Sensor and detection method for a test substance characterized by low potential detection

Abstract

To provide a test substance sensor suitable for use in vivo. [Solution] A test substance sensor that responds to a low working electrode potential may include an active region on the surface of the working electrode, the active region comprising a polymer, a redox mediator covalently bonded to the polymer, and at least one enzyme that responds to the test substance and is covalently bonded to the polymer. A particular redox mediator that reacts at a low potential may have the structure of (I), where G is a bonding group that covalently bonds the redox mediator to the polymer. A mass transfer limiting membrane permeable to the test substance may cover the active region. In some sensor configurations, the mass transfer limiting membrane may comprise a membrane polymer crosslinked with a branched crosslinking agent containing three or more crosslinking groups, such as polyethylene glycol tetraglycidyl ether. JPEG2026083107000020.jpg35170

Description

[Technical Field]

[0001] This disclosure relates to a sensor for a substance to be tested and a detection method. [Background technology]

[0002] The detection of various test substances within an individual can be important for monitoring their health status. Deviations from normal test substance levels can indicate many physiological conditions. Glucose levels are particularly important for detection and monitoring in individuals with diabetes, for example. By monitoring glucose levels with sufficient regularity, individuals with diabetes can take corrective action before significant physiological problems occur (e.g., by injecting insulin to lower glucose levels or by eating to raise glucose levels). For other physiological conditions, it may be desirable to monitor other test substances. In some cases, monitoring multiple test substances may be desirable, especially for comorbid conditions where dysregulation of two or more test substances occurs simultaneously in combination.

[0003] When appropriate detection chemistry can be identified, many test substances become interesting targets for physiological analysis. For this purpose, current-measuring sensors configured to continuously assay glucose in vivo have been developed and improved in recent years, proving useful for health monitoring in individuals with diabetes. Other test substances that commonly cause dysregulation simultaneously with glucose in individuals with diabetes include, for example, lactate, oxygen, pH, A1c, and ketones. It may also be desirable to monitor these along with other test substances independent of glucose dysregulation. While test substance sensors configured to detect non-glucose test substances in vivo are known, they are not yet sufficiently improved. Low sensitivity to small amounts of test substances can be a particular problem.

[0004] Individual marker monitoring can be performed periodically or continuously over a period of time. Periodic marker monitoring can be performed by taking samples of bodily fluids such as blood or urine at set time intervals and analyzing them in vitro. Periodic in vitro marker monitoring is sufficient to determine the physiological state of many individuals. However, in vitro marker monitoring can be inconvenient or painful in some cases. Furthermore, if marker measurements cannot be obtained at the appropriate time, there is no way to compensate for the missing data. Continuous marker monitoring may be performed using one or more sensors that remain at least partially implanted in the individual's tissue, such as in the skin, subcutaneously, or intravenously, so that analysis can be performed in vivo. Depending on the individual's specific health needs and / or previously measured marker levels, implantable sensors can collect marker data on demand, on a fixed schedule, or continuously. Marker monitoring using in vivo implantable sensors is a more desirable approach for individuals with severe marker dysregulation and / or rapidly fluctuating marker levels. However, it is equally beneficial for other individuals. Since implantable test substance sensors often remain within an individual's tissues for extended periods, there is a strong desire to manufacture such sensors from stable materials that exhibit high biocompatibility.

[0005] To improve biocompatibility, a test substance sensor may include a membrane placed over the entire embedded portion of the sensor, particularly a membrane covering at least the active area of ​​the sensor. In addition to promoting biocompatibility, the membrane may be permeable or semipermeable to the test substance of interest and restrict the total test substance flux into the active area of ​​the sensor. Such a mass transfer restricting membrane can help avoid overloading (saturation) of the detected component within the active area, thereby improving sensor performance and accuracy. For example, in the case of a sensor performing enzyme-based detection, restricting the mass transfer of the test substance into the active area can make the chemical reaction kinetics of the detection process enzyme-restricted rather than enzyme-restricted, thereby making it easier to correlate the sensor output with the amount of test substance present.

[0006] One problem associated with equipping a sample sensor with a membrane is that the sample flux across the membrane can vary significantly with temperature and / or the length of time the sample sensor is implanted in tissue. Some membrane materials are less susceptible to temperature-dependent sample flux fluctuations than others. If necessary, calibration coefficients or equations can be employed to assess the temperature-dependent variability of the sample flux, but this can significantly complicate the use of the sensor. Changes in membrane composition can occur over time, particularly due to extraction loss and / or membrane degradation or metabolism of various membrane components, and the degree of these compositional changes can cause differences in sample permeability values. Quantitatively addressing compositional changes that result in different membrane permeability values ​​can be difficult, and determining when membrane permeability will stabilize sufficiently for accurate measurement of sample concentration can be challenging. Often, when a new sample sensor is implanted in a living organism, it may take several hours or more for the sample flux across the membrane to stabilize. [Brief explanation of the drawing]

[0007] The following drawings are included to illustrate certain aspects of the present disclosure and should not be regarded as exclusive embodiments. The disclosed subject matter is capable of numerous modifications, variations, combinations, and equivalents in form and function without departing from the scope of the present disclosure.

[0008] [Figure 1] A schematic diagram of an exemplary detection system incorporating the analyte sensor of the present disclosure is shown.

[0009] [Figure 2A] A cross-sectional view of an analyte sensor including a single active region is shown. [Figure 2B] A cross-sectional view of an analyte sensor including a single active region is shown. [Figure 2C] A cross-sectional view of an analyte sensor including a single active region is shown.

[0010] [Figure 3A] A cross-sectional view of an analyte sensor including two active regions is shown. [Figure 3B] A cross-sectional view of an analyte sensor including two active regions is shown. [Figure 3C] A cross-sectional view of an analyte sensor including two active regions is shown. [Figure 4] A cross-sectional view of an analyte sensor including two active regions is shown.

[0011] [Figure 5A] A perspective view of an analyte sensor including two active regions on separate working electrodes is shown. [Figure 5B] A perspective view of an analyte sensor including two active regions on separate working electrodes is shown. [Figure 5C] A perspective view of an analyte sensor including two active regions on separate working electrodes is shown.

[0012] [Figure 6] A, B, C show schematic diagrams of enzyme systems that can be used to detect ketones in an analyte sensor.

[0013] [Figure 7] The cyclic voltammograms of unbound and polymer-bound low-potential redox mediators are shown.

[0014] [Figure 8] The current-time plots for various working electrode potentials of a ketone sensor incorporating a low-potential redox mediator are shown.

[0015] [Figure 9] The graph shows current versus ketone concentration at various working electrode potentials of a ketone sensor incorporating a low-potential redox mediator.

[0016] [Figure 10] The following shows UV-VIS absorbance measurement data of extracts obtained from membrane polymers crosslinked with polyethylene glycol tetraglycidyl ether, compared to membrane polymers crosslinked with polyethylene glycol diglycidyl ether. [Figure 11] The following shows UV-VIS absorbance measurement data of extracts obtained from membrane polymers crosslinked with polyethylene glycol tetraglycidyl ether, compared to membrane polymers crosslinked with polyethylene glycol diglycidyl ether.

[0017] [Figure 12] This shows a plot of sensor output versus time for a substance sensor covered with a membrane polymer crosslinked with polyethylene glycol tetraglycidyl ether, compared to a substance sensor covered with a membrane polymer crosslinked with polyethylene glycol diglycidyl. [Figure 13] This shows a plot of sensor output versus time for a substance sensor covered with a membrane polymer crosslinked with polyethylene glycol tetraglycidyl ether, compared to a substance sensor covered with a membrane polymer crosslinked with polyethylene glycol diglycidyl. [Modes for carrying out the invention]

[0018] This disclosure generally describes a test substance sensor suitable for use in vivo, and more specifically, the test substance sensor comprises components including low-potential operating capability and / or stabilized membrane material features. Depending on the sensor configuration, the test substance sensor of this disclosure may be configured to detect one or more test substances simultaneously or nearly simultaneously. A series of dip coating operations may be performed to introduce different membrane compositions at specific locations on the test substance sensor.

[0019] Various components of a test substance sensor can cause specific problems when monitoring several test substances or combinations of test substances. Redox mediators used to facilitate electron transfer to the working electrode may require the test substance sensor to operate at relatively high potentials, and high-potential operation can lead to electrochemical side reactions that complicate the detection of small amounts of the test substance. Changes in the composition of the mass transfer limiting membrane within the test substance sensor can lead to undesirable changes in the permeability of the test substance during the period the sensor is implanted in a living organism, particularly during long-term sensor implantation. Furthermore, when analyzing multiple test substances using a single test substance sensor, different test substance permeability characteristics may necessitate the use of different mass transfer limiting membranes at various locations.

[0020] To address the aforementioned needs, this disclosure provides redox mediators for facilitating electron transfer at working electrode potentials lower than those conventionally used. The use of such “low-potential” redox mediators can reduce the occurrence of electrochemical side reactions by enabling the detection of a test substance at a potential lower than would otherwise be possible. By reducing the occurrence of electrochemical side reactions and the associated signal noise, the detection of small amounts of a test substance, such as ketones, can be made easier than would otherwise be possible at higher working electrode potentials. Such low-potential redox mediators may be advantageous when used in conjunction with the detection of multiple test substances, as will be discussed further below. Redox mediators capable of facilitating the detection of test substances at low working electrode potentials are described further below.

[0021] Various crosslinked polyvinylimidazole and polyvinylpyridine membrane polymers can be used in analyte sensors to improve biocompatibility and provide properties that restrict mass transfer. Functionalization on the membrane material may be chosen to modify the permeability of the analyte and to limit temperature-dependent changes in analyte permeability. Such membrane polymers can be crosslinked to linear glycidyl ethers having two crosslinking groups, such as polyethylene glycol diglycidyl ether (PEGDGE), but an equilibrium period may be required after sensor implantation to allow the analyte flux to stabilize. While not bound by theory or mechanism, it is believed that the composition of the membrane can change during the equilibrium period, during which small amounts of extractable material are released from the membrane, thereby altering the permeability of the analyte. Surprisingly, this disclosure shows that branched crosslinkers containing three or more crosslinking groups, such as branched polyethylene glycol glycidyl ether, more specifically polyethylene glycol tetraglycidyl ether, can reduce the amount of extractable material released from the membrane after sensor implantation. A reduction in the generation of extractable substances from the membrane can be achieved even if the crosslinking density and the amount (mass) of the crosslinking agent are substantially the same as when provided by a linear glycidyl ether having two crosslinking groups. That is, a given amount of crosslinking groups from a branched crosslinking agent such as polyethylene glycol tetraglycidyl ether can result in a reduction of extractable substances for a given membrane material compared to the amount of extractable substances resulting from a substantially similar amount of crosslinking groups obtained from a linear glycidyl ether crosslinking agent such as polyethylene glycol diglycidyl ether. Advantageously, as disclosed herein, the reduction in extractable substances may originate from the membrane material crosslinked with the branched crosslinking agent, thereby giving an improved toxicity profile. Furthermore, the reduction in compositional changes brought about by the membrane materials disclosed herein may result in a shorter sensor equilibration time after the sensor is embedded, potentially extending the sensor's wear life.

[0022] Before further detailing the test substance sensors and their components of this disclosure, an overview of suitable in vivo test substance sensor configurations and sensor systems using those test substance sensors is first provided so that embodiments of this disclosure may be better understood. Figure 1 shows a diagram of an exemplary detection system that may incorporate the test substance sensors of this disclosure. As shown in the figure, the detection system 100 includes a sensor control unit 102 and a reader 120 configured to communicate with each other wired or wirelessly, unidirectional or bidirectional, and via an encrypted or unencrypted local communication path or link. The reader 120 may, according to some embodiments, constitute an output device for viewing the concentration of the test substance and warnings or notifications determined by the sensor 104 or associated processor, and also allow input from one or more users. The reader 120 may be a multipurpose smartphone or a dedicated electronic reading device. Only one reader 120 is shown, but multiple reader 120 may be present in particular cases. The reader 120 may also communicate with the remote terminal 170 and / or the trusted computer system 180 via wired or wireless, unidirectional or bidirectional, encrypted or unencrypted communication paths / links 141 and / or 142, respectively. In addition to or instead, the reader 120 may communicate with a network 150 (e.g., a mobile telephone network, the Internet, or a cloud server) via a communication path / link 151. The network 150 may further be communicably connected to the remote terminal 170 via a communication path / link 152 and / or the trusted computer system 180 via a communication path / link 153. Alternatively, the sensor 104 may communicate directly with the remote terminal 170 and / or the trusted computer system 180 without the presence of the intervening reader 120.For example, according to some embodiments, as described in U.S. Patent Application Publication No. 2011 / 0213225, which is incorporated herein by reference in its entirety, the sensor 104 may communicate with a remote terminal 170 and / or a trusted computer system 180 via a direct communication link to a network 150. Any suitable electronic communication protocol may be used for each communication path or link, such as Near Field Communication (NFC), Radio Frequency Identification (RFID), Bluetooth® or Bluetooth® Low Energy Protocol, or Wi-Fi. According to some embodiments, the remote terminal 170 and / or the trusted computer system 180 may be accessible by an individual other than the first user who is interested in the user's test substance level. The reader 120 may include a display unit 122 and an optional input element 121. According to some embodiments, the display unit 122 may include a touchscreen interface.

[0023] The sensor control device 102 includes a sensor housing 103 and may house electrical circuits and a power supply for operating the sensor 104. Optionally, the power supply and / or active electrical circuits may be omitted. A processor (not shown) may be connected to the sensor 104 in a communicative manner, and the processor is physically integrated into the sensor housing 103 or the reader 120. The sensor 104 protrudes from the underside of the sensor housing 103 and extends through an adhesive layer 105, and according to some embodiments, is suitable for adhering the sensor housing 103 to a skin-like tissue surface.

[0024] Sensor 104 is adapted to be inserted at least partially into the target tissue, such as the dermis or subcutaneous layer of the skin. Sensor 104 may include a sensor tail of sufficient length to be inserted to a desired depth in a given tissue. The sensor tail may include at least one working electrode. In certain configurations, it will be further discussed that the sensor tail may include a ketone-responsive active region and, in certain cases, a low-potential redox mediator. A counter electrode may be present in combination with at least one working electrode. Specific electrode configurations on the sensor tail are described in further detail below.

[0025] One or more mass transfer restriction membranes, particularly those crosslinked by branched glycidyl ethers such as polyethylene glycol tetraglycidyl ether, may cover active regions, as will be described in more detail below. Active regions may be configured to detect specific test substances. For example, a glucose-responsive active region may contain a glucose-responsive enzyme, a lactate-responsive active region may contain a lactate-responsive enzyme, and a ketone-responsive active region may contain an enzyme system comprising at least two enzymes that can work together to facilitate the detection of ketones. Suitable enzyme systems for detecting ketones are described further below with reference to Figures 6A to 6C. According to various embodiments, each active region may contain a polymer covalently bonded to at least several enzymes.

[0026] In any embodiment of the present disclosure, one or more test substances may be monitored in a bodily fluid of interest, such as skin fluid, interstitial fluid, plasma, blood, lymph, synovial fluid, cerebrospinal fluid, saliva, bronchoalveolar lavage, or amniotic fluid. In certain embodiments, the test substance sensors of the present disclosure may be adapted for dermal or interstitial fluid assays to determine the concentration of one or more test substances in vivo.

[0027] Referring further to Figure 1, the sensor 104 may automatically transfer data to the reader 120. For example, test substance concentration data (i.e., glucose concentration and / or ketone concentration) may be communicated automatically and periodically using data stored in memory until the data is transmitted (e.g., every minute, every 5 minutes, or other predetermined time intervals) at a specific frequency or after a specific period of time has elapsed since data was obtained. In other embodiments, the sensor 104 may communicate with the reader 120 in a non-automatic manner and without following a set schedule. For example, data may be communicated from the sensor 104 using RFID technology when the sensor electronics are brought into the communication range of the reader 120. The data may remain stored in the sensor 104's memory until it is communicated to the reader 120. Therefore, the user does not need to constantly stay near the reader 120, but can instead upload data at a convenient time. In yet another embodiment, a combination of automatic and non-automatic data transfer may be performed. For example, data transfer may continue automatically until the reader 120 is no longer within the communication range of the sensor 104.

[0028] An introducer may be present temporarily to facilitate the introduction of the sensor 104 into the tissue. In exemplary embodiments, the introducer may comprise a needle or a similar sharp portion. It should be recognized that other types of introducers, such as a sheath or blade, may be present in alternative embodiments. More specifically, the needle or other introducer may be present temporarily near the sensor 104 before insertion into the tissue and then withdrawn. While present, the needle or other introducer may facilitate the insertion of the sensor 104 into the tissue by opening an access path for the sensor 104 to follow. For example, according to one or more embodiments, the needle may facilitate penetration of the epithelium as an access path to the dermis to make the implantation of the sensor 104 feasible. After opening the access path, the needle or other introducer may be withdrawn to avoid posing a hazard due to its sharp tip. In exemplary embodiments, a suitable needle may be solid or hollow, acute or non-acute, and / or round or non-round in cross-section. In more specific embodiments, a suitable needle may have a cross-sectional diameter of approximately 250 microns (250 μm), comparable to an acupuncture needle in terms of cross-sectional diameter and / or tip design. However, it should be recognized that a suitable needle may have a larger or smaller cross-sectional diameter if required for a particular application.

[0029] In some embodiments, the tip of the needle (while present) may be angled over the end of the sensor 104 so that the needle first penetrates the tissue and opens an access path for the sensor 104. In other exemplary embodiments, the sensor 104 may be located in the lumen or groove of the needle so that the needle similarly opens an access path for the sensor 104. In any case, the needle is then withdrawn after facilitating the insertion of the sensor.

[0030] Sensor configurations including a single active region configured to detect a corresponding single test substance may employ a two-electrode or three-electrode detection motif, as further described here with reference to Figures 2A to 2C. Sensor configurations including two different active regions for the detection of different test substances, either on separate working electrodes or on the same working electrode, will be described separately later with reference to Figures 3A to 5C. Sensor configurations with multiple working electrodes may be particularly advantageous for incorporating two different active regions within the same sensor tail, as the signals contributed by each active region can be more easily determined. Furthermore, the deposition of different film compositions on each active region can be easily carried out by a series of dip-coating operations when the active region is located on a second working electrode.

[0031] When a single working electrode is present in the sensor for the substance being tested, a three-electrode sensor configuration may include a working electrode, a counter electrode, and a reference electrode. A related two-electrode sensor configuration may include a working electrode and a second electrode, the second electrode may function as both a counter electrode and a reference electrode (i.e., a counter / reference electrode). The various electrodes may be at least partially stacked on each other (layered) and / or spaced apart laterally on the sensor tail. A suitable sensor configuration may be substantially flat or substantially cylindrical. In any sensor configuration disclosed herein, the various electrodes may be electrically insulated from each other by a dielectric material or similar insulator.

[0032] A sensor for a substance being tested, comprising multiple working electrodes, may similarly include at least one additional electrode. If one additional electrode is present, it may function as a pair / reference electrode for each of the multiple working electrodes. If two additional electrodes are present, one of the additional electrodes may function as a pair electrode for each of the multiple working electrodes, and the other additional electrode may function as a reference electrode for each of the multiple working electrodes.

[0033] Figure 2A shows a schematic diagram of an exemplary two-electrode substance sensor configuration suitable for use in the disclosure herein. As shown in the figure, the substance sensor 200 includes a substrate 212 positioned between a working electrode 214 and a pair / reference electrode 216. Alternatively, the working electrode 214 and the pair / reference electrode 216 may be positioned on the same side of the substrate 212, together with a dielectric material interposed between them (configuration not shown). An active area 218 is positioned as at least one layer on at least a portion of the working electrode 214. The active area 218 may include multiple spots or a single spot configured to detect the substance at a low working electrode potential, as further discussed herein.

[0034] Referring further to Figure 2A, according to some embodiments, the film 220 may cover at least the active area 218 and optionally some or all of the working electrode 214 and / or the pair / reference electrode 216, or the entire substance sensor 200. One or both sides of the substance sensor 200 may be covered with the film 220. The film 220 may comprise one or more polymer film materials capable of restricting the substance flux to the active area 218 (i.e., the film 220 is a mass transfer limiting film that is permeable to the substance of interest). According to the disclosure herein, the film 220 may be crosslinked using a branching crosslinking agent in certain sensor configurations. The composition and thickness of the film 220 may be modified to promote a desired substance flux to the active area 218, thereby providing a desired signal intensity and stability. The test substance sensor 200 is capable of assaying the test substance by any electrochemical detection technique, such as coulometry, amperometry, voltammetry, or potentiometry.

[0035] Figures 2B and 2C show schematic diagrams of exemplary three-electrode substance sensor configurations, which are also suitable for use in the disclosure herein. The three-electrode substance sensor configurations may be similar to the configuration shown as substance sensor 200 in Figure 2A, except that they include an additional electrode 217 in substance sensors 201 and 202 (Figures 2B and 2C). When the additional electrode 217 is present, the pair / reference electrode 216 may then function as either the pair electrode or the reference electrode, and the additional electrode 217 performs the function of the other electrode that is not configured. The working electrode 214 continues to perform its original function. The additional electrode 217 may be positioned on either the working electrode 214 or the electrode 216 with a dielectric material separation layer in between. For example, as shown in Figure 2B, dielectric layers 219a, 219b, and 219c separate electrodes 214, 216, and 217 from each other and provide electrical insulation. Alternatively, at least one of the electrodes 214, 216, and 217 may be arranged on both sides of the substrate 212, as shown in Figure 2C. Thus, in some embodiments, electrodes 214 (working electrode) and 216 (counter electrode) may be arranged on both sides of the substrate 212, and electrode 217 (reference electrode) may be placed on one of electrodes 214 or 216, spaced apart from each other with a dielectric material in between. A reference material layer 230 (e.g., Ag / AgCl) may be present on electrode 217, and the arrangement of the reference material layer 230 is not limited to the arrangements shown in Figures 2B and 2C. Similar to the sensor 200 shown in Figure 2A, the active area 218 in the substance-of-test sensors 201 and 202 may include multiple spots or a single spot. Furthermore, the test substance sensors 201 and 202 can be operated to assay the test substance by any coulometry, amperometry, voltammetry, or potentiometry electrochemical detection technique.

[0036] In substance sensors 201 and 202, as in the substance sensor 200, the membrane 220 may also cover the active area 218, similar to other sensor configurations, thereby acting as a mass transfer limiting membrane. An additional electrode 217 may be covered by the membrane 220 in some embodiments. Figures 3B and 2C show all electrodes 214, 216 and 217 covered by the membrane 220, although it should be noted that in some embodiments, only the working electrode 214 may be covered. Furthermore, the thickness of the membrane 220 at each of electrodes 214, 216 and 217 may be the same or different, and / or the composition of the membrane may differ locally. As in the two-electrode substance sensor configuration (Figure 2A), one or both sides of the substance sensors 201 and 202 may be covered by the membrane 220 in the sensor configurations of Figures 2B and 2C, or the entire substance sensors 201 and 202 may be covered. Furthermore, it should be understood that the three-electrode sensor configurations shown in Figures 2B and 2C are not limited to the embodiments disclosed herein, and alternative electrode and / or layer configurations remain within the scope of this disclosure.

[0037] Figure 3A shows an exemplary configuration of a sensor 203 having a single working electrode with two different active regions positioned on a single working electrode. Figure 3A is similar to Figure 2A except that the two active regions are located on the working electrode 214, the first active region 218a and the second active region 218b respond to different test substances and are spaced laterally apart from each other on the surface of the working electrode 214. The active regions 218a and 218b may contain multiple spots or a single spot configured for the detection of each test substance. The composition of the film 220 may differ or be compositionally the same in the active regions 218a and 218b. The first active region 218a and the second active region 218b may be configured to detect the corresponding test substances at different working electrode potentials, as will be discussed further below.

[0038] Figures 3B and 3C show, respectively, cross-sectional views of exemplary three-electrode sensor configurations for sensors 204 and 205, featuring a single working electrode having a first active region 218a and a second active region 218b located on a single working electrode. Figures 3B and 3C are otherwise similar to Figures 2B and 2C, which can be better understood by reference. As with Figure 3A, the composition of the film 220 may differ in the active regions 218a and 218b, or may be compositionally the same.

[0039] Exemplary sensor configurations having multiple working electrodes, particularly two working electrodes, are described in further detail with reference to Figures 4 to 5C. The following description primarily focuses on sensor configurations having two working electrodes, but it should be acknowledged that configurations with more than two working electrodes may be incorporated through extensions of the disclosure herein. Additional working electrodes may be used to provide the test substance sensor with additional detection capabilities beyond just the first and second test substances.

[0040] Figure 4 shows a cross-sectional view of an exemplary substance sensor configuration having two working electrodes, a reference electrode, and a counter electrode, suitable for use in the disclosure herein. As shown in the figure, the substance sensor 300 includes working electrodes 304 and 306 arranged on opposite sides of a substrate 302. A first active area 310a is located on the surface of the working electrode 304, and a second active area 310b is located on the surface of the working electrode 306. The counter electrode 320 is electrically insulated from the working electrode 304 by a dielectric layer 322, and the reference electrode 321 is electrically insulated from the working electrode 306 by a dielectric layer 323. Outer dielectric layers 330 and 332 are located on the reference electrode 321 and the counter electrode 320, respectively. The film 340 may, according to various embodiments, cover at least the active areas 310a and 310b, and similarly, other components of the substance sensor 300 or the entire substance sensor 300 may optionally be covered by the film 340. Furthermore, if necessary, the membrane 340 may be compositionally different in the active regions 310a and 310b to provide permeability values ​​suitable for differentially adjusting the flux of the test substance at each location. For example, the membrane 340 may be homogeneous in the area covering the active region 310a and heterogeneous in the area covering the active region 310b.

[0041] Alternative sensor configurations having multiple working electrodes, different from the configuration shown in Figure 4, may have a single pair / reference electrode instead of separate pair and reference electrodes 320, 321, and / or be characterized by a different arrangement of layers and / or films from the clearly shown arrangement. For example, the arrangement of pair electrode 320 and reference electrode 321 may be reversed from the arrangement shown in Figure 4. Furthermore, working electrodes 304 and 306 do not necessarily have to be present on both sides of the substrate 302 in the arrangement shown in Figure 4. Instead, working electrodes 304 and 306 may be present on the same surface of the substrate 302, or separated from each other by a gap. In particular, working electrode 304 may be positioned towards the more distal (tip) end (front) of the substance sensor, or towards the more proximal end, compared to working electrode 306. If a sufficiently large gap exists between the working electrodes 304 and 306, the film 340 may be deposited on the working electrodes 304 and 306 by a series of dip coating operations, and the composition of the film 340 may differ locally. In particular, the first dip coating operation may deposit a first film polymer on both the working electrodes 304 and 306, and the second dip coating operation may deposit a second film polymer with a different composition only on the working electrode 304, thereby defining two layers on the working electrode 304 and leaving a uniform film on the working electrode 306. Thus, the lower layer of the two-layer film and the uniform film may contain the same film polymer. Alternatively, to define the two-layer film and the uniform film, the first dip coating operation may deposit a first film polymer on the working electrode 304, and the second dip coating operation may deposit a second film polymer with a different composition on both the working electrodes 304 and 306. In this case, the upper layer of the two-layer film and the uniform film may contain the same film polymer.

[0042] The size of the gap between working electrodes 304 and 306 is such that there is sufficient margin to provide at least electrical insulation between the two electrodes, and more typically, it is such that a series of dip coating operations can be sufficiently facilitated (for example, by allowing the substance sensor to be immersed to different depths so that one of the working electrodes is preferentially covered in at least one immersion step). In other words, the size of the gap can provide room for error in lowering the substance sensor to a particular depth in a particular dip coating agent in order to facilitate film formation on one of the working electrodes in preference to the other.

[0043] Appropriate sensor configurations may feature substantially flat electrodes, including flat sensor configurations with spaced-apart working electrodes; however, it should be understood that sensor configurations featuring non-flat electrodes may be advantageous and particularly suitable for use in the disclosure herein. In particular, cylindrical electrodes arranged concentrically and spaced apart along the length of the sensor tail can facilitate the deposition of mass transfer limiting films of different compositions, as will be further described below. In particular, concentric working electrodes spaced apart along the length of the sensor tail can facilitate deposition by a series of dip-coating operations, similar to the method described above for substantially flat sensor configurations. Figures 5A to 5C show perspective views of a substance sensor with two concentrically arranged working electrodes. It should also be understood that sensor configurations without a second working electrode but with a concentric electrode arrangement are also possible in this disclosure.

[0044] Figure 5A shows a perspective view of an exemplary sensor configuration in which multiple electrodes are substantially cylindrical and arranged concentrically with respect to a central substrate. As shown in the figure, the substance sensor 400 includes a central substrate 402 in which all electrodes and dielectric layers are arranged concentrically with respect to each other. In particular, the working electrode 410 is located on the surface of the central substrate 402, and the dielectric layer 412 is located on a portion of the working electrode 410 distal to the sensor tip 404. The working electrode 420 is located on the dielectric layer 412, and the dielectric layer 422 is located on a portion of the working electrode 420 distal to the sensor tip 404. The counter electrode 430 is located on the dielectric layer 422, and the dielectric layer 432 is located on a portion of the counter electrode 430 distal to the sensor tip 404. The reference electrode 440 is located on the dielectric layer 432, and the dielectric layer 442 is located on a portion of the reference electrode 440 distal to the sensor tip 404. Thus, the exposed surfaces of the working electrode 410, the working electrode 420, the counter electrode 430, and the reference electrode 440 are spaced apart from each other along the longitudinal axis B of the substance sensor 400. This electrode arrangement may allow the film 450 to be deposited by a series of dip coating operations, which may allow for a bilayer film portion to be placed on the working electrode 410 and a uniform film portion to be placed on the working electrode 420, as will be discussed below. The bilayer film portion and the uniform film portion may be adjacent to each other.

[0045] Referring further to Figure 5A, the first active area 414a and the second active area 414b, which respond to different test substances, are located on the exposed surfaces of the working electrodes 410 and 420, respectively, thereby enabling contact with the fluid to be detected. Although the active areas 414a and 414b are shown as three separate spots in Figure 5A, it should be understood that alternative sensor configurations may include a continuous layer of active areas and may have fewer or more spots than three.

[0046] In Figure 5A, the sensor 400 is partially coated with a film 450 on the working electrodes 410 and 420 and the active areas 414a and 414b located thereon. Figure 5B shows an alternative sensor configuration in which substantially the entire sensor 401 is covered with the film 450. The film 450 may be compositionally the same or different in the active areas 414a and 414b. For example, the film 450 may include a bilayer portion covering the active area 414a and a uniform portion covering the active area 414b. The film 450 may be deposited by a series of dip-coating operations to deposit the bilayer portion on the working electrode 410 and the active area 414a, and the uniform portion on the working electrode 420 and the active area 414b.

[0047] It should be further understood that various electrode arrangements in Figures 5A and 5B may differ from the arrangements clearly shown. For example, the arrangement of the counter electrode 430 and the reference electrode 440 may be reversed from the configurations shown in Figures 5A and 5B. Similarly, the arrangement of the working electrodes 410 and 420 is not limited to the arrangements clearly shown in Figures 5A and 5B. Figure 5C shows an alternative sensor configuration to the configuration shown in Figure 5B, in which the sensor 405 includes the counter electrode 430 and the reference electrode 440 positioned more proximal to the sensor tip 404, and the working electrodes 410 and 420 positioned more distal to the sensor tip 404. A sensor configuration in which the working electrodes 410 and 420 are positioned more distal to the sensor tip 404 may be advantageous by providing a larger surface area for deposition of the active areas 414a and 414b (in Figure 5C, five separate detection spots are shown exemplarily), thereby facilitating signal intensity enhancement in some cases. Furthermore, by having working electrodes 410 and 420 that are concentric with each other and spaced apart along the sensor tail, a series of dip-coating operations may result in the two-layer film being deposited on the working electrode located near the sensor tip 404, or the uniform film being deposited on the working electrode located further away from the sensor tip 404. Similarly, the central substrate 402 may be omitted in any concentric sensor configuration disclosed herein, and the furthest electrode may instead be supported following the deposited layer.

[0048] It should also be understood that a low-potential operating substance sensor according to the following disclosure may include a mass transfer limiting film having a bilayer portion and a homogeneous film portion. A series of dip-coating operations may be advantageous for deposition such as a mass transfer limiting film. However, this disclosure also considers a substance sensor further equipped with a mass transfer limiting film including a bilayer portion and a homogeneous film portion, although detection does not necessarily have to be performed at low potential.

[0049] As a result, according to this disclosure, a low-potential operating substance sensor may include a sensor tail comprising at least a first working electrode; a first active region positioned on the surface of the first working electrode and responding to a first substance at a low potential, the first active region comprising a first polymer, a first redox mediator covalently bonded to the first polymer, and at least one enzyme covalently bonded to the first polymer and responding to the first substance; and a mass transfer limiting membrane permeable to the first substance covering at least the first active region. As used herein, the term “low potential” refers to a potential higher than the redox potential of the first redox mediator, and, when measured in comparison to an Ag / AgCl reference electrode, a potential less than approximately +100mV, less than approximately -50mV, less than approximately -80mV, or less than approximately +200mV, including less than approximately -100mV. Exemplary redox potentials of a first redox mediator that can facilitate operation at potentials such as the working electrode potential may be less than approximately -200mV, such as approximately -400mV to approximately -200mV, or approximately -350mV to approximately -250mV, or approximately -300mV to approximately -250mV, when measured in comparison to the Ag / AgCl reference potential.

[0050] A suitable example of a first redox mediator capable of promoting operation at low potential may have a structure represented by Equation 1. [ka] In the formula, M is osmium, ruthenium, vanadium, cobalt, or iron, and L 1 From L 6 is an independent heteroaromatic ligand that coordinately binds to M, and L 1 From L 6 Two or more of these may combine arbitrarily to form a ligand with two, three, or more loci, L 1 From L 6 At least one of these contains a binding group that attaches the first redox mediator to the first polymer, L 1 From L 6At least one of them is functionalized by an electron-donating group. The electron-donating group is separated from and different from the linking group.

[0051] Particularly suitable bidentate ligands characterized by a heteroaromatic ligand included in a low-potential redox mediator include optionally substituted 2,2'-biimidazole ligands, 2-(2-pyridyl)imidazole ligands and 2,2'-bipyridine ligands.

[0052] Examples of 2,2'-biimidazole ligands are represented by Formula 2,

Chemical formula

[0053] Examples of 2-(2-pyridyl)imidazole ligands may have a structure represented by Formula 3,

Chemical formula

[0054] An example of a 2,2'-bipyridine ligand may have the structure represented by formula 4. [ka] In the formula, Q is any substitution bonded to one or more carbon atoms of the pyridine ring (p=0, 1, 2, 3, or 4), and any substitution of Q may, in some embodiments, be an electron-donating group or a bonding group. Suitable electron-donating groups containing one or more Qs include, for example, alkyl, alkoxy, hydroxyl, amino, alkylamino, or dialkylamino. If Q is absent, the carbon atom has a hydrogen atom.

[0055] A particularly suitable example of a redox mediator capable of promoting electron transfer at low potential may have a structure represented by equation 5, L 1 and L 2 , L 3 and L 4 , and L 5 and L 6 Both are bonded together to form a bidentate ligand, and one of the bidentate ligands has a bonding group G that covalently bonds the redox mediator to the polymer, L 1 -L 2 , L 3 -L 4 , and L 5 -L 6At least one of the groups has an electron-donating group. The electron-donating group is separate from and distinct from the bonding group. The electron-donating group may be on the same bidentate ligand containing the bonding group G, or on different bidentate ligands. 1 -L 2 , L 3 -L 4 , and L 5 -L 6 This may include, for example, one or more bidentate ligands represented by equations 2 to 4. [ka]

[0056] In some embodiments, the redox mediator may be positively charged (e.g., in the range of +1 to +5). Alternatively, if the ligand or backbone is derivatized by a sufficient number of negatively charged functional groups, such as carboxylic acid groups, phosphate groups, or sulfonic acid groups, the redox mediator will be negatively charged (e.g., in the range of -1 to -5). One or more counterions are used to balance the charge. Examples of suitable counterions include anions such as halide ions (e.g., fluoride ions, chloride ions, bromide ions, or iodide ions), sulfate ions, phosphate ions, hexafluorophosphate ions, and tetrafluoroborate ions, and cations, particularly monovalent cations such as lithium ions, sodium ions, potassium ions, tetraalkylammonium ions, and ammonium ions.

[0057] In more specific examples, the redox mediator in the first active region may have a structure represented by formula 6. [ka] In the formula, G is a bonding group that covalently bonds the redox mediator to the polymer in the active region, and D is an electron-donating group. Specific examples of suitable electron-donating groups include, for example, hydroxyl groups, alkoxy groups (e.g., methoxy or ethoxy groups), amino groups, or alkyl or dialkylamino groups (e.g., methylamino, ethylamino, dimethylamino, or diethylamino groups). In more specific examples, the electron-donating group may be located at the 4-position of the pyridine ring, as shown in Formula 7. [ka] In any embodiment of this specification, a redox mediator capable of promoting electron transfer at low potential may have a structure represented by Equation 8. [ka]

[0058] In at least one embodiment, the binding group G may include a reactive group to promote covalent bonding to the polymer. This reactive group reacts with a complementary reactive group located on or within the polymer precursor to promote covalent bonding to it. In some embodiments, an amide group may be present within the binding group G.

[0059] The main chain of any suitable polymer may be present in the active region to facilitate the detection of the test substance at low potentials by covalent bonding of redox mediators and enzymes to it. Examples of suitable polymers in the active region include poly(4-vinylpyridine) and poly(N-vinylimidazole) or their copolymers, where, for example, quaternized pyridine and quaternized imidazole groups act as binding sites for redox mediators or enzymes. Other suitable polymers that may be present in the active region include, but are not limited to, polymers described in U.S. Patent No. 6,605,200, all of which are incorporated herein by reference, such as poly(acrylic acid), styrene / maleic anhydride copolymer, methyl vinyl ether / maleic anhydride copolymer (GANTREZ polymer), poly(vinyl benzyl chloride), poly(allylamine), polylysine, poly(4-vinylpyridine) quaternized with carboxypentyl groups, and poly(4-sodium styrenesulfonate).

[0060] In the first active region, it is not inconceivable that the enzyme covalently bound to the polymer capable of facilitating detection at low potentials is particularly limited. Suitable enzymes may include those capable of detecting glucose, lactate, ketones, or creatinine, etc. In some cases, at least one enzyme covalently bound to the polymer in the first active region may include multiple enzymes that respond collectively to the test substance at low potentials. Enzyme systems may be particularly desirable for detecting ketones and creatinine.

[0061] In more specific embodiments, the first active region may include an enzyme system capable of detecting ketones. As previously mentioned, ketones typically exist in small biological amounts and can benefit from detection at low potentials as disclosed herein. Now, referring to Figures 6A to 6C, specific enzyme systems that may be used to detect ketones are described in further detail. In the enzymatic reaction shown, β-hydroxybutyrate acts as a substitute for ketones formed in vivo. As shown in Figure 6A, a pair of synergistic enzymes that may be used to detect ketones as disclosed herein are β-hydroxybutyrate dehydrogenase (HBDH) and diaphorase, which may be deposited within a ketone-responsive active region on the surface of at least one working electrode, as further described here. If the ketone-responsive active region includes this pair of synergistic enzymes, β-hydroxybutyrate dehydrogenase reacts with β-hydroxybutyrate and oxidized nicotinamide adenine dinucleotide (NAD + ) are converted to acetacetate and reduced nicotinamide adenine dinucleotide (NADH), respectively. Enzyme cofactor NAD + NADH plays a role in facilitating the coordinated enzymatic reaction disclosed herein. NADH may then undergo oxidation via diaphorase, and the transfer of electrons between these processes provides a basis for ketone detection at the working electrode. Thus, there is a 1:1 molar correspondence between the amount of electrons transferred to the working electrode and the amount of β-hydroxybutyrate converted, thereby providing a basis for ketone detection and quantification based on the measurement of the current at the working electrode. The electron transfer resulting in NADH oxidation at the working electrode may be carried out by a redox mediator capable of facilitating operation at low potentials. Albumin may be present as a stabilizer along with this pair of coordinated enzymes. According to certain embodiments, β-hydroxybutyrate dehydrogenase and diaphorase may be covalently bonded to a polymer within the ketone-responsive active region of the test substance sensor. + It may or may not be covalently bonded to the polymer, NAD + If covalent bonding does not occur, NAD can physically remain within the ketone-responsive active region. The membrane surrounding the ketone-responsive active region contains NAD within the ketone-responsive active region. +This helps to retain the ketone and allows sufficient internal diffusion of the ketone to enable ketone detection.

[0062] Other suitable chemistry for enzymatic detection of ketones is shown in Figures 6B and 6C. In both cases, there is a further 1:1 molar correspondence between the amount of electrons transferred to the working electrode and the amount of β-hydroxybutyrate converted, thereby providing a basis for ketone detection.

[0063] As shown in Figure 6B, β-hydroxybutyrate dehydrogenase (HBDH) further processes β-hydroxybutyrate and NAD. + These can be converted to acetacetate and NADH, respectively. Instead of electron transfer to the working electrode, which is completed by diaphorase (shown in Figure 6A) and a suitable redox mediator, the reduced form of NADH oxidase (NADHOx(Red)) undergoes the reaction to form the corresponding oxidized form (NADHOx(Ox)). NADHOx(Red) is then reformed by a reaction using molecular oxygen to produce superoxide, which can then be converted to hydrogen peroxide via superoxide dismutase (SOD). Hydrogen peroxide may then be oxidized at the working electrode, providing a signal that may correlate with the amount of ketone initially present. SOD may be covalently bonded to the polymer in the ketone-responsive active region according to various embodiments. As in the enzyme system shown in Figure 6A, β-hydroxybutyrate dehydrogenase and NADH oxidase may be covalently bonded to the polymer in the ketone-responsive active region, and NAD may or may not be covalently bonded to the polymer in the ketone-responsive active region. + If covalent bonding does not occur, NAD is present in the ketone-responsive active region. + The ketone-responsive active region may be physically retained using a membrane polymer that promotes retention.

[0064] As shown in Figure 6C, the detection chemistry of another enzyme against ketones is β-hydroxybutyrate and NAD +β-hydroxybutyrate dehydrogenase (HBDH) may be utilized to convert acetacetate and NADH, respectively. In this case, the electron transfer cycle is completed at the working electrode by oxidation of 1,10-phenanthroline-5,6-dione to reformate NAD. 1,10-phenanthroline-5,6-dione may or may not be covalently bonded to the polymer in the ketone-responsive active region. As in the enzyme system shown in Figure 6A, β-hydroxybutyrate dehydrogenase may be covalently bonded to the polymer in the ketone-responsive active region, and NAD may or may not be covalently bonded to the polymer in the ketone-responsive active region. The inclusion of albumin in the active region can provide a remarkable improvement in reaction stability. Suitable membrane polymers may contain NAD in the ketone-responsive active region. + This may promote retention.

[0065] The substance sensor of this disclosure may further be configured to analyze a second or subsequent substance in addition to a substance detectable at low potential in the first active region. To facilitate the detection of the second substance, the substance sensor of this disclosure further includes a second working electrode and a second active region located on the surface of the second working electrode and responding to a second substance different from the first substance, the second active region comprising a second polymer, a second redox mediator covalently bonded to the second polymer and different from the first redox mediator, and at least one enzyme covalently bonded to the second polymer and responding to the second substance. A second portion of a mass transfer limiting membrane may cover the second active region. The at least one enzyme responding to the second substance may comprise an enzyme system comprising a plurality of enzymes that respond collectively to the second substance. In the second active region, the second redox mediator does not necessarily have to be able to facilitate electron transfer at low potential, but may be able to facilitate electron transfer at low potential.

[0066] Suitable redox mediators included in the second active area may include, but are not limited to, osmium complexes and other transition metal complexes, as described in U.S. Patents No. 6,134,461 and 6,605,200, all of which are incorporated herein by reference. Additional examples of suitable redox mediators include those described in U.S. Patents No. 6,736,957, 7,501,053 and 7,754,093, all of which are also incorporated herein by reference in each disclosure. Other suitable redox mediators included in the second active area may include metal compounds or metal complexes of ruthenium, osmium, iron (e.g., polyvinylferrocene or hexacyanoferric acid), or cobalt, including, for example, its metallocene compounds. Suitable ligands for metal complexes may include, for example, bipyridine, biimidazole, phenanthroline, or pyridyl(imidazole), for example, ligands with two or more locates. Other suitable bidentate ligands may include, for example, amino acids, oxalic acid, acetylacetone, diaminoalkanes, or o-diaminoarenes. Any combination of monodentate, bidentate, tridentate, tetradentate, or more-dentate ligands may be present in the metal complex to achieve a complete coordination sphere.

[0067] The active regions for facilitating the detection of a test substance as disclosed herein may include polymers to which the redox mediator is covalently bonded. Suitable examples of polymer-bonded redox mediators are described in U.S. Patents 8,444,834, 8,268,143, and 6,605,201, all of which are incorporated herein by reference. Suitable polymers to be included in the active regions may include, but are not limited to, polyvinylpyridine (e.g., poly(4-vinylpyridine)), polyvinylimidazole (e.g., poly(1-vinylimidazole)), or any copolymer thereof. Suitable exemplary copolymers to be included in the active regions may include, for example, those containing monomer units such as styrene, acrylamide, methacrylamide, or acrylonitrile. The polymers in each active region may be the same or different.

[0068] In certain examples, the second active region may be configured to detect glucose in combination with a test substance detectable at a low potential in the first active region. Thus, in certain embodiments of this disclosure, the second enzyme may be glucose oxidase. Furthermore, in more specific examples, the first test substance may be a ketone detectable by an enzymatic system as described herein, and the second test substance may be glucose detectable by glucose oxidase.

[0069] The detection of each test substance may involve applying separate potentials to each working electrode so that separate signals are obtained from each test substance. The signals obtained from each test substance can then be correlated with the concentration of the test substance using a calibration curve or function, or by using a lookup table. The correlation between the test substance signal and the test substance concentration may, in certain examples, be derived by the use of a processor.

[0070] In other test substance sensor configurations, the first and second active regions may be located on a single working electrode. The first signal may be acquired at a low potential from the first active region, while the second signal, which includes signal contributions from both active regions, may be acquired at a higher potential. Subtracting the first signal from the second signal may then allow for the determination of the signal contribution originating from the second test substance. Then, similar to the methods described for sensor configurations with multiple working electrodes, the signal contribution from each test substance may be associated with the test substance concentration.

[0071] As a result, the present disclosure provides a test substance sensor comprising: a sensor tail including at least a first working electrode; a first active region disposed on the surface of the first working electrode and responding to a first test substance at a low potential, wherein the first active region comprises a first polymer; a first redox mediator covalently bonded to the first polymer; and at least one enzyme covalently bonded to the first polymer and responding to the first test substance, wherein the redox mediator has a structure represented by any one of the above formulas 1 to 8, where G is a binding group covalently bonded to the first redox mediator to the first polymer; and a mass transfer limiting membrane permeable to the first test substance that covers at least the first active region; and a method comprising: applying a low potential to the first working electrode; obtaining a first signal that is proportional to the concentration of the first test substance in a fluid in contact with the first active region and is greater than or equal to the oxidation-reduction potential of the first active region; and correlating the concentration of the first test substance in the fluid with the first signal.

[0072] When measured relative to an Ag / AgCl reference electrode, low potentials include approximately less than +100mV, less than -50mV, less than -80mV, or less than -100mV, and may also be less than approximately +200mV. Low potentials may also be higher than the redox potential of the first redox mediator. Exemplary redox potentials of the first redox mediator that can facilitate operation at low working electrode potentials may be less than approximately -200mV, such as approximately -400mV to approximately -200mV, or approximately -350mV to approximately -250mV, or approximately -300mV to approximately -250mV, when measured relative to an Ag / AgCl reference electrode.

[0073] The substance sensor disclosed herein further includes a substance-permeable mass transfer limiting membrane covering at least a first active area. If multiple active areas are present, the mass transfer limiting membrane may cover each active area with different compositions on different active areas, such that a two-layer membrane portion on the working electrode near the sensor tip can be achieved by a series of dip-coating operations. The mass transfer limiting membrane may include a membrane polymer, such as a homopolymer or copolymer of polyvinylpyridine or polyvinylimidazole, which may be further crosslinked with a suitable crosslinking agent. In certain embodiments, the membrane polymer may include a copolymer of vinylpyridine and styrene. In more specific examples, the membrane polymer covering one or more active areas may be crosslinked with a branched crosslinking agent containing three or more crosslinking groups, such as polyethylene glycol tetraglycidyl ether, which may dramatically reduce the amount of extractable material obtained from the mass transfer limiting membrane as described above. More specifically, the mass transfer-restricting membrane may comprise a copolymer of vinylpyridine and styrene crosslinked with polyvinylpyridine or a branched glycidyl ether crosslinking agent containing three crosslinking groups, such as polyethylene glycol tetraglycidyl ether. In particular, the epoxy group of polyethylene glycol tetraglycidyl ether may react with a pyridine nitrogen atom or an imidazole nitrogen atom, and the covalent bonding of the crosslinking group may be promoted by ring-opening of the epoxide ring. A hydroxyalkyl group may be provided that crosslinks the crosslinking agent body to the heterocycle of the membrane polymer.

[0074] Suitable copolymers of vinylpyridine and styrene may contain styrene in the range of about 0.01% to about 50% mol%, or about 0.05% to about 45% mol%, or about 0.1% to about 40% mol%, or about 0.5% to about 35% mol%, or about 1% to about 30% mol%, or about 2% to about 25% mol%, or about 5% to about 20% mol%. Substituted styrene may be used in a similar manner and in similar amounts. Suitable copolymers of vinylpyridine and styrene may have molecular weights of 5 kDa or more, or about 10 kDa or more, or about 15 kDa or more, or about 20 kDa or more, or about 25 kDa or more, or about 30 kDa or more, or about 40 kDa or more, or about 50 kDa or more, or about 75 kDa or more, or about 90 kDa or more, or about 100 kDa or more. In a limited number of examples, suitable copolymers of vinylpyridine and styrene may have molecular weights ranging from about 5 kDa to about 150 kDa, or from about 10 kDa to about 125 kDa, or from about 15 kDa to about 100 kDa, or from about 20 kDa to about 80 kDa, or from about 25 kDa to about 75 kDa, or from about 30 kDa to about 60 kDa.

[0075] As a result, at least some of the test substance sensors described herein may include a sensor tail including at least a first working electrode, a first active region disposed on the surface of the first working electrode, and a mass transfer limiting membrane permeable to the first test substance that covers at least the first active region. The first active region includes a first polymer and at least one enzyme covalently bonded to the first polymer and responsive to the first test substance. The mass transfer limiting membrane includes a membrane polymer crosslinked with a branched glycidyl ether crosslinking agent containing three or more crosslinking groups, such as polyethylene glycol tetraglycidyl ether.

[0076] Crosslinking can occur intermolecularly in more specific embodiments. Polyethylene glycol tetraglycidyl ether used to facilitate intermolecular crosslinking between two or more membrane polymer backchains can exhibit a broad range of suitable molecular weights. Up to four polymer backchains can be crosslinked by a single molecule of polyethylene glycol tetraglycidyl ether crosslinking agent. In specific examples, the molecular weight of polyethylene glycol tetraglycidyl ether may range from about 1000 g / mol to about 5000 g / mol. The number of repeating ethylene glycol units in each arm of the polyethylene glycol tetraglycidyl ether may be the same or different, and may vary over a given range of samples, usually giving an average molecular weight. The structure of polyethylene glycol tetraglycidyl ether before crosslinking is represented by the following formula 9: [ka] In the formula, n1, n2, n3, and n4 are all non-negative integers, usually 1 or greater, and n1, n2, n3, and n4 may be the same or different. The sum of n1, n2, n3, and n4 may be chosen such that the molecular weight of the polyethylene glycol tetraglycidyl ether falls within the above range. In other words, to produce a polyethylene glycol tetraglycidyl ether having a molecular weight within the above range, the sum of n1, n2, n3, and n4 may be in the range of about 14 to about 110, or about 15 to about 104, including any subrange between these values, and n1, n2, n3, and n4 may be any non-negative integer or an integer of 1 or greater separately.

[0077] Crosslinking density indicates the number of membrane polymer side chains that have a crosslinking agent bound to them. For example, membrane polymers crosslinked with branched glycidyl ethers such as polyethylene glycol tetraglycidyl ether or similar polyethylene oxide crosslinking agents having three or more crosslinking groups may have crosslinking densities that vary over a wide range. In certain examples, the portion of the side chain that may have a crosslinking agent attached to the side chain may be about 0.1% or more of the available heterocycles in the film polymer, or about 0.2% or more of the available heterocycles in the film polymer, or about 0.3% or more of the available heterocycles in the film polymer, or about 0.4% or more of the available heterocycles in the film polymer, or about 0.5% or more of the available heterocycles in the film polymer, or about 0.6% or more of the available heterocycles in the film polymer, or about 0.7% or more of the available heterocycles in the film polymer, or about 0.8% or more of the available heterocycles in the film polymer, or about 0.9% or more of the available heterocycles in the film polymer, or about 1.0% or more of the available heterocycles in the film polymer, or about 1.2% or more of the available heterocycles in the film polymer, or available heterocycles in the film polymer It may be about 1.4% or more of the available heterocycles, or about 1.6% or more of the available heterocycles in the film polymer, or about 1.8% or more of the available heterocycles in the film polymer, or about 2.0% or more of the available heterocycles in the film polymer, or about 2.2% or more of the available heterocycles in the film polymer, or about 2.4% or more of the available heterocycles in the film polymer, or about 2.6% or more of the available heterocycles in the film polymer, or about 2.8% or more of the available heterocycles in the film polymer, or about 3.0% or more of the available heterocycles in the film polymer, or about 3.5% or more of the available heterocycles in the film polymer, or about 4.0% or more of the available heterocycles in the film polymer, or about 4.5% or more of the available heterocycles in the film polymer, or about 5% of the available heterocycles in the film polymer.It may be 0% or more, or about 5.5% or more of the available heterocycles in the film polymer, or about 6.0% or more of the available heterocycles in the film polymer, or about 6.5% or more of the available heterocycles in the film polymer, or about 7.0% or more of the available heterocycles in the film polymer, or about 7.5% or more of the available heterocycles in the film polymer, or about 8.0% or more of the available heterocycles in the film polymer, or about 8.5% or more of the available heterocycles in the film polymer, or about 9.0% or more of the available heterocycles in the film polymer, or about 9.5% or more of the available heterocycles in the film polymer, or about 10% or more of the available heterocycles in the heterocycle polymer. In more specific embodiments, the crosslinking agent may be added between approximately 1% and approximately 20% of the available heterocycles in the film polymer, or between approximately 2% and approximately 10%, or between approximately 3% and approximately 8%, or between approximately 4% and approximately 9%, or between approximately 5% and approximately 12%.

[0078] A suitable membrane polymer may further comprise one or more polyether arms (side chains) bonded to the nitrogen atom of a pyridine or imidazole monomer unit. Some membrane polymers disclosed herein may further comprise one or more polyether arms. Polyether arms are distinguished from crosslinking groups formed from polyethylene glycol tetraglycidyl ether or similar crosslinking agents, and polyether arms do not extend between other polymer chains or terminate within a molecule within a single polymer chain. Thus, polyether arms are separate from and distinct from crosslinking groups formed from crosslinking agents. Polyether arms may comprise polyethylene oxide blocks and polypropylene oxide blocks, and in particular, polyether arms having polypropylene oxide blocks are inserted between two polyethylene oxide blocks. Bonding between polyether arms and the nitrogen atom of a heterocycle may occur by any reactive functional group in the membrane polymer capable of forming a bond with the nitrogen atom of a heterocycle. Bonding between polyether arms and the nitrogen atom of a heterocycle may occur by alkyl groups, hydroxyl-functionalized alkyl groups, or carbonyl groups. In other specific cases, the polyether arm may also contain an amine group detached from the nitrogen atom of the heterocycle, or it may be amine-free.

[0079] The polyether arm of the membrane polymer may comprise at least one polyethylene oxide block and at least one polypropylene oxide block, thereby providing at least a diblock arrangement of monomer units of polyethylene oxide and polypropylene oxide bonded to the nitrogen atom of the heterocyclic ring by spacers. Either the polyethylene oxide block or the polypropylene oxide block may be bonded to a spacer. In other more specific embodiments, the polyether arm may comprise, in order, a spacer, a first polyethylene oxide block, a polypropylene oxide block, and a second polyethylene oxide block (i.e., an ABA repeating pattern) or, in order, a spacer, a first polypropylene oxide block, a polyethylene oxide block, and a second polypropylene oxide block (i.e., a BAB repeating pattern). Amine groups may mediate between the polyethylene oxide block and the polypropylene oxide block in the amine-containing polyether arm. Thus, the polyether arm in the membrane polymer disclosed herein may have a structure generally defined by the following formulas 10 to 13: [ka] In the formula, PE represents a polyethylene oxide block, PP represents a polypropylene oxide block, A is an amino group, and J is a spacer group. The spacer group J may be bonded to the heterocycle of the film polymer. Suitable spacer groups J may include, but are not limited to, alkyl groups, hydroxy-functionalized alkyl groups, carbonyl groups, carboxylic acid esters, and carboxamides. The variable q, r, s, and t are positive integers that determine the number of monomer units in each block and the number of repetitions of the block, and under the condition of a diblock sequence, the variable t may be 0 and the variable s may be 1. According to some embodiments, the variable q is an integer in the range of about 2 to about 50 or about 6 to about 20, the variable r is an integer in the range of about 2 to about 60 or about 10 to about 40, and the variable t is an integer in the range of about 2 to about 50 or about 10 to about 30. According to some other various embodiments, the variable s is an integer in the range between 1 and about 20 or between 1 and about 10. In some embodiments, the variable s is 1.

[0080] According to more specific embodiments of the present disclosure, an amine-free polyether arm having a triblock arrangement of polyethylene oxide, polypropylene oxide, and polyethylene oxide (similar to Formula 10) arms may have a structure defined by Formula 14. [ka] In the formula, R is an alkyl group, particularly a methyl group; variable w is 0 or 1; variable x is an integer in the range of about 4 to about 24 or about 6 to about 20; variable y is an integer in the range of about 8 to about 60 or about 10 to about 40; and variable z is an integer in the range of about 6 to about 36 or about 10 to about 30. In more specific embodiments, variable x may be in the range of about 8 to about 16 or about 9 to about 12; variable y may be in the range of about 10 to about 32, or about 16 to about 30, or about 12 to about 20; and variable z may be in the range of about 10 to about 20 or about 14 to about 18. In some embodiments, variable x may be smaller than variable z so that the second polyethylene oxide block is longer (larger) than the first polyethylene oxide block. When the variable w is 0, the amine-free polyether arm is directly bonded to the film polymer by a two-carbon alkyl group, although long alkyl groups are also considered in this disclosure.

[0081] In other specific embodiments of the present disclosure, a polyether arm (similar to Formula 12) having a triblock arrangement of polyethylene oxide, polypropylene oxide, and polyethylene oxide, and having an amine group mediating between the polyethylene oxide block and the polypropylene oxide block, may have a structure defined by Formula 15. [ka] In the formula, w, x, y, z, and R are defined in Formula 14 above. When the variable w is 0, the polyether arm is directly bonded to the film polymer by a two-carbon alkyl group, although long alkyl groups are also considered in this disclosure.

[0082] The polyether arms disclosed herein may be bonded to the nitrogen atom of the heterocycle as a reactive functional group in the polyether arm precursor. Suitable reactive functional groups may include, for example, halogens or epoxides. As shown in Formulas 14 and 15 above (n=1 in Formulas 14 and 15), for example, epoxides lead to the formation of a hydroxyalkyl spacer group that bonds the polyether arm to the nitrogen atom of the heterocycle of the film polymer. In contrast, halogen-functionalized polyether arm precursors may lead to alkyl spacers (n=0 in Formulas 14 and 15), and suitable alkyl groups may be linear or branched and may contain 2 to about 20 carbon atoms.

[0083] In some embodiments, sulfonic acid-containing arms may be added as side chains in at least a portion of the membrane polymers disclosed herein. Sulfonic acid-containing arms may be present in any suitable ratio in combination with polyether arms and / or crosslinking agents. Any membrane polymer disclosed herein may contain a larger amount of polyether arms or crosslinking groups than sulfonic acid-containing arms. Sulfonic acid-containing arms are added to the membrane polymer by alkyl groups. According to various embodiments, alkyl groups may contain carbon atoms between 1 and about 6, or carbon atoms between 2 and about 4. Suitable reagents for introducing sulfonic acid-containing arms into the membrane polymers disclosed herein may include halosulfonic acid compounds such as chloromethanesulfonic acid or bromoethanesulfonic acid, or cyclic sulfonic acids (sultones).

[0084] Polydimethylsiloxane (PDMS) may be incorporated into some of the mass transfer limiting membranes disclosed herein.

[0085] When a first active region and a second active region, configured to assay different test substances, are located on separate working electrodes, the mass transfer limiting membrane may have different permeability values ​​for the first and second test substances. The membrane thickness and / or size of the active region on each working electrode can be modified to ensure uniform sensitivity for each test substance; however, this approach can significantly complicate the fabrication of the test substance sensor. As a solution, the mass transfer limiting membrane covering at least one of the active regions may include a mixed membrane of the first and second membrane polymers or a bilayer membrane of the first and second membrane polymers. A uniform membrane may cover active regions not covered by a mixed or bilayer membrane, and the uniform membrane may contain only one of the first or second membrane polymers. Advantageously, the structure of the test substance sensor disclosed herein readily accommodates a continuous film having a uniform film portion located on a first active area of ​​the test substance sensor and a multi-component film portion located on a second active area, thereby homogenizing the permeability values ​​for each test substance and simultaneously improving sensitivity and detection accuracy. In certain embodiments, the formation of the continuous film can be carried out by a series of dip coating operations.

[0086] Embodiments disclosed herein include the following:

[0087] A. A test substance sensor capable of detecting a test substance at a low potential. The test substance sensor comprises a sensor tail portion including at least a first working electrode, and a first active region disposed on the surface of the first working electrode and responding to a first test substance at a low potential, wherein the first active region comprises a first polymer, a first redox mediator covalently bonded to the first polymer, and at least one enzyme covalently bonded to the first polymer and responding to the first test substance, wherein the first redox mediator, [ka] A test substance sensor comprising: a first active region having the structure, wherein G is a bonding group that covalently bonds the first redox mediator to the first polymer; and a mass transfer limiting membrane that is permeable to the first test substance and covers at least the first active region.

[0088] B. A method for detecting a test substance using a test substance sensor capable of low-potential detection. The sensor comprises a tail portion including at least a first working electrode, and a first active region disposed on the surface of the first working electrode and responding to the first test substance at a low potential, wherein the first active region includes a first polymer, a first redox mediator covalently bonded to the first polymer, and at least one enzyme covalently bonded to the first polymer and responding to the first test substance, and the first redox mediator is [ka] A sensor for a test substance comprising: a first active region having the structure, wherein G is a bonding group that covalently bonds the first redox mediator to the first polymer; and a mass transfer limiting membrane that is permeable to the first test substance and covers at least the first active region; and a method comprising: applying a low potential to the first working electrode; obtaining a first signal that is proportional to the concentration of the first test substance in the fluid in contact with the first active region and is greater than or equal to the oxidation-reduction potential of the first active region; and correlating the concentration of the first test substance in the fluid with the first signal.

[0089] B1. A method for detecting a test substance using a test substance sensor capable of low-potential detection. The sensor comprises a tail portion including at least a first working electrode, and a first active region disposed on the surface of the first working electrode and responding to the first test substance at a low potential, wherein the first active region includes a first polymer, a first redox mediator covalently bonded to the first polymer, and at least one enzyme covalently bonded to the first polymer and responding to the first test substance, and the first redox mediator is [ka] A method comprising: exposing a substance sensor having a structure such that G is a bonding group that covalently bonds the first redox mediator to the first polymer, a first active region, and a mass transfer limiting membrane permeable to the first substance to be tested that covers at least the first active region, to a fluid containing the first substance to be tested; applying a low potential to the first working electrode; obtaining a first signal that is proportional to the concentration of the first substance to be tested in the fluid and is greater than or equal to the redox potential of the first active region; and correlating the concentration of the first substance to be tested in the fluid with the first signal.

[0090] C. A substance sensor comprising a mass transfer limiting membrane crosslinked with a branched glycidyl ether crosslinking agent. A substance sensor comprising: a sensor tail portion comprising at least a first working electrode; a first active region disposed on the surface of the first working electrode, the first active region comprising a first polymer and at least one enzyme covalently bonded to the first polymer and responding to a first substance; and a mass transfer limiting membrane covering at least the first active region, the mass transfer limiting membrane comprising a membrane polymer crosslinked with a branched crosslinking agent comprising three or more crosslinking groups.

[0091] D. A method for detecting a test substance using a test substance sensor comprising a mass transfer limiting membrane crosslinked with a branched glycidyl ether crosslinking agent. The method provides a test substance sensor comprising: a sensor tail comprising at least a first working electrode; a first active area disposed on the surface of the first working electrode, the first active area comprising a first polymer and at least one enzyme covalently bonded to the first polymer and responding to a first test substance; and a mass transfer limiting membrane covering at least the first active area, the mass transfer limiting membrane comprising a membrane polymer crosslinked with a branched crosslinking agent comprising three or more crosslinking groups; and a method comprising: applying a potential to the first working electrode; obtaining a first signal that is proportional to the concentration of the first test substance in a fluid in contact with the first active area and is greater than or equal to the redox potential of the first active area; and correlating the concentration of the first test substance in the fluid with the first signal.

[0092] Embodiments A to D may have one or more of the following elements in any combination:

[0093] Element 1: The at least one enzyme comprises an enzyme system comprising a plurality of enzymes that respond collectively to the first test substance.

[0094] Element 2: The first test substance contains one or more ketones.

[0095] Element 3: The mass transfer limiting membrane comprises a membrane polymer crosslinked with a branched crosslinking agent containing three or more crosslinking groups.

[0096] Element 4: The film polymer comprises polyvinylpyridine or polyvinylimidazole.

[0097] Element 5: The film polymer comprises a copolymer of vinylpyridine and styrene.

[0098] Element 6: The branched crosslinking agent comprises polyethylene glycol tetraglycidyl ether.

[0099] Element 7: The test substance sensor further includes a second working electrode, a second active region disposed on the surface of the second working electrode and responding to a second test substance different from the first test substance, the second active region comprising a second polymer, a second redox mediator covalently bonded to the second polymer and different from the first redox mediator, and at least one enzyme covalently bonded to the second polymer and responding to the second test substance, and a second portion of a mass transfer limiting membrane covering the second active region.

[0100] Element 8: The at least one enzyme that responds to the second test substance comprises an enzyme system comprising a plurality of enzymes that respond collectively to the second test substance.

[0101] Element 9: The second test substance contains glucose.

[0102] Element 10: The low potential is higher than the redox potential of the first redox mediator and less than approximately -80 mV relative to the Ag / AgCl reference electrode.

[0103] Element 10A: The oxidation-reduction potential of the first redox mediator is in the range of approximately -200mV to -400mV relative to the Ag / AgCl reference electrode.

[0104] Element 11: The polyethylene glycol tetraglycidyl ether has a molecular weight in the range of about 1000 g / mol to about 5000 g / mol.

[0105] As an example without limitation, typical combinations applicable to A and B include, but are not limited to, 1 and 2; 1 through 3; 1, 3 and 6; 1 and 4; 1 and 7; 1 and 9; 1 and 10 or 10A; 2 and 3; 2, 3 and 6; 2 and 7; 2 and 9; 2 and 10 or 10A; 3 and 4; 3 and 6; 3, 4 and 6; 3, 5 and 6; 3 and 7; 3 and 9; 3 and 10 or 10A; 4 and 7; 5 and 7; 4 or 5, and 9; and 4 or 5, and 10 or 10A. As an example without limitation, typical combinations applicable to C and D include, but are not limited to, 6 and 11; 4 and 6; 5 and 6; 6 and 7; 6 through 8; 4, 6 and 11; 5, 6 and 11; 6, 7 and 11; and 6 through 8 and 11.

[0106] Further embodiments disclosed herein include:

[0107] A': A method for forming a mass transfer limiting film by dip coating. The method provides a sensor for a test substance comprising: a sensor tail including a first working electrode and a second working electrode spaced apart from each other at least along the length of the sensor tail; a first active area disposed on the surface of the first working electrode and a second active area disposed on the surface of the second working electrode, the first active area and the second active area responding to different test substances; and a method for depositing a mass transfer limiting film on the first active area and the second active area by a series of dip coating operations, wherein the mass transfer limiting film comprises a two-layer film portion covering the first active area and a uniform film portion covering the second active area.

[0108] B': A substance sensor having a dip-coated mass transfer limiting film. The substance sensor comprises a sensor tail including a first working electrode and a second working electrode spaced apart from each other at least along the length of the sensor tail; a first active area and a second active area disposed on the surface of the first working electrode and the surface of the second working electrode, the first active area and the second active area responding to different substances; and a mass transfer limiting film dip-coated on the first active area and the second active area, the dip-coated mass transfer limiting film including a dip-coated double-layer film portion covering the first active area and a dip-coated uniform film portion covering the second active area.

[0109] Embodiments A' and B' may have one or more of the following elements in any combination:

[0110] Element 1': The two-layer film portion and the uniform film portion are adjacent to each other.

[0111] Element 2': The first working electrode and the first active area are located closer to the tip of the test substance sensor than the second working electrode and the second active area.

[0112] Element 3': The upper layer of the two-layer film portion and the uniform film portion contains the same film polymer.

[0113] Element 4': The first dip coating operation deposits a first film polymer on the first active area, and the second dip coating operation deposits a second film polymer on both the first and second active areas, defining the two-layer film portion on the first active area and the uniform film portion on the second active area, wherein the first and second film polymers are different from each other.

[0114] Element 5': The lower layer of the two-layer film portion and the uniform film portion contains the same film polymer.

[0115] Element 6': The first dip coating operation deposits the first film polymer on both the first active area and the second active area, and the second dip coating operation deposits the second film polymer on the first active area, defining the two-layer film portion on the first active area, wherein the first film polymer and the second film polymer are different from each other.

[0116] Element 7': The first active region responds to glucose, lactate, ketone, or creatinine.

[0117] Element 8': The first active region responds to ketones.

[0118] Element 9': The second active region responds to glucose.

[0119] Element 10': At least a portion of the mass transfer limiting membrane comprises a homopolymer or copolymer of crosslinked polyvinylpyridine.

[0120] Element 11': At least a portion of the mass transfer limiting membrane comprises a membrane polymer crosslinked with a branched crosslinking agent containing three or more crosslinking groups.

[0121] Element 12': The branched crosslinking agent comprises polyethylene glycol tetraglycidyl ether.

[0122] Element 13': The dip-coated two-layer film portion and the dip-coated uniform film portion are adjacent to each other.

[0123] Element 14': The first working electrode and the first active area are located closer to the tip of the test substance sensor than the second working electrode and the second active area.

[0124] Element 15': The upper layer of the dip-coated bilayer film portion and the dip-coated uniform film portion contains the same film polymer.

[0125] Element 16': The lower layer of the dip-coated bilayer film portion and the dip-coated uniform film portion contains the same film polymer.

[0126] Element 17': The first active region responds to ketones.

[0127] Element 18': The second active region responds to glucose.

[0128] As an example that is not limited, typical combinations applicable to A' include, but are not limited to, 1' and 2'; 1' to 3'; 1' to 4'; 1', 2' and 5'; 1', 2', 5' and 6'; 1' and 7'; 1' and 8'; 1', 8' and 9'; 1' and 10'; 2' and 3'; 2' to 4'; 2' to 5'; 2', 5' and 6'; 2' and 7'; 2' and 8'; 2', 3 Including ', 4' and 8'; 2', 3', 4', 8' and 9'; 2', 5', 6' and 8'; 2', 5', 6', 8' and 9'; 3' and 4'; 3', 4' and 7'; 3', 4' and 8'; 3', 4', 8' and 9'; 3' and 7'; 3' and 8'; 3', 8' and 9'; 5' and 6'; 5', 6' and 8'; 5', 6', 8' and 9'; and 8' and 9'. B' Typical applicable combinations include, but are not limited to, 13' and 14'; 13', 14' and 15'; 13', 14' and 16'; 13', 14' and 17'; 13', 14', 17' and 18'; 14' and 15'; 14' and 16'; 14' and 17'; 14' and 18'; 15' and 17'; 15', 17' and 18'; 16' and 17'; 16', 17' and 18'; and 17' and 18'.

[0129] To facilitate understanding of the disclosure herein, various representative embodiments are given below. The scope of the invention should not be limited or defined by reading the following embodiments.

[0130] Examples Example 1: Detection of ketones at low potential using a test substance sensor having synergistic diaphorase and β-hydroxybutyrate dehydrogenase. In this example, the enzyme system in Figure 6A is used to facilitate the detection of ketones using either an unbound formula 8 transition metal complex (Table 1) or a formula 8 transition metal complex bound to polyvinylpyridine-co-styrene (Table 2) as a redox mediator, with the active region formulations described in Tables 1 and 2 below (HBHD = β-hydroxybutyrate dehydrogenase; HSA = human serum albumin; PEGDGE400 = polyethylene glycol diglycidyl ether). The detection active region formulations shown in Tables 1 and 2 are approximately 0.2 mm 2 The carbon-coated working electrode was coated by depositing a single spot having a certain area. The active area was cured at 25°C for 24 hours. After curing, a polyvinylpyridine film having the formulation described in Table 3 was applied to the active area by a total of four dip coatings. After the application of the film, the sensor was cured at 25°C for 24 hours, and then at 56°C for 48 hours. [Table 1] [Table 2] [Table 3]

[0131] Figure 7 shows cyclic voltammograms for the transition metal complex of formula 8 or polymer-bound form of the formula 8 transition metal complex. Cyclic voltammograms were obtained in a 100 mM PBS solution at pH 7.4, deoxygenated by nitrogen bubbling, and maintained at a temperature of 33°C. Investigations were performed from -0.5 V to 0.1 V at an investigation rate of 5 mV / s using a carbon counter electrode and an Ag / AgCl reference electrode. From the cyclic voltammograms, the E of the transition metal complex of formula 8 was determined. 1 / 2It was measured to -0.29V for Ag / AgCl, and is the E of polymer-bound type 8 transition metal complexes. 1 / 2 It was measured at -0.24V relative to Ag / AgCl. The comparative redox mediator lacking the N,N-dimethylamino substitution showed a much smaller negative E at -0.08V relative to Ag / AgCl. 1 / 2 The value was shown (no data available).

[0132] Figure 8 shows current-to-time plots of a ketone sensor containing a polymer-bound type 8 transition metal complex at various working electrode potentials. The sensor maintained one of four potentials ranging from +40 mV to -200 mV in 100 mM PBS at pH 7.4, and various amounts of β-hydroxybutyrate were titrated at 33°C until a final ketone concentration of 8 mM. As shown in the figure, the sensor reaction stabilized rapidly after the addition of the ketone at each potential. All four potentials investigated showed a linear response with respect to ketone concentration (shown in Figure 9).

[0133] Example 2: Extractables from membranes crosslinked with polyethylene glycol tetraglycidyl ether. Polymer membrane samples crosslinked with polyethylene glycol tetraglycidyl ether (molecular weight ~2500) were prepared. The base membrane polymer was a copolymer of vinylpyridine and styrene containing an amine-free polyether arm bonded to at least a portion of the pyridine moiety in the base membrane polymer. Comparative polymer membrane samples were prepared using the same membrane polymer crosslinked with polyethylene glycol diglycidyl ether (molecular weight ~1000). The comparative polymer membrane samples were crosslinked with polyethylene glycol diglycidyl ether in a similar amount to the crosslinking agent used for polyethylene glycol tetraglycidyl ether in the other samples, thereby providing a similar crosslinking density.

[0134] The membrane polymer was given the role of a film on the bottom of a sample vial using 0.5 mL of a solution containing the membrane polymer. The solvent was evaporated, and then the film was cured at 25°C for 48 hours and at 56°C for 56 hours. Water or 95% ethanol (3 mL) was added to the cast polymer film, and extraction was performed at room temperature for 72 hours (water) or 144 hours (ethanol). The vial was stirred on a rocker during the extraction period. The water extract was analyzed undiluted by UV-Vis spectrophotometric analysis (Figure 10), and the ethanol extract was diluted 1:9 before UV-Vis analysis (Figure 11). In both cases, indicated by the intensity of UV-Vis absorbance, samples crosslinked with polyethylene glycol tetraglycidyl ether showed a smaller amount of extractable material.

[0135] Example 3: Equilibrium time for sensors with membranes crosslinked with polyethylene glycol tetraglycidyl ether. Glucose-responsive test substance sensors were coated with a membrane polymer crosslinked with polyethylene glycol tetraglycidyl ether (shown in Example 2). A comparative sensor was prepared using a comparative membrane polymer crosslinked with polyethylene glycol diglycidyl ether. Each membrane-coated sensor was placed in a 30 mM glucose solution in 1000 mM PBS (pH=7.4) at 37°C. Figure 12 shows a comparative plot of sensor output versus time over 10 hours for a test substance sensor coated with a membrane polymer crosslinked with polyethylene glycol tetraglycidyl ether compared to a test substance sensor coated with a membrane polymer crosslinked with polyethylene glycol diglycidyl ether. As shown in the figure, both membranes provided stable sensor output after equilibrium of approximately 1 hour. Figure 13 shows an enlarged plot of sensor equilibrium at 1 hour, showing that the membrane crosslinked with polyethylene glycol tetraglycidyl ether provided a faster and more stable response than the membrane crosslinked with polyethylene glycol diglycidyl ether.

[0136] Unless otherwise indicated, all figures representing quantities, etc., in this specification and related claims are understood to be modified in all cases by the term “approximately.” Therefore, unless otherwise indicated, the numerical parameters described in the following specification and appended claims are approximations that may vary depending on the desired properties to be obtained by embodiments of the invention. Each numerical parameter should be interpreted by applying common rounding techniques, taking into account at least the number of significant figures reported, and not in any attempt to limit the application of the principle of equivalence to the scope of the claims.

[0137] One or more exemplary embodiments incorporating various features are presented herein. Not all features of a physical implementation are described or shown herein for clarity. It is understood that in developing a physical implementation incorporating an embodiment of the present invention, a number of implementation-specific decisions must be made to achieve the developer's objectives, such as compliance with system, business, government, and other constraints, and these will vary by implementation and by circumstances. While the developer's efforts may be time-consuming, such efforts are still routine for those skilled in the art and will be of interest to this disclosure.

[0138] While various systems, tools, and methods are described herein in terms of "equipping" various components or processes, systems, tools, and methods can also be described as "essentially consisting of" or "made of" various components or processes.

[0139] As used here, the phrase "at least one of" with the terms "and" or "or" that precede a series of items and separate some of the items modifies the entire list rather than each member of the list (i.e., each item). The phrase "at least one of" allows meanings including at least one of any one of the items, and / or at least one of any combination of the items, and / or at least one of each of the items. For example, the phrases "at least one of A, B, and C" or "at least one of A, B, or C" both indicate A only, B only, or C only; any combination of A, B, and C; and / or at least one of each of A, B, and C.

[0140] Accordingly, the disclosed systems, tools, and methods are fully applicable to achieve the purposes and benefits mentioned, as well as those inherent. The teachings of this disclosure may be modified and implemented in different but equivalent ways, which will be obvious to those skilled in the art who are interested in the teachings of this specification, and the specific embodiments disclosed above are merely examples. Furthermore, except as described in the following claims, no limitation is intended on the details of the configurations or designs shown herein. Accordingly, the specific embodiments disclosed above may be changed, combined, or modified, and it is obvious that all such changes will be considered within the scope of this disclosure. The systems, tools, and methods disclosed exemplary herein may be adequately implemented without any elements not specifically disclosed herein and / or without any elements disclosed herein. While systems, tools, and methods are described in terms of "equipping," "containing," or "including" various components or processes, systems, tools, and methods can also be "essentially made of" or "consisting of" various components or processes. All the numbers and scopes disclosed above may vary in some way. Whenever a numerical range with lower and upper limits is disclosed, any number that falls within that range and any range that it includes are specifically disclosed. In particular, all ranges of values ​​disclosed herein (in the form of "about a to about b," or equivalently "about a to b," or equivalently "about a to b") are understood to include all numbers and ranges that fall within a broader range of values. Furthermore, in the claims, terms have plain and ordinary meanings unless explicitly and clearly defined by the patentee. Moreover, it is hereby defined that the indefinite article "a" or "an" used in the claims means one or more elements than one of the elements it introduces. If there are any inconsistencies in the use of a word or term between this specification and one or more patent documents or other documents incorporated herein by reference, the definition consistent with this specification should be adopted.

Claims

1. An electrochemical test substance sensor for detecting at least one first test substance in a living organism, (a) Ag / AgCl reference electrode, (b) First working electrode and (c) A first active region disposed on the surface of the first working electrode and responding to the first test substance, wherein the first active region comprises a first polymer, a first redox mediator covalently bonded to the first polymer, and at least one enzyme that responds to the first test substance, the first test substance being a ketone, (d) comprising a mass transfer limiting membrane that covers at least the first active area and is permeable to the first test substance, The first redox mediator described above has the following structure: 【Chemistry 1】 During the ceremony, G is a bonding group that covalently bonds the first redox mediator to the first polymer. D is a hydroxyl group, alkoxy group, amino group, alkyl group, or dialkylamino group. M is osmium, ruthenium, vanadium, cobalt, or iron. The substance sensor is a substance sensor that can be partially inserted into the user's skin so that the substance sensor comes into contact with interstitial fluid in order to detect the first substance in the living body.

2. The sensor for testing a substance according to claim 1, wherein D is a dialkylamino group.

3. The aforementioned dialkylamino group is a group consisting of a dimethylamino group and a diethylamino group. A sensor for testing a substance, selected from the following, according to claim 2.

4. The sensor for testing a substance, according to claim 3, wherein the dialkylamino group is a dimethylamino group.

5. The test substance sensor according to claim 1, wherein the at least one enzyme comprises an enzyme system comprising a plurality of enzymes that cooperatively respond to ketones.

6. The test substance sensor according to claim 5, wherein the enzyme system comprises β-hydroxybutyrate dehydrogenase and diaphorase.

7. The substance sensor according to claim 1, wherein the substance transfer limiting membrane comprises a membrane polymer crosslinked with a branched crosslinking agent containing three or more crosslinking groups.

8. The sensor for testing a substance according to claim 7, wherein the membrane polymer comprises polyvinylpyridine or polyvinylimidazole.

9. The sensor for testing a substance according to claim 8, wherein the branched crosslinking agent comprises polyethylene glycol tetraglycidyl ether.

10. (e) Second working electrode and (f) The test substance sensor according to claim 6, further comprising: (f) a second active region disposed on the surface of the second working electrode and responding to a second test substance different from the first test substance.

11. The test substance sensor according to claim 10, wherein the second active region comprises a second polymer, an enzyme that responds to the second test substance, and a second redox mediator different from the first redox mediator.

12. The sensor for testing a substance according to claim 11, wherein the second redox mediator is covalently bonded to the second polymer.

13. The test substance sensor according to claim 12, wherein the second test substance is glucose.

14. The substance sensor for testing according to claim 10, wherein the substance transfer limiting film is a two-layer film.

15. The sensor for testing a substance according to claim 14, wherein the two-layer film includes an inner layer covering the first active area and an outer layer covering the second active area.

16. The sensor for testing a substance according to claim 1, wherein the first active region responds to one or more ketones at an operating electrode potential of -400 mV to -80 mV relative to an Ag / AgCl reference potential.

17. A method for controlling a test substance sensor according to any one of claims 1 to 16, which has already been introduced into an organization, Applying the low potential to the first working electrode, To obtain a first signal that is proportional to the ketone concentration in the fluid in contact with the first active region, at a redox potential above that of the first active region, The first signal is correlated with the ketone concentration in the fluid. A method for controlling a sensor for a substance to be tested, comprising the above components.

18. (i) A test substance sensor according to any one of claims 1 to 16, the test substance sensor which acquires a first signal indicating the concentration of the first test substance in the user's interstitial fluid and transmits the signal indicating the concentration of the first test substance to a processor, (ii) The processor for determining the concentration of the first test substance based on the signal and A sensor control device equipped with the following features.

19. (i) A test substance sensor according to any one of claims 1 to 16, the test substance sensor which acquires a first signal indicating the concentration of the first test substance in the user's interstitial fluid and transmits the signal indicating the concentration of the first test substance to a processor, (ii) The processor determines the concentration of the first test substance based on the signal and transmits the concentration of the first test substance to a reader so as to be displayed. A monitoring system for the substance being tested, equipped with the following features.

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