile Arg Val Asn Asn Ile Gly Pro Gly Ala Ile Asn 180 185 190 Thr Thr Ile Asn Ala Glu Lys Phe Ala Asp Pro Lys Gln Lys Ala Asp 195 200 205 Val Glu Ser Met Ile Pro Met Gly Tyr Ile Gly Glu Pro Glu Glu Ile 210 215 220 Ala Ala Val Ala Ala Trp Leu Ala Ser Lys Glu Ala Ser Tyr Val Thr 225 230 235 240 Gly Ile Thr Leu Phe Ala Asp Gly Gly Met Thr Leu Tyr Pro Ser Phe 245 250 255 Gln Ala Gly Arg Gly 260 <210> 12 <211> 261 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 12 Met Tyr Pro Asp Leu Lys Gly Lys Val Val Ala Ile Thr Gly Ala Ala 1 5 10 15 Ser Gly Leu Gly Lys Ala Met Ala Ile Arg Phe Gly Lys Glu Gln Ala 20 25 30 Lys Val Val Ile Asn Tyr Tyr Ser Asn Lys Gln Asp Pro Asn Glu Val 35 40 45 Lys Glu Glu Val Ile Lys Ala Gly Gly Glu Ala Val Val Val Gln Gly 50 55 60 Asp Val Thr Lys Glu Glu Asp Val Lys Asn Ile Val Gln Thr Ala Ile 65 70 75 80 Lys Glu Phe Gly Thr Leu Asp Ile Met Ile Asn Asn Ala Gly Leu Phe 85 90 95 Asn Pro Val Pro Ser His Glu Met Pro Leu Lys Asp Trp Asp Lys Val 100 105 110 Ile Gly Thr Asn Leu Thr Gly Ala Phe Leu Gly Ser Arg Glu Ala Ile 115 120 125 Lys Tyr Phe Val Glu Asn Asp Ile Lys Gly Asn Val Ile Asn Met Ser 130 135 140 Ser Val His Glu Val Ile Pro Trp Pro Leu Ala Ser His Tyr Thr Ala 145 150 155 160 Ser Lys Gly Gly Met Lys Leu Met Thr Lys Thr Leu Gly Leu Glu Tyr 165 170 175 Ala Pro Lys Gly Ile Arg Val Asn Asn Ile Gly Pro Gly Ala Ile Asn 180 185 190 Thr Thr Ile Asn Ala Glu Lys Phe Ala Asp Pro Lys Gln Lys Ala Asp 195 200 205 Val Glu Ser Met Ile Pro Met Gly Tyr Ile Gly Glu Pro Glu Glu Ile 210 215 220 Ala Ala Val Ala Ala Trp Leu Ala Ser Lys Glu Ala Ser Tyr Val Thr 225 230 235 240 Gly Ile Thr Leu Phe Ala Asp Gly Gly Met Thr Leu Tyr Pro Ser Phe 245 250 255 Gln Ala Gly Arg Gly 260 <210> 13 <211> 261 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 13 Met Tyr Pro Asp Leu Lys Gly Lys Val Val Ala Ile Thr Gly Ala Ala 1 5 10 15 Ser Gly Leu Gly Lys Ala Met Ala Ile Arg Phe Gly Lys Glu Gln Ala 20 25 30 Lys Val Val Ile Asn Tyr Tyr Ser Asn Lys Gln Asp Pro Asn Glu Val 35 40 45 Lys Glu Glu Val Ile Lys Glu Gly Gly Glu Ala Val Val Val Gln Gly 50 55 60 Asp Val Thr Lys Glu Glu Asp Val Lys Asn Ile Val Gln Thr Ala Ile 65 70 75 80 Lys Glu Phe Gly Thr Leu Asp Ile Met Ile Asn Asn Ala Gly Leu Phe 85 90 95 Asn Pro Val Pro Ser His Glu Met Pro Leu Lys Asp Trp Asp Lys Val 100 105 110 Ile Gly Thr Asn Leu Thr Gly Ala Phe Leu Gly Ser Arg Glu Ala Ile 115 120 125 Lys Tyr Phe Val Glu Asn Asp Ile Lys Gly Asn Val Ile Asn Met Ser 130 135 140 Ser Val His Glu Val Ile Pro Trp Pro Leu Ala Ser His Tyr Thr Ala 145 150 155 160 Ser Lys Gly Gly Met Lys Leu Met Thr Lys Thr Leu Ala Leu Glu Tyr 165 170 175 Ala Pro Lys Gly Ile Arg Val Asn Asn Ile Gly Pro Gly Ala Ile Asn 180 185 190 Thr Thr Ile Asn Ala Glu Lys Phe Ala Asp Pro Lys Gln Lys Ala Asp 195 200 205 Val Glu Ser Met Ile Pro Met Gly Tyr Ile Gly Glu Pro Glu Glu Ile 210 215 220 Ala Ala Val Ala Ala Trp Leu Ala Ser Lys Glu Ala Ser Tyr Val Thr 225 230 235 240 Gly Ile Thr Leu Phe Ala Asp Gly Gly Met Thr Leu Tyr Pro Ser Phe 245 250 255 Gln Ala Gly Arg Gly 260 <210> 14 <211> 261 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 14 Met Tyr Pro Asp Leu Lys Gly Lys Val Val Ala Ile Thr Gly Ala Ala 1 5 10 15 Ser Gly Leu Gly Lys Ala Met Ala Ile Arg Phe Gly Lys Glu Gln Ala 20 25 30 Lys Val Val Ile Asn Tyr Tyr Ser Asn Lys Gln Asp Pro Asn Glu Val 35 40 45 Lys Glu Glu Val Ile Lys Ala Gly Gly Glu Ala Val Val Val Gln Gly 50 55 60 Asp Val Thr Lys Glu Glu Asp Val Lys Asn Ile Val Gln Thr Ala Ile 65 70 75 80 Lys Glu Phe Gly Thr Leu Asp Ile Met Ile Asn Asn Ala Gly Leu Phe 85 90 95 Asn Pro Val Pro Ser His Glu Met Pro Leu Lys Asp Trp Asp Lys Val 100 105 110 Ile Gly Thr Asn Leu Thr Gly Ala Phe Leu Gly Ser Arg Glu Ala Ile 115 120 125 Lys Tyr Phe Val Glu Asn Asp Ile Tyr Gly Asn Val Ile Asn Met Ser 130 135 140 Ser Val His Glu Val Ile Pro Trp Pro Leu Ala Ser His Tyr Thr Ala 145 150 155 160 Ser Lys Gly Gly Met Lys Leu Met Thr Lys Thr Leu Ala Leu Glu Tyr 165 170 175 Ala Pro Lys Gly Ile Arg Val Asn Asn Ile Gly Pro Gly Ala Ile Asn 180 185 190 Thr Thr Ile Asn Ala Glu Lys Phe Ala Asp Pro Lys Gln Lys Ala Asp 195 200 205 Val Glu Ser Met Ile Pro Met Gly Tyr Ile Gly Glu Pro Glu Glu Ile 210 215 220 Ala Ala Val Ala Ala Trp Leu Ala Ser Lys Glu Ala Ser Tyr Val Thr 225 230 235 240 Gly Ile Thr Leu Phe Ala Asp Gly Gly Met Thr Leu Tyr Pro Ser Phe 245 250 255 Gln Ala Gly Arg Gly 260 <210> 15 <211> 261 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 15 Met Tyr Pro Asp Leu Lys Gly Lys Val Val Ala Ile Thr Gly Ala Ala 1 5 10 15 Ser Gly Leu Gly Lys Ala Met Ala Ile Arg Phe Gly Lys Glu Gln Ala 20 25 30 Lys Val Val Ile Asn Tyr Tyr Ser Asn Lys Gln Asp Pro Asn Glu Val 35 40 45 Lys Glu Glu Val Ile Lys Ala Gly Gly Glu Ala Val Val Val Gln Gly 50 55 60 Asp Val Thr Lys Glu Glu Asp Val Lys Asn Ile Val Gln Thr Ala Ile 65 70 75 80 Lys Glu Phe Gly Thr Leu Asp Ile Met Ile Asn Asn Ala Gly Leu Phe 85 90 95 Asn Pro Val Pro Ser His Glu Met Pro Leu Lys Asp Trp Asp Lys Val 100 105 110 Ile Gly Thr Asn Leu Thr Gly Ala Phe Leu Gly Ser Arg Glu Ala Ile 115 120 125 Lys Tyr Phe Val Glu Asn Asp Ile Lys Gly Asn Val Ile Asn Met Ser 130 135 140 Ser Val His Glu Val Ile Pro Trp Pro Leu Ala Ser His Tyr Thr Ala 145 150 155 160 Ser Lys Gly Gly Met Lys Leu Met Thr Lys Thr Leu Ala Leu Glu Tyr 165 170 175 Ala Pro Lys Gly Ile Arg Val Asn Asn Ile Gly Pro Gly Ala Ile Asn 180 185 190 Thr Thr Ile Asn Ala Glu Lys Phe Ala Asp Pro Lys Gln Lys Ala Asp 195 200 205 Val Glu Ser Met Ile Pro Met Gly Tyr Ile Gly Glu Pro Glu Glu Ile 210 215 220 Ala Ala Val Ala Ala Trp Leu Ala Ser Lys Glu Ala Ser Tyr Val Thr 225 230 235 240 Gly Ile Thr Leu Phe Ala Asp Gly Gly Met Thr Val Tyr Pro Ser Phe 245 250 255 Gln Ala Gly Arg Gly 260 <210> 16 <211> 261 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 16 Met Tyr Pro Asp Leu Lys Gly Lys Val Val Ala Ile Thr Gly Ala Ala 1 5 10 15 Ser Gly Leu Gly Lys Ala Met Ala Ile Arg Phe Gly Lys Glu Gln Ala 20 25 30 Lys Val Val Ile Asn Tyr Tyr Ser Asn Lys Gln Asp Pro Asn Glu Ala 35 40 45 Lys Glu Glu Val Ile Lys Ala Gly Gly Glu Ala Val Val Val Gln Gly 50 55 60 Asp Val Thr Lys Glu Glu Asp Val Lys Asn Ile Val Gln Thr Ala Ile 65 70 75 80 Lys Glu Phe Gly Thr Leu Asp Ile Met Ile Asn Asn Ala Gly Leu Phe 85 90 95 Asn Pro Val Pro Ser His Glu Met Pro Leu Lys Asp Trp Asp Lys Val 100 105 110 Ile Gly Thr Asn Leu Thr Gly Ala Phe Leu Gly Ser Arg Glu Ala Ile 115 120 125 Lys Tyr Phe Val Glu Asn Asp Ile Lys Gly Asn Val Ile Asn Met Ser 130 135 140 Ser Val His Glu Val Ile Pro Trp Pro Leu Ala Ser His Tyr Thr Ala 145 150 155 160 Ser Lys Gly Gly Met Lys Leu Met Thr Lys Thr Leu Ala Leu Glu Tyr 165 170 175 Ala Pro Lys Gly Ile Arg Val Asn Asn Ile Gly Pro Gly Ala Ile Asn 180 185 190 Thr Thr Ile Asn Ala Glu Lys Phe Ala Asp Pro Lys Gln Lys Ala Asp 195 200 205 Val Glu Ser Met Ile Pro Met Gly Tyr Ile Gly Glu Pro Glu Glu Ile 210 215 220 Ala Ala Val Ala Ala Trp Leu Ala Ser Lys Glu Ala Ser Tyr Val Thr 225 230 235 240 Gly Ile Thr Leu Phe Ala Asp Gly Gly Met Thr Leu Tyr Pro Ser Phe 245 250 255 Gln Ala Gly Arg Gly 260 <210> 17 <211> 261 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 17 Met Tyr Pro Asp Leu Lys Gly Lys Val Val Ala Ile Thr Gly Ala Ala 1 5 10 15 Ser Gly Leu Gly Lys Ala Met Ala Ile Arg Phe Gly Lys Glu Gln Ala 20 25 30 Lys Val Val Ile Asn Tyr Tyr Ser Asn Lys Gln Asp Pro Asn Glu Val 35 40 45 Lys Glu Glu Val Ile Lys Ala Gly Gly Glu Ala Val Val Val Gln Gly 50 55 60 Asp Val Thr Lys Glu Glu Asp Val Lys Asn Ile Val Gln Thr Ala Ile 65 70 75 80 Lys Glu Phe Gly Thr Leu Asp Ile Met Ile Asn Asn Ala Gly Leu Phe 85 90 95 Asn Pro Val Pro Ser His Glu Met Pro Leu Lys Asp Trp Asp Lys Val 100 105 110 Ile Gly Thr Asn Leu Thr Gly Ala Phe Leu Gly Ser Arg Glu Ala Ile 115 120 125 Lys Tyr Phe Val Glu Asn Asp Ile Lys Gly Asn Val Ile Asn Met Ser 130 135 140 Ser Val His Glu Val Ile Pro Trp Pro Leu Ala Ser His Tyr Thr Ala 145 150 155 160 Ser Lys Gly Gly Met Lys Leu Met Thr Lys Thr Leu Ala Leu Glu Tyr 165 170 175 Ala Pro Lys Gly Ile Arg Val Ser Asn Ile Gly Pro Gly Ala Ile Asn 180 185 190 Thr Thr Ile Asn Ala Glu Lys Phe Ala Asp Pro Lys Gln Lys Ala Asp 195 200 205 Val Glu Ser Met Ile Pro Met Gly Tyr Ile Gly Glu Pro Glu Glu Ile 210 215 220 Ala Ala Val Ala Ala Trp Leu Ala Ser Lys Glu Ala Ser Tyr Val Thr 225 230 235 240 Gly Ile Thr Leu Phe Ala Asp Gly Gly Met Thr Leu Tyr Pro Ser Phe 245 250 255 Gln Ala Gly Arg Gly 260 <210> 18 <211> 261 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 18 Met Tyr Pro Asp Leu Lys Gly Lys Val Val Ala Ile Thr Gly Ala Ala 1 5 10 15 Ser Gly Leu Gly Lys Ala Met Ala Ile Arg Phe Gly Lys Glu Gln Ala 20 25 30 Lys Val Val Ile Asn Tyr Tyr Ser Asn Lys Gln Asp Pro Asn Glu Val 35 40 45 Lys Glu Glu Val Ile Lys Ala Gly Gly Glu Ala Val Val Val Gln Gly 50 55 60 Asp Val Thr Lys Glu Glu Asp Val Lys Asn Ile Val Gln Thr Ala Ile 65 70 75 80 Lys Glu Phe Gly Thr Leu Asp Ile Met Ile Asn Asn Ala Gly Leu Phe 85 90 95 Asn Pro Val Pro Ser His Glu Met Pro Leu Lys Asp Trp Asp Lys Val 100 105 110 Ile Gly Thr Asn Leu Thr Gly Ala Phe Leu Gly Ser Arg Glu Ala Ile 115 120 125 Lys Tyr Phe Val Glu Asn Asp Ile Lys Gly Asn Val Ile Asn Met Ser 130 135 140 Ser Val His Glu Val Ile Pro Trp Pro Leu Ala Ser His Tyr Thr Ala 145 150 155 160 Ser Lys Gly Gly Met Ser Leu Met Thr Lys Thr Leu Ala Leu Glu Tyr 165 170 175 Ala Pro Lys Gly Ile Arg Val Asn Asn Ile Gly Pro Gly Ala Ile Asn 180 185 190 Thr Thr Ile Asn Ala Glu Lys Phe Ala Asp Pro Lys Gln Lys Ala Asp 195 200 205 Val Glu Ser Met Ile Pro Met Gly Tyr Ile Gly Glu Pro Glu Glu Ile 210 215 220 Ala Ala Val Ala Ala Trp Leu Ala Ser Lys Glu Ala Ser Tyr Val Thr 225 230 235 240 Gly Ile Thr Leu Phe Ala Asp Gly Gly Met Thr Leu Tyr Pro Ser Phe 245 250 255 Gln Ala Gly Arg Gly 260 <210> 19 <211> 261 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 19 Met Tyr Pro Asp Leu Lys Gly Lys Val Val Ala Ile Thr Gly Ala Ala 1 5 10 15 Ser Gly Leu Gly Lys Ala Met Ala Ile Arg Phe Gly Lys Glu Gln Ala 20 25 30 Lys Val Val Ile Asn Tyr Tyr Ser Asn Lys Gln Asp Pro Asn Glu Val 35 40 45 Lys Glu Glu Val Ile Lys Ala Gly Gly Glu Ala Val Val Val Gln Gly 50 55 60 Asp Val Thr Lys Glu Glu Asp Val Lys Asn Ile Val Gln Thr Ala Ile 65 70 75 80 Lys Glu Phe Gly Thr Leu Asp Ile Met Ile Asn Asn Ala Gly Leu Phe 85 90 95 Asn Pro Val Pro Ser His Glu Met Pro Leu Lys Asp Trp Asp Lys Val 100 105 110 Ile Gly Thr Asn Leu Thr Gly Ala Phe Leu Gly Ser Arg Glu Ala Ile 115 120 125 Lys Tyr Phe Val Glu Asn Asp Ile Lys Gly Asn Val Ile Asn Met Ser 130 135 140 Ser Val His Glu Val Ile Pro Trp Pro Leu Ala Ser His Tyr Thr Ala 145 150 155 160 Ser Lys Gly Gly Met Lys Leu Met Thr Lys Thr Leu Ala Leu Glu Tyr 165 170 175 Ala Pro Lys Gly Ile Arg Val Asn Asn Ile Gly Pro Gly Ala Ile Asn 180 185 190 Thr Thr Ile Asn Ala Glu Lys Phe Ala Asp Pro Lys Gln Lys Ala Asp 195 200 205 Val Glu Ser Met Ile Pro Met Gly Tyr Ile Gly Glu Pro Glu Glu Ile 210 215 220 Ala Ala Val Ala Ala Trp Leu Ala Ser Lys Glu Ala Ser Tyr Val Gln 225 230 235 240 Gly Ile Thr Leu Phe Ala Asp Gly Gly Met Thr Leu Tyr Pro Ser Phe 245 250 255 Gln Ala Gly Arg Gly 260 <210> 20 <211> 261 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 20 Met Tyr Pro Asp Leu Lys Gly Lys Val Val Ala Ile Thr Gly Ala Ala 1 5 10 15 Ser Gly Leu Gly Lys Ala Met Ala Ile Arg Phe Gly Lys Glu Gln Ala 20 25 30 Lys Val Val Ile Asn Tyr Tyr Ser Asn Lys Gln Asp Pro Asn Glu Val 35 40 45 Lys Glu Glu Val Ile Lys Ala Gly Gly Glu Ala Val Val Val Gln Gly 50 55 60 Asp Val Thr Lys Glu Glu Asp Val Lys Asn Ile Val Gln Thr Ala Ile 65 70 75 80 Lys Glu Phe Gly Thr Leu Asp Ile Met Ile Asn Asn Ala Gly Leu Glu 85 90 95 Asn Pro Val Pro Ser His Glu Met Pro Leu Lys Asp Trp Asp Lys Val 100 105 110 Ile Gly Thr Asn Leu Thr Gly Ala Phe Leu Gly Ser Arg Glu Ala Ile 115 120 125 Lys Tyr Phe Val Glu Asn Asp Ile Lys Gly Asn Val Ile Asn Met Ser 130 135 140 Ser Val His Glu Val Ile Pro Trp Pro Leu Phe Val His Tyr Ala Ala 145 150 155 160 Ser Lys Gly Gly Met Lys Leu Met Thr Glu Thr Leu Ala Leu Glu Tyr 165 170 175 Ala Pro Lys Gly Ile Arg Val Asn Asn Ile Gly Pro Gly Ala Ile Asn 180 185 190 Thr Thr Ile Asn Ala Gly Lys Phe Ala Asp Pro Lys Gln Lys Ala Asp 195 200 205 Val Glu Ser Met Ile Pro Met Gly Tyr Ile Gly Glu Pro Glu Glu Ile 210 215 220 Ala Ala Val Ala Ala Trp Leu Ala Ser Lys Glu Ala Ser Tyr Val Thr 225 230 235 240 Gly Ile Thr Leu Phe Ala Asp Gly Gly Met Thr Gln Tyr Pro Ser Phe 245 250 255 Gln Ala Gly Arg Gly 260

Claims

1. A protein switch, which is a glucose dehydrogenase polypeptide and contains at least 7 mutations at amino acid positions 96, 155, 156, 159, 170, 198, and 252 of SEQ ID NO:

20. The polypeptide has: (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 reactant, wherein (i) the analyte binding to the analyte-binding domain is different from the reactant binding to or reacting with the oxidase or dehydrogenase domain; and (ii) the mutations cause the oxidase or dehydrogenase activity to change when the analyte binds to the analyte-binding domain. wherein the protein switch comprises: 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; 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.

2. The protein switch according to claim 1, which further comprises one or more of the following: 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; alanine, glycine, isoleucine, or leucine at amino acid position 48 of SEQ ID NO:20; 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; Phenylalanine, tryptophan, or tyrosine at amino acid position 137 of SEQ ID NO:20; Alanine, glycine, leucine, or valine at amino acid position 141 of SEQ ID NO:20; Alanine, glycine, isoleucine, or leucine at amino acid position 149 of SEQ ID NO:20; 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.

3. A protein switch, which consists of the amino acid sequence of any one of SEQ ID NO:1-19.

4. A composition, which comprises at least one protein switch as described in claim 1 or 3 and at least one reactant.

5. The composition as described in claim 4, wherein the reactant is glucose or lactate.

6. A method for detecting an analyte, the method comprising: Provide a protein switch as claimed in claim 1 or 3; Contact the protein switch with a fluid comprising a reactant specific for the protein switch, wherein the analyte binding domain binds the analyte in the fluid, whereby the oxidase or dehydrogenase activity is altered; and Detect the change in the rate of breakdown of the reactant by the oxidase or dehydrogenase domain of the protein switch.

7. The method as described in claim 6, wherein the protein switch consists of the amino acid sequence of any one of SEQ ID NO:1-19.

8. The method as described in claim 6, wherein the oxidase is glucose oxidase or lactate oxidase.

9. The method as described in claim 6, wherein the dehydrogenase is glucose dehydrogenase or lactate dehydrogenase.

10. The method according to claim 6, wherein the reactant is glucose or lactic acid.

11. The method according to any one of claims 6-10, wherein the analyte is warfarin, cortisol, methotrexate or triiodothyronine.

12. A system for detecting or monitoring the concentration of an analyte, comprising a sensor control device and a signal detection device, wherein the sensor control device comprises at least one sensor, and the at least one sensor comprises the protein switch according to claim 1 or 3.

13. The system according to claim 12, wherein the oxidase is glucose oxidase or lactate oxidase.

14. The system according to claim 12, wherein the dehydrogenase is glucose dehydrogenase or lactate dehydrogenase.

15. The system according to any one of claims 12-14, wherein the protein switch consists of the amino acid sequence of any one of SEQ ID NO: 1-19.

16. An analyte monitoring system, comprising: A sensor, the sensor comprising a substrate, a working electrode and a protein switch as claimed in claim 1 or 3, at least a portion of the sensor being adapted for implantation and in intimate contact with a body fluid, the sensor being configured and arranged to generate a signal representative of the level of analyte in the body fluid; And signal detection means for receiving the signal, wherein the signal is generated by the contact of the analyte with the protein switch.

17. The analyte monitoring system according to claim 16, wherein the oxidase is glucose oxidase or lactate oxidase.

18. The analyte monitoring system according to claim 16, wherein the dehydrogenase is glucose dehydrogenase or lactate dehydrogenase.

19. The analyte monitoring system according to claim 16, wherein the protein switch consists of the amino acid sequence of any one of SEQ ID NO: 1-19.

20. The analyte monitoring system according to any one of claims 16-19, wherein the analyte is warfarin, cortisol, methotrexate or triiodothyronine.

21. A kit, comprising at least one protein switch according to claim 1 or 3 and at least one reactant.

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