A sensor for a test substance equipped with one or more detection enhancements.

Abstract

To address such a problem that analyte sensors are being increasingly employed for monitoring various analytes in vivo, analyte sensors configured to monitor a plurality of analytes are also in development, however, sufficient sensitivity for low-abundance analytes and multiple analytes having differing membrane permeability values may complicate analyte detection in some cases.SOLUTION: Analyte sensors may feature enhancements to address one or both of these issues. Some analyte sensors may comprise a carbon working electrode comprising a dielectric substrate, one or more apertures extending through the dielectric substrate and filled with a carbon conductor pillar, a carbon conductor coating on a first face of the dielectric substrate in direct contact with each carbon conductor pillar, and one or more active areas on a second face of the dielectric substrate in electrical communication with the carbon conductor pillars. Photopolymerized mass transport limiting membranes may be used in combination with such carbon working electrodes or with other working electrode types.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This invention relates to a sensor for detecting a substance. [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, for example, can be particularly important for detection and monitoring in individuals with diabetes. By monitoring glucose levels with sufficient regularity, individuals with diabetes can take corrective action (e.g., by injecting insulin to lower glucose levels or by eating to raise glucose levels) before significant physiological problems occur. Monitoring other test substances may be desirable for various other physiological conditions. In some cases, monitoring multiple test substances may be desirable, particularly for comorbid conditions that result in simultaneous dysregulation of two or more test substances in combination.

[0003] When appropriate detection chemicals can be identified, many test substances become interesting targets for physiological analysis. For this purpose, in vivo test substance sensors configured to assay various physiological test substances have been developed and improved in recent years, many of which utilize enzyme-based detection strategies to improve detection specificity. In fact, in vivo test substance sensors that utilize glucose-responsive enzymes to monitor blood glucose levels are now commonly used among individuals with diabetes. In vivo test substance sensors for other test substances, including in vivo test substance sensors that can monitor multiple test substances, are in various stages of development. Low sensitivity to small amounts of test substance can be a particular problem for some test substance sensors, especially due to excessive background signals resulting from the interaction of interfering substances with the carbon working electrode.

[0004] To improve biocompatibility, in vivo spectroscopy sensors may include a membrane placed over the embedded portion of the sensor, particularly a membrane covering at least the active region(s) of the spectroscopy sensor. In addition to promoting biocompatibility, the membrane may be permeable or semipermeable to the spectroscopy of interest and restrict the total spectroscopy flux to the active region(s) of the spectroscopy sensor. Such a mass transfer-restricting membrane can help avoid overloading (saturation) of the detected component within the active region(s), thereby improving sensor performance and accuracy. By restricting the mass transfer of the spectroscopy, the chemical reaction kinetics of the detection process can be made spectroscopy-restricted rather than enzyme-restricted, thereby allowing the sensor output to be easily correlated with the amount of spectroscopy present.

[0005] Another problem associated with spectrometry monitoring is that different spectrometry substances may exhibit different permeability to a given mass transfer limiting membrane. The most common mass transfer limiting membranes used in in vivo spectrometry sensors are polyvinylpyridine or polyvinylimidazole polymers and copolymers, which can be readily applied to the working electrode by dip-coating techniques. Polyurethane is also commonly used to form mass transfer limiting membranes. Within this limited range of polymers, identifying the appropriate membrane chemistry to promote sufficient permeability for a particular spectrometry substance can sometimes be challenging. The problem of different spectrometry permeability can be further complicated in spectrometry sensors configured to assay multiple spectrometry substances, where compositionally different mass transfer limiting membranes may be required on different portions of the sensor tail to provide sufficient permeability and / or a stable response for each spectrometry substance. Different membrane permeability values ​​can result in very different response sensitivities between two spectrometry substances, which can complicate detection. While dip-coating processes for applying mass transfer limiting membranes with compositionally different portions onto the spectrometry sensor tail are feasible, they can significantly increase manufacturing complexity. Furthermore, membrane chemicals other than those based on polyvinylpyridine or polyvinylimidazole may not be readily extendable to dip-coating processes. While different sensitivities to multiple test substances can sometimes be partially compensated for by using active regions of different sizes (e.g., smaller active regions for highly sensitive / permeable test substances, and larger active regions for less sensitive / permeable test substances), this approach can present significant manufacturing challenges and is not always applicable. Therefore, a lack of available compositions for forming mass transfer-limiting membranes can complicate the detection of specific test substances. [Brief explanation of the drawing]

[0006] The following figures are included to illustrate specific aspects of the disclosure and should not be considered exclusive embodiments. The disclosed subject matter can be modified, altered, combined, and equivalent in form and function without departing from the scope of the disclosure.

[0007] [Figure 1] A schematic diagram of an exemplary detection system into which the substance sensor of this disclosure may be incorporated is shown. [Figure 2A] This shows a cross-sectional view of a test substance sensor with a single active region. [Figure 2B] This shows a cross-sectional view of a test substance sensor with a single active region. [Figure 2C] This shows a cross-sectional view of a test substance sensor with a single active region. [Figure 3A] A cross-sectional view of a test substance sensor with two active regions is shown. [Figure 3B] A cross-sectional view of a test substance sensor with two active regions is shown. [Figure 3C] A cross-sectional view of a test substance sensor with two active regions is shown. [Figure 4] This diagram shows a cross-sectional view of a test substance sensor equipped with two working electrodes, each having an active region on which it is located. [Figure 5] This is a schematic diagram showing a top view of a conventional carbon working electrode with an active region at the top. [Figure 6A] A cross-sectional view of a first configuration of a carbon working electrode suitable for use in the substance sensor of this disclosure is shown. [Figure 6B] The corresponding top view is shown. [Figure 7] A schematic diagram of an exemplary process in which a carbon working electrode can be fabricated within a dielectric substrate is shown. [Figure 8A] A cross-sectional view of a second configuration of a carbon working electrode suitable for use in the substance sensor of this disclosure is shown. [Figure 8B] The corresponding top view is shown. [Figure 9A] A cross-sectional view of a third configuration of a carbon working electrode suitable for use in the substance sensor of this disclosure is shown. [Figure 9B]Shows the corresponding top view. [Figure 10A] Shows an enzyme system configured to detect glucose. [Figure 10B] Shows an enzyme system configured to detect glucose. [Figure 10C] Shows an enzyme system configured to detect glucose. [Figure 11A] Shows an enzyme system configured to detect ketone. [Figure 11B] Shows an enzyme system configured to detect ketone. [Figure 11C] Shows an enzyme system configured to detect ketone. [Figure 12] Shows an enzyme system configured to detect creatinine. [Figure 13] Shows plots of the sensor responses of several glucose sensors covered with a mass transfer limiting membrane formed from various ratios of HEMA:POMA. [Figure 14] Shows the corresponding plots of the sensor responses as a function of glucose concentration. [Figure 15A] Shows plots of the sensor responses of several ketone sensors covered with a mass transfer limiting membrane formed from various ratios of HEMA:POMA. [Figure 15B] Shows plots of the sensor responses of several ketone sensors covered with a mass transfer limiting membrane formed from various ratios of HEMA:POMA. [Figure 16A] Shows the corresponding plots of the sensor responses as a function of ketone concentration. [Figure 16B] Shows the corresponding plots of the sensor responses as a function of ketone concentration. [Figure 17] Shows plots of the sensor responses of several ketone sensors covered with a mass transfer limiting membrane formed from various ratios of thiol-ene polymer. [Figure 18] Shows the corresponding plots of the sensor responses as a function of ketone concentration.

MODE FOR CARRYING OUT THE INVENTION

[0008] This disclosure generally describes a test substance sensor suitable for in vivo use, and more specifically, a test substance sensor characterized by one or more enhancements to facilitate improved detection sensitivity, as well as methods for manufacturing and using the same. Such enhancements may include reducing the amount of foreign carbon on the surface of the carbon working electrode and / or expanding the range of membrane chemicals available for the deposition of a mass transfer limiting film. In particular, the range of chemicals available for the mass transfer limiting film may be expanded through in-situ polymerization reactions such as photopolymerization to provide selective film deposition on the active region(s) of the working electrode. The selectivity provided by in-situ photopolymerization may allow mass transfer limiting films with different compositions to be deposited on selected regions of the working electrode, which may be particularly beneficial when assaying multiple test substances using a single test substance sensor capable of detecting multiple test substances. Specific details and further advantages of each type of enhancement are described in more detail herein. Depending on the specific needs, the test substance sensors of this disclosure may be configured to detect one or more test substances simultaneously or nearly simultaneously.

[0009] Enzyme-based detection sensors are commonly used to assay single spectrometry substances, such as glucose, due to the frequent specificity of enzymes to specific substrates or classes of substrates. For this purpose, spectrometry sensors employing both single enzymes and enzyme systems comprising multiple enzymes acting in coordination may be used. As used herein, the term “coordinated” refers to a conjugated enzyme reaction in which the product of a first enzymatic reaction becomes a substrate for a second enzymatic reaction, and the second or subsequent enzymatic reaction serves as the basis for measuring the concentration of the spectrometry substance. The use of in vivo spectrometry sensors featuring enzymes or enzyme systems to facilitate detection may be particularly advantageous in avoiding the frequent withdrawal of body fluids that may otherwise be required to perform spectrometry monitoring.

[0010] While various active regions are currently known that are suitable for assaying a wide range of test substances, there are sometimes difficulties associated with detecting specific test substances or combinations of test substances. For example, sensor sensitivity may be insufficient for small amounts of test substances. Carbon working electrodes can provide relatively high background signals that can complicate the accurate detection of some small amounts of test substances. Significantly different membrane permeability values ​​can also be a problem when assaying multiple test substances. This disclosure provides a test substance sensor enhancement that can improve the detection sensitivity for both single test substances and multiple test substances combined, either alone or in combination, as will be described in more detail below. Specifically, this disclosure provides a test substance sensor having a carbon working electrode that can reduce background signals and expand the range of suitable membrane chemicals that can be selectively deposited on the test substance sensor. While certain aspects of this disclosure focus on the enhancement of carbon working electrodes, it should be understood that other types of electrodes can be similarly enhanced according to the disclosures herein. Types of electrodes that can be enhanced through the use of the disclosures herein also include gold, platinum, and PEDOT, etc.

[0011] Before further detailing the test substance sensors and their enhancements in this disclosure, a brief overview of the configuration of a suitable in vivo test substance sensor and a sensor system using the test substance sensor is first provided so that embodiments of this disclosure may be better understood. Figure 1 shows a schematic diagram of an exemplary detection system into which the test substance sensor of this disclosure may be incorporated. As shown, the detection system 100 includes a sensor control device 102 and a reading device 120 configured to communicate with each other via a local communication path or link 140, which may be wired or wireless, unidirectional or bidirectional, and encrypted or unencrypted. According to some embodiments, the reading device 120 may constitute an output medium for viewing the test substance concentration and warnings or notifications determined by the sensor 104 or its associated processor, and for enabling one or more user inputs. The reading device 120 may be a multipurpose smartphone or a dedicated electronic reading device. Although only one reading device 120 is shown, multiple reading devices 120 may be present in specific examples. The reading device 120 can also communicate with the remote terminal 170 and / or the trusted computer system 180 via communication paths / links 141 and / or 142, respectively, which may also be wired or wireless, unidirectional or bidirectional, and encrypted or unencrypted. The reading device 120 can also, or instead, communicate with the network 150 (e.g., a cellular network, the internet, or a cloud server) via communication path / link 151. The network 150 may further be communicably connected to the remote terminal 170 via communication path / link 152 and / or the trusted computer system 180 via communication path / link 153. Alternatively, the sensor 104 can communicate directly with the remote terminal 170 and / or the trusted computer system 180 without the presence of the intervening reading device 120. For example, according to some embodiments, sensor 104 may communicate with a remote terminal 170 and / or a trusted computer system 180 via a direct communication link to network 150, as described in U.S. Patent Application Publication No. 2011 / 0213225, which is incorporated herein by reference in its entirety.Any suitable electronic communication protocol may be used for each of the communication paths or links, such as Near Field Communication (NFC), Radio Frequency Identification (RFID), Bluetooth® or Bluetooth® Low Energy Protocol, or Wi-Fi. The remote terminal 170 and / or trusted computer system 180 may, according to some embodiments, be accessible by individuals other than the primary user who are interested in the user's test substance levels. The reading device 120 may comprise a display unit 122 and an optional input component 121. The display unit 122 may, according to some embodiments, comprise a touchscreen interface.

[0012] The sensor control device 102 includes a sensor housing 103 capable of housing a circuit and power supply for operating the sensor 104. Optionally, the power supply and / or active circuit may be omitted. A processor (not shown) may be communicatively connected to the sensor 104, and the processor is physically located within the sensor housing 103 or the reading device 120. The sensor 104 protrudes from the underside of the sensor housing 103 and penetrates an adhesive layer 105, which, according to some embodiments, is adapted to adhere the sensor housing 103 to a skin-like tissue surface.

[0013] Sensor 104 is adapted to be at least partially inserted into the target tissue, such as within the dermis or subcutaneous layer of the skin. Alternatively, sensor 104 may be adapted to penetrate the epidermis. Alternatively, sensor 104 may be placed on a surface and not penetrate the tissue, such as when assaying one or more test substances in sweat on the skin. Sensor 104 may have a sensor tail of sufficient length to be inserted to a desired depth in a given tissue. The sensor tail may comprise at least one working electrode and an active region comprising an enzyme or enzyme system configured to assay one or more test substances of interest. Suitable enzymes and enzyme systems are discussed in more detail herein. A counter electrode may be present in combination with at least one working electrode, and optionally in combination with a reference electrode. Specific electrode configurations on the sensor tail are described in more detail below with reference to Figures 2A to 4. According to various embodiments, one or more enzymes within the active region(s) may be covalently bonded to a polymer comprising the active region(s). Alternatively, the enzyme may be non-covalently associated within the active region(s) by encapsulation or physical entrainment, etc. One or more test substances may be monitored in any bodily fluid of the subject, 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 sensor of this disclosure may be adapted to assay skin fluid or interstitial fluid to determine the concentration of the test substance in vivo.

[0014] One or more mass transfer limiting films may cover an active region(s). When different types of active regions are present, the mass transfer limiting films may be compositionally identical or compositionally different in each type of active region. To facilitate the detection of multiple spectroscopy substances, sensor architectures are available for incorporating different mass transfer limiting films on each type of active region via dip-coating techniques, as needed. Different film thicknesses or film architectures may also be used to facilitate the detection of multiple spectroscopy substances. As further described herein, mass transfer limiting films having compositional variations in one or more active regions may also be prepared by in-situ polymerization, such as in-situ photopolymerization. Living polymerization techniques may also be used to form mass transfer limiting films on active regions. A simpler sensor architecture may be used if a single mass transfer limiting film provides sufficient permeability to both spectroscopy substances detectable by a multi-spectroscopy detector. In-situ photopolymerization can also extend the range of suitable film chemicals beyond those suitable only for deposition by dip-coating techniques. In some cases, films of better quality can also be formed compared to dip-coating techniques.

[0015] Referring again to Figure 1, the sensor 104 may automatically transfer data to the reading device 120. For example, the concentration data of a test substance may be automatically and periodically communicated at a specific frequency when the data is acquired, or after a specific period of time has elapsed, and stored in memory until transmitted (e.g., every minute, every five minutes, or at other predetermined intervals). Data associated with different test substances may be transferred at the same or different frequencies and / or using the same or different communication protocols. In other embodiments, the sensor 104 may communicate with the reading device 120 non-automatically 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 within the communication range of the reading device 120. The data may remain stored in the sensor 104's memory until it is communicated to the reading device 120. Thus, the user does not need to maintain constant proximity to the reading device 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 implemented. For example, data transfer may continue automatically until the reading device 120 is out of the communication range of the sensor 104.

[0016] To facilitate the introduction of the sensor 104 into the tissue, an introducer may be temporarily present. In exemplary embodiments, the introducer may comprise a needle or a similar sharp object, or a combination thereof. It should be noted 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 temporarily present near the sensor 104 before tissue insertion 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 epidermis as an access path to the dermis, allowing the implantation of the sensor 104 to be carried out. After opening the access path, the needle or other introducer may be withdrawn so as not to pose a sharp hazard. In exemplary embodiments, a suitable needle may be solid or hollow, beveled or non-beveled, and / or have a circular or non-circular 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. For example, needles with cross-sectional diameters ranging from approximately 300 microns (300 μm) to approximately 400 microns (400 μm) may be used.

[0017] In some embodiments, the tip of the needle (while present) may be angled across 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 within the lumen or groove of the needle, and the needle similarly opens an access path for the sensor 104. In any case, the needle may be withdrawn after facilitating sensor insertion.

[0018] Sensor configurations featuring a single active region configured for the detection of a single corresponding test substance may use a two-electrode or three-electrode detection motif, as further described herein with reference to Figures 2A to 2C. Sensor configurations featuring two different active regions for the detection of a different test substance, either on a different working electrode or on the same working electrode, are described separately later with reference to Figures 3A to 4. Sensor configurations having multiple working electrodes may be particularly advantageous for incorporating two different active regions within the same sensor tail, as the signal contribution from each active region can be more easily determined through the separate responses of each working electrode. Each active region may be covered with a mass transfer limiting film, which may be introduced by dip coating or generated by in situ photopolymerization, as described in the further disclosures below.

[0019] When a single working electrode is present within the sensor for the substance being tested, a three-electrode sensor configuration may comprise a working electrode, a counter electrode, and a reference electrode. A related two-electrode sensor configuration may comprise 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 (layered) with respect to each other and / or spaced laterally apart from each other on the sensor tail. In any of the sensor configurations disclosed herein, the various electrodes may be electrically insulated from each other by a dielectric material or similar insulator.

[0020] A sensor for a substance being tested, characterized by multiple working electrodes, may similarly have at least one additional electrode. When one additional electrode is present, it may function as a pair / reference electrode for each of the multiple working electrodes. When 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.

[0021] Figure 2A shows a schematic diagram of an exemplary two-electrode substance sensor configuration suitable for use in the disclosure herein. As shown, the substance sensor 200 comprises 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 with a dielectric material in between (configuration not shown). An active region 218 is positioned as at least one layer on at least a portion of the working electrode 214. The active region 218 may comprise a plurality of spots or a single spot configured for the detection of the substance, as will be discussed further herein.

[0022] Referring further to Figure 2A, according to some embodiments, the membrane 220 may cover at least the active region 218 and optionally some or all of the working electrode 214 and / or the pair / reference electrode 216, or the entire test substance sensor 200. One or both sides of the test substance sensor 200 may be covered by the membrane 220. The membrane 220 may comprise one or more polymer membrane materials having the ability to restrict the flux of the test substance to the active region 218 (i.e., the membrane 220 is a mass transfer restricting membrane having some permeability to the test substance of interest). The composition and thickness of the membrane 220 may be varied to promote the desired flux of the test substance to the active region 218, thereby providing the desired signal intensity and stability. The test substance sensor 200 may be operable to assay the test substance by any of the electrochemical detection techniques of coulometry, amperometry, voltammetry, or potentiometry.

[0023] 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 those shown as substance sensor 200 in Figure 2A, except that they include an additional electrode 217 within substance sensors 201 and 202 (Figures 2B and 2C). With the additional electrode 217, the pair / reference electrode 216 may then function as either the pair electrode or the reference electrode, and the additional electrode 217 may perform other electrode functions not otherwise described. The working electrode 214 continues to perform its original function. The additional electrode 217 may be placed on either the working electrode 214 or electrode 216 with a dielectric material isolation layer in between. For example, as shown in Figure 2B, dielectric layers 219a, 219b and 219c isolate electrodes 214, 216 and 217 from each other and provide electrical insulation. Alternatively, at least one of electrodes 214, 216, and 217 may be positioned on the opposite side of the substrate 212, as shown in Figure 2C. Thus, in some embodiments, electrodes 214 (working electrode) and 216 (counter electrode) may be positioned on the opposite side of the substrate 212, and electrode 217 (reference electrode) may be positioned on one of electrodes 214 or 216 and separated therefrom by a dielectric material. A reference material layer 230 (e.g., Ag / AgCl) may be present on electrode 217, and the position of the reference material layer 230 is not limited to the locations shown in Figures 2B and 2C. Similar to the sensor 200 shown in Figure 2A, the active region 218 in the substance-of-test sensors 201 and 202 may comprise multiple spots or a single spot. In addition, the test substance sensors 201 and 202 may similarly be capable of assaying the test substance by any of the following electrochemical detection techniques: coulometry, amperometry, voltammetry, or potentiometry.

[0024] Similar to the substance sensor 200, the film 220 may also cover the active region 218 and other sensor components in the substance sensors 201 and 202, thereby functioning as a mass transfer limiting film. An additional electrode 217 may be covered by the film 220 in some embodiments. The film 220 may further be manufactured by dip coating or in situ photopolymerization, and its composition may vary at different locations. Figures 2B and 2C show electrodes 214, 216, and 217 all covered by the film 220, but it should be noted that in some embodiments, only the working electrode 214 or the active region 218 may be covered. Furthermore, the thickness of the film 220 on each of electrodes 214, 216, and 217 may be the same or different. 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 film 220 in the sensor configurations of Figures 2B and 2C, or the entire substance sensors 201 and 202 may be covered. Accordingly, the three-electrode sensor configurations shown in Figures 2B and 2C should be understood as not limiting the embodiments disclosed herein, and alternative electrode and / or layer configurations remain within the scope of this disclosure.

[0025] Figure 3A shows an exemplary configuration of a sensor 203 having a single working electrode on which two different active regions are positioned. Figure 3A is similar to Figure 2A except that there are two active regions on the working electrode 214, namely a first active region 218a and a second active region 218b, which 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 comprise multiple spots or a single spot configured for the detection of each test substance. The composition of the film 220 may vary in the active regions 218a and 218b, or may be compositionally the same. The first active region 218a and the second active region 218b may be configured to detect their corresponding test substances at different working electrode potentials, as will be discussed further below.

[0026] Figures 3B and 3C show cross-sectional views of exemplary three-electrode sensor configurations for sensors 204 and 205, respectively, each featuring a single working electrode having a first active region 218a and a second active region 218b positioned on top. Figures 3B and 3C are otherwise similar to Figures 2B and 2C and can be better understood by referring to them. As with Figure 3A, the composition of the film 220 may vary in the active regions 218a and 218b, or may be compositionally the same.

[0027] An exemplary sensor configuration having multiple working electrodes, specifically two working electrodes, is described in more detail with reference to Figure 4. While the following description primarily concerns a sensor configuration with two working electrodes, it should be understood that, with extensions of the disclosure herein, more than two working electrodes may be incorporated. Additional working electrodes may be used to impart additional detection capabilities to the test substance sensor, not only for the first and second test substances, but also for other test substances. That is, a test substance sensor containing more than two working electrodes may be suitable for detecting a corresponding number of additional test substances.

[0028] 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, the substance sensor 300 includes working electrodes 304 and 306 located on opposite sides of the substrate 302. A first active region 310a is located on the surface of the working electrode 304, and a second active region 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 regions 310a and 310b, and other components of the substance sensor 300 or the entire substance sensor 300 may also be optionally covered by the film 340. Furthermore, the membrane 340 may have its composition changed in the active regions 310a and 310b as needed, providing appropriate permeability values ​​to individually adjust the flux of the test substance at each location.

[0029] Alternative sensor configurations having multiple working electrodes, different from the configuration shown in Figure 4, may feature different pair / reference electrodes instead of another pair electrode and reference electrodes 320, 321, and / or may feature different layer and / or film arrangements than those explicitly shown. For example, the positions of the pair electrode 320 and reference electrode 321 may be opposite to those shown in Figure 4. Furthermore, the working electrodes 304 and 306 do not necessarily have to be located on the opposite side of the substrate 302 as shown in Figure 4.

[0030] A carbon working electrode can appropriately comprise one working electrode(s) in any of the test substance sensors disclosed herein. Although carbon working electrodes are very commonly used in electrochemical detection, their use in electrochemical detection is not without its challenges. In particular, a current related to the target test substance is generated only when the active region interacts with the test substance, transferring electrons to the portion of the carbon working electrode adjacent to the active region. The fluid containing the target test substance also interacts with the carbon surface of the carbon working electrode that is not covered by the active region, and does not contribute to the test substance signal because there are no enzymes or enzymatic systems at these locations that facilitate electron transfer from the test substance to the working electrode. However, interfering substances can be oxidized in the portion of the working electrode lacking the active region, contributing to background for the overall signal. Ascorbic acid is an example of an interfering substance commonly found in bodily fluids that can generate background signals at carbon working electrodes. Therefore, carbon working electrodes with foreign carbon regions on the electrode surface do not significantly contribute to the test substance signal and may, in some cases, result in excessive background. Other electrodes with excessive surface area that do not directly detect the target substance may experience similar problems and can be enhanced through improvements to the disclosures herein.

[0031] Figure 5 is a schematic diagram showing a top view of a conventional carbon working electrode 412 having active regions 418 arranged as multiple spots on top. When a test substance interacts with the active regions 418, only the portion of the carbon working electrode 412 below the active regions 418 contributes to the signal related to the test substance. Exotic carbon regions 410 are not directly covered by the active regions 418 and do not contribute to the signal related to the test substance, but may generate background signals related to one or more interfering substances.

[0032] This disclosure shows how the foreign carbon region can be reduced in a carbon working electrode while still retaining functionality for generating a signal associated with the substance under investigation. In particular, this disclosure provides a substance sensor comprising an improved carbon working electrode at least partially formed in a dielectric substrate such as a polymer block, where the dielectric substrate replaces most or all of the foreign carbon region on the electrode surface not covered by the active region. More specifically, the carbon working electrodes described herein can be obtained by filling one or more openings with a carbon conductor, as described in more detail below. Alternative electrodes comprising metal or PEDOT can similarly be formed in a dielectric substrate according to the disclosure herein.

[0033] Figure 6A shows a cross-sectional view of a first configuration of a carbon working electrode suitable for use in a substance sensor of the present disclosure. As shown in Figure 6A, the carbon working electrode 500 includes a dielectric substrate 510 having carbon conductor pillars 512 that penetrate the dielectric substrate 510 and extend between a first surface 514 and a second surface 516. The carbon conductor pillars 512 can be formed by filling vias or similar openings with carbon conductor ink and subsequently curing, as will be described in more detail below with reference to Figure 7. A carbon conductor coating 520 is placed on the first surface 514 and in direct contact with each of the carbon conductor pillars 512, thereby establishing electrical conductivity with each of the carbon conductor pillars 512. A dielectric coating 522 is placed on the carbon conductor coating 520. Thus, the only carbon surface available for electron transfer is located on the second surface 516 through which the carbon conductor pillars 512 penetrate the dielectric substrate 510. The active region 530 is located on a second surface 516 that is in direct contact with the carbon conductor pillar 512.

[0034] Substitutes for carbon working electrodes can be formed by depositing similar conductive inks in openings penetrating the dielectric substrate 510. For example, metal-containing inks can be similarly deposited and cured to provide metal conductor pillars. Metal conductor coatings may interconnect the metal conductor pillars and may also be similarly formed from metal-containing inks. Thus, any electrode structure described herein in relation to carbon working electrodes can similarly be formed using alternative conductive materials.

[0035] Figure 6B shows the corresponding top view of the carbon working electrode in Figure 6A. As shown, the active region 530 covers the exposed surface of the carbon conductor pillar 512 (not visible in the shown figure), thereby leaving very little foreign carbon region available to generate an electrochemical signal when interacting with interfering material. While Figures 6A and 6B show a complete overlay of the active region 530 on the carbon conductor pillar 512, it should be understood that the active region 530 may also be slightly offset from the carbon conductor pillar 512 and / or not completely cover the carbon conductor pillar 512, thereby leaving a small amount of foreign carbon region that can be exposed to interfering material. Nevertheless, it should be understood that the amount of foreign carbon present in the carbon working electrode 500 is far less than the amount in a conventional carbon working electrode having a surface substantially formed of carbon conductors. It should also be understood that the number and spacing of the carbon conductor pillars 512 and active region 530 are illustrative and non-limiting. In exemplary embodiments, the number of openings (vias) may range from about 1 to about 20, or from about 3 to about 15, or from about 2 to about 10, and the diameter of the openings may range from about 25 μm to about 200 μm. Non-circular openings may also be present. A corresponding number of active regions may be present on the carbon conductor pillars, or the number of active regions may be less than the number of carbon conductor pillars. If fewer than all of the carbon conductor pillars are covered by the active regions, some foreign carbon remains present for potential exposure to interfering materials.

[0036] Figure 7 shows a schematic diagram of an exemplary process in which carbon working electrodes can be manufactured within a dielectric substrate. First, the dielectric substrate 510 is provided with a number of vias 511 penetrating it. Numerous methods exist for introducing the vias 511 into the dielectric substrate 510, such as perforation, laser perforation, or similar techniques well known to those skilled in the art. The carbon conductive ink is then deposited by screen printing or similar deposition techniques to form a carbon conductive coating 520 on a first surface 514. The vias 511 can be at least partially filled during this process. If necessary, completion of the filling of the vias 511 with carbon conductive ink to provide carbon conductive pillars 512 after the ink dries can be performed from a second surface 516. Suitable carbon conductive inks are well known to those skilled in the art.

[0037] Next, the dielectric coating 522 is deposited on the carbon conductor coating 520, leaving the carbon conductor pillar surface 528 exposed on the second surface 516 of the dielectric substrate 510. Finally, the active region 530 is deposited on the exposed carbon conductor pillar surface 528 to provide a carbon working electrode 500 that has the capability to detect the target substance. Although Figure 7 describes via filling, it should be understood that other types of openings can be filled with carbon conductor ink to form similar types of carbon conductor pillars therein.

[0038] Accordingly, a method for forming a substance sensor according to the present disclosure may include: providing a dielectric substrate having one or more openings that penetrate the dielectric substrate and extend between a first surface and a second surface; filling one or more openings with a carbon conductor to form carbon conductor pillars therein; depositing a carbon conductor coating on the first surface of the dielectric substrate and bringing the carbon conductor coating into direct contact with each carbon conductor pillar; depositing a dielectric coating on the carbon conductor coating; forming one or more active regions on the second surface of the dielectric substrate that are electrically conductive with the carbon conductor pillars in one or more openings and respond to a substance; and depositing a mass transfer limiting film on at least one of the active regions.

[0039] Figure 8A shows a cross-sectional view of a second configuration of a carbon working electrode suitable for use in a substance sensor of the present disclosure. The carbon working electrode 501 differs from the carbon working electrode 500 shown in Figures 6A and 6B in that a monolithic carbon conductor pillar 513 penetrates the dielectric substrate 510 between a first surface 514 and a second surface 516, thereby replacing a plurality of smaller carbon conductor pillars 512. The monolithic carbon conductor pillar 513 can be formed by filling slots or similar openings with carbon conductor ink in a manner similar to that described above with reference to Figure 7, and then drying the ink. Each of the active regions 530 is located on the second surface 516 in direct contact with the monolithic carbon conductor pillar 513.

[0040] Figure 8B shows the corresponding top view of the carbon working electrode in Figure 8A. As shown, the active region 530 covers the exposed surface 540 of the monolithic carbon conductor pillar 513. Although the exposed surface 540 is not completely covered by the active region 530 within the carbon working electrode 501, the amount of foreign carbon is far less than that present in conventional carbon working electrodes having surfaces substantially formed of carbon conductors. Furthermore, it should be understood that the number and spacing of the active regions 530 are exemplary and non-limiting. In exemplary embodiments, the slots may range in width from about 25 μm to about 200 μm and in length from about 100 μm to about 2000 μm.

[0041] Figure 9A shows a cross-sectional view of a third configuration of a carbon working electrode suitable for use in a substance sensor of the present disclosure. The carbon working electrode 502 differs from the carbon working electrode 500 shown in Figures 6A and 6B in that the carbon conductor pillars 512 are interconnected by carbon conductor strips 550 disposed on a second surface 516 of the dielectric substrate 510. The active region 530 is located directly on the carbon strips 550 rather than on the individual carbon conductor pillars 512 within the carbon working electrode 501. The use of carbon conductor strips 550 ensures that the entire active region 530 is electrically connected to the carbon conductor coating 520. For example, if, due to manufacturing issues, incomplete via filling exists when forming one or more of the carbon conductor pillars 512, the conductivity of a given active region 530 to the carbon conductor coating 520 can still be maintained by the carbon conductor strips 550. Furthermore, in electrode configurations using carbon conductor strips 550, the active region 530 does not need to directly cover each carbon conductor pillar 512.

[0042] Figure 9B shows the corresponding top view of the carbon working electrode in Figure 9A. The top view of the carbon working electrode 502 is similar to the top view of the carbon working electrode 501, as shown in Figure 8B, except that the carbon conductor strip 550 covers the individual carbon conductor pillars 512, rather than the monolithic carbon conductor pillars 513 that provide the entirety of the foreign carbon on the second surface 516 and the deposition surface for the active region 530. Similar to the carbon working electrode 501, the carbon conductor strip 550 is not completely covered by the active region 530 within the carbon working electrode 502, but the amount of foreign carbon is much less than that present in conventional carbon working electrodes that have a surface substantially formed of carbon conductors. Furthermore, it should be understood that the number and spacing of the carbon conductor pillars 512 and the active region 530 are illustrative and non-limiting. In exemplary embodiments, the number of openings (vias) may range from about 1 to about 20, or from about 3 to about 15, or from about 2 to about 10, and the diameter of the openings may range from about 25 μm to about 200 μm. The carbon conductor strips interconnected with the carbon conductor pillars may have a width of about 25 μm to about 200 μm and a length of about 100 μm to about 2000 μm.

[0043] Accordingly, the present disclosure provides a substance sensor comprising a dielectric substrate, one or more openings that penetrate the dielectric substrate and fill carbon conductor pillars extending between a first surface and a second surface of the dielectric substrate, and a carbon conductor coating disposed on the first surface of the dielectric substrate in direct contact with each carbon conductor pillar. One or more active regions are located on the second surface of the dielectric substrate and are electrically conductive with each of the carbon conductor pillars, and one or more active regions respond to the substance to be tested. A mass transfer limiting film may cover at least one or more active regions.

[0044] One or more openings penetrating the dielectric substrate may be in the form of multiple vias penetrating the dielectric substrate or slots penetrating the dielectric substrate. Carbon conductor pillars may be arranged within the vias or slots and may completely fill the vias or slots. The active region may be directly located on the carbon conductor pillar(s) arranged within any type of opening on a second surface of the dielectric substrate.

[0045] Optionally, multiple vias penetrating the dielectric substrate may be arranged on a second surface of the dielectric substrate and electrically interconnected via a carbon conductor strip covering the multiple vias. The active region may be located directly on the carbon conductor strip and does not necessarily need to cover the corresponding vias. The carbon conductor strip introduces some foreign carbon to the electrode surface, but the improvement in manufacturing reliability may outweigh the enhancement of sensor performance in some cases. The thickness of the carbon conductor strip may range, for example, from about 1 micron (1 μm) to about 20 microns (20 μm).

[0046] The active region in any of the test substance sensors disclosed herein may comprise one or more test substance-responsive enzymes acting individually or in coordination within an enzyme system. The one or more enzymes, as well as one or more electron transfer agents located within the active region, may be covalently bonded to the polymer comprising the active region.

[0047] Examples of suitable polymers within each active region may include poly(4-vinylpyridine) and poly(N-vinylimidazole) or copolymers thereof, where, for example, quaternated pyridine and imidazole groups function as electron transfer agents or binding sites for enzymes(s). Other suitable polymers that may be present in the active regions(s) include, but are not limited to, those described in U.S. Patent No. 6,605,200 (which is incorporated herein by reference in its entirety), such as poly(acrylic acid), styrene / maleic anhydride copolymer, methyl vinyl ether / maleic anhydride copolymer (GANTREZ polymer), poly(vinylbenzyl chloride), poly(allylamine), polylysine, quaternated poly(4-vinylpyridine) with carboxypentyl groups, and poly(sodium 4-styrenesulfonate).

[0048] The enzyme covalently bonded to the polymer in an active region capable of facilitating the detection of a test substance is not considered particularly limited. Suitable enzymes may include those capable of detecting glucose, lactate, ketones, or creatinine, etc. Any of these test substances may be detected in combination with each other in a test substance sensor capable of detecting multiple test substances. Suitable enzymes and enzyme systems for detecting these test substances are described below.

[0049] In some embodiments, the test substance sensor may have a glucose-responsive active region comprising a glucose-responsive enzyme located on the sensor tail. Suitable glucose-responsive enzymes may include, for example, glucose oxidase or glucose dehydrogenase (e.g., pyrroloquinoline quinone (PQQ) or cofactor-dependent glucose dehydrogenase, e.g., flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase or nicotinamide adenine dinucleotide (NAD)-dependent glucose dehydrogenase). Glucose oxidases and glucose dehydrogenases are distinguished by their ability to utilize oxygen as an electron acceptor when oxidizing glucose; glucose oxidases may utilize oxygen as an electron acceptor, while glucose dehydrogenases transfer electrons to natural or artificial electron acceptors such as enzyme cofactors. Exemplary enzyme-based detection schemes for analyzing glucose are further shown in Figures 10A to 10C, which utilize glucose oxidase or glucose dehydrogenase to facilitate detection. Both glucose oxidase and glucose dehydrogenase may be covalently bonded to a polymer having a glucose-responsive active region, or they may exchange electrons with an electron transfer agent (e.g., an osmium (Os) complex or a similar transition metal complex), which may also be covalently bonded to the polymer. Suitable electron transfer agents are described in further detail below. Glucose oxidase can directly exchange electrons with an electron transfer agent (Figure 10A), while glucose dehydrogenase can improve electron exchange with an electron transfer agent by utilizing a cofactor (Figures 10B and 10C). The FAD cofactor can directly exchange electrons with an electron transfer agent, as shown in Figure 10B. In contrast, the NAD cofactor can facilitate electron transfer from the cofactor to the electron transfer agent by utilizing a diaphorase, as shown in Figure 10C. Further details regarding glucose-responsive active regions incorporating glucose oxidase or glucose dehydrogenase, and glucose detection thereby, can be found, for example, in U.S. Patent No. 8,268,143.

[0050] Figures 11A to 11C show an enzyme system configured to detect ketones. Further details regarding the ketone-responsive enzyme system can be found in the shared U.S. Patent Application No. 16 / 774,835, entitled "Analyte Sensors and Sensing Methods Featuring Dual Detection of Glucose and Ketones," filed on 28 January 2020 and published as U.S. Patent Application Publication No. [number missing]. In the enzyme system shown in Figure 11A, β-hydroxybutyrate functions as a substitute for ketones formed in vivo, which, upon reaction with an enzyme system comprising β-hydroxybutyrate dehydrogenase (HBDH) and diaphorase, facilitates ketone detection within a ketone-responsive active region located on the surface of at least one working electrode, as further described herein. Within the ketone-responsive active region, β-hydroxybutyrate dehydrogenase reacts with β-hydroxybutyrate and oxidized nicotinamide adenine dinucleotide (NAD + ) can be converted to acetoacetic acid and reduced nicotinamide adenine dinucleotide (NADH), respectively. The term "nicotinamide adenine dinucleotide (NAD)" should be understood to include the phosphate-bound form of the aforementioned enzyme cofactor. That is, the use of the term "NAD" herein means that NAD + Both phosphate and NADH phosphate refer to diphosphates that combine two nucleotides, one containing an adenine nucleic acid base and the other containing a nicotinamide nucleic acid base. +The NADH enzyme cofactor assists in facilitating the cooperative enzymatic reactions disclosed herein. Once formed, NADH may undergo oxidation mediated by diaphorase, and the electrons transferred during this process provide 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. The transfer of electrons to the working electrode may occur under further mediation by electron transfer agents such as osmium (Os) compounds or similar transition metal complexes, as described in more detail below. Albumin may further be present as a stabilizer within the active region. β-hydroxybutyrate dehydrogenase and diaphorase may be covalently bonded to a polymer having a ketone-responsive active region. + It may or may not be covalently bonded to the polymer, and NAD + If covalent bonding does not occur, the ketone-responsive active region is covered by a mass transfer limiting membrane that is also permeable to ketones, and NAD + It may be physically retained within the ketone-responsive active region.

[0051] Other suitable chemicals for enzymatically detecting ketones are shown in Figures 11B and 11C. In both cases, a 1:1 molar correspondence again exists between the amount of electrons transferred to the working electrode and the amount of β-hydroxybutyrate converted, thereby providing a basis for ketone detection.

[0052] As shown in Figure 11B, β-hydroxybutyrate dehydrogenase (HBDH) again converts β-hydroxybutyrate and NAD +can be converted to acetoacetic acid and NADH, respectively. Instead of the electron transfer to the working electrode completed by diaphorase (see Figure 11A) and an appropriate redox mediator, the reduced form of NADH oxidase (NADHOx(Red)) undergoes a reaction to form the corresponding oxidized form (NADHOx(Ox)). Next, NADHOx(Red) may be reformed through reaction with molecular oxygen to produce superoxide, which can undergo the next conversion to hydrogen peroxide under the mediation of superoxide dismutase (SOD). Next, hydrogen peroxide undergoes oxidation at the working electrode to provide a signal that can be correlated to the amount of ketone initially present. SOD can be covalently bound to the polymer in the ketone-responsive active region according to various embodiments. Similar to the enzyme system shown in Figure 11A, β-hydroxybutyrate dehydrogenase and NADH oxidase may be covalently bound to the polymer in the ketone-responsive active region, and NAD + may or may not be covalently bound to the polymer in the ketone-responsive active region. If NAD+ is not covalently bound, it may be physically retained within the ketone-responsive active region, and the membrane polymer promotes the retention of NAD + within the ketone-responsive active region.

[0053] As shown in Figure 11C, another enzymatic detection chemical for ketones utilizes β-hydroxybutyrate dehydrogenase (HBDH) to convert β-hydroxybutyric acid and NAD + to acetoacetic acid and NADH, respectively. The electron transfer cycle in this case is completed by the oxidation of NADH by 1,10-phenanthroline-5,6-dione to reform NAD + , and 1,10-phenanthroline-5,6-dione then transfers electrons to the working electrode. 1,10-phenanthroline-5,6-dione may or may not be covalently bound to the polymer within the ketone-responsive active region. Similar to the enzyme system shown in Figure 11A, β-hydroxybutyrate dehydrogenase may be covalently bound to the polymer in the ketone-responsive active region, and NAD +Albumin 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 remarkable improvements in response stability. Suitable membrane polymers provide NAD within the ketone-responsive active region. + This may promote retention.

[0054] In some embodiments, the test substance sensor may further comprise a creatinine-responsive active region comprising an enzyme system that works in coordination to facilitate the detection of creatinine. A suitable enzyme system that may be used to detect creatinine in the test substance sensors disclosed herein is shown in Figure 12 and described in further detail below. Further details relating to the creatinine-responsive enzyme system were filed on September 25, 2019, and published in the United States Patent Application No. This can be found in the shared U.S. Patent Application No. 16 / 774,835, entitled "Analyte Sensors and Sensing Methods for Detecting Creatine," which was published as such.

[0055] As shown in Figure 12, creatinine can react reversibly and hydrolytically in the presence of creatinine amidohydrolase (CNH) to form creatine. Creatine can then undergo catalytic hydrolysis in the presence of creatine amidohydrolase (CRH) to form sarcosine. Neither of these reactions generates an electron flow (e.g., oxidation or reduction) that would provide a basis for the electrochemical detection of creatinine.

[0056] Referring further to Figure 12, sarcosine produced via hydrolysis of creatine may be oxidized in the presence of oxidized sarcosine oxidase (SOX-ox) to form glycine and formaldehyde, thereby generating reduced sarcosine oxidase (SOX-red) during the process. Hydrogen peroxide may also be generated in the presence of oxygen. The reduced sarcosine oxidase may then be reoxidized in the presence of an oxidized electron transfer agent (e.g., an Os(III) complex), thereby generating a corresponding reduced electron transfer agent (e.g., an Os(II) complex) that delivers a flow of electrons to the working electrode.

[0057] Oxygen can interfere with the coordinated series of reactions used to detect creatinine in accordance with the above disclosure. Specifically, reduced sarcosine oxidase may react with oxygen to reform its corresponding oxidized form, but without exchanging electrons with an electron transfer agent. When the reaction with oxygen occurs, the enzyme remains fully active, but no electrons flow to the working electrode. While not bound by theory or mechanism, the competitive reaction with oxygen is thought to be due to kinetic effects; that is, the oxidation of reduced sarcosine oxidase by oxygen is thought to occur faster than the oxidation facilitated by an electron transfer agent. Hydrogen peroxide is also formed in the presence of oxygen.

[0058] The desired reaction pathway for facilitating creatinine detection, as shown in Figure 12, can be facilitated by including an oxygen scavenger adjacent to the enzyme system. Various oxygen scavengers and their arrangements, including oxidase enzymes such as glucose oxidase, may be suitable. Small molecule oxygen scavengers may also be suitable, but they may be completely consumed before the sensor lifetime is fully exhausted. In contrast, enzymes can undergo reversible oxidation and reduction, thereby resulting in a longer sensor lifetime. By preventing the oxidation of the reduced form of sarcosine oxidase by oxygen, a slower electron exchange reaction with the electron transfer agent may occur, thereby enabling the generation of a current at the working electrode. The magnitude of the current generated is proportional to the amount of creatinine initially reacted.

[0059] The oxygen scavenger used to facilitate the desired reaction pathway in Figure 12 may be an oxidase enzyme in any embodiment of this disclosure. Any oxidase enzyme may be used in proximity to the enzyme system to facilitate oxygen capture, provided that a suitable substrate is also available, thereby providing a reagent for reacting with oxygen in the presence of the oxidase enzyme. Oxidase enzymes that may be suitable for oxygen capture in this disclosure include, but are not limited to, glucose oxidase, lactate oxidase, and xanthine oxidase. Glucose oxidase may be a particularly desirable oxidase enzyme for facilitating oxygen capture because glucose is readily available in various body fluids. Reaction 1 below shows an enzymatic reaction facilitated by glucose oxidase, resulting in oxygen removal. [ka] Although the concentration of lactate available in the body is lower than that of glucose, it is still sufficient to promote oxygen capture.

[0060] Oxidase enzymes, such as glucose oxidase, can be placed at any suitable location in the test substance sensors disclosed herein to facilitate oxygen capture. For example, glucose oxidase may be placed on the sensor tail so that it is functional and / or nonfunctional in order to facilitate glucose detection. When nonfunctional in order to facilitate glucose detection, glucose oxidase may be placed on the sensor tail so that electrons generated during glucose oxidation are prevented from reaching the working electrode, for example, by electrically insulating glucose oxidase from the working electrode.

[0061] In some embodiments, the test substance sensor may comprise a lactate-responsive active region comprising a lactate-responsive enzyme located on the sensor tail. Suitable lactate-responsive enzymes may include, for example, lactate oxidase. Lactate oxidase or other lactate-responsive enzymes may be covalently bonded to the polymer comprising the lactate-responsive active region, or they may exchange electrons with an electron transfer agent (e.g., an osmium (Os) complex or a similar transition metal complex), which may also be covalently bonded to the polymer. Suitable electron transfer agents are described in more detail below. Albumin, such as human serum albumin, may be present within the lactate-responsive active region to stabilize the sensor response, as described in whole by reference in shared U.S. Patent Application Publication No. 20190320947. Lactate levels may change in response to a number of environmental or physiological factors, including, for example, feeding, stress, exercise, sepsis or septic shock, infection, hypoxia, or the presence of cancerous tissue.

[0062] In some embodiments, the test substance sensor may include an active region that responds to pH. A suitable test substance sensor configured to determine pH is incorporated herein by reference and described in shared U.S. Patent Application Publication No. 20200060592. Such a test substance sensor may also include a sensor tail comprising a first working electrode and a second working electrode, wherein the first active region located on the first working electrode comprises a substance having a pH-dependent redox chemistry, and the second active region located on the second working electrode comprises a substance having a redox chemistry that is substantially invariant with respect to pH. By obtaining the difference between the first signal and the second signal, this difference can be correlated to the pH of the fluid to which the test substance sensor is exposed.

[0063] Two different types of active regions may be arranged on a single working electrode, such as the carbon working electrode described above, and spaced apart from each other. Each active region may have a redox potential, and the redox potential of the first active region is sufficiently separated from the redox potential of the second active region to allow for the independent generation of a signal from one of the active regions. In non-limiting examples, the redox potentials may differ by at least about 100 mV, or at least about 150 mV, or at least about 200 mV. The upper limit of the separation between redox potentials is determined by the working electrochemical window in the body. By sufficiently separating the redox potentials of the two active regions in magnitude, an electrochemical reaction can occur in one of the two active regions (i.e., the first or second active region) without substantially involving the electrochemical reaction in the other active region. Thus, a signal from one of the first or second active regions can be generated independently at a redox potential greater than or equal to its corresponding redox potential (the lower redox potential) but less than the redox potential of the other active region. Difference signals can allow for the separation of signal contributions from each test substance.

[0064] Some or other embodiments of the substance sensor disclosed herein may feature two distinct active regions located on the surface of another working electrode. Such a substance sensor may comprise a sensor tail comprising at least a first working electrode and a second working electrode, a first active region located on the surface of the first working electrode, and a second active region located on the surface of the second working electrode that responds to a different substance. A membrane may cover at least one of the first and second active regions. The membrane may be a mass transfer limiting membrane and may comprise a multicomponent membrane covering at least one of the active regions. The multicomponent membrane may comprise a bilayer of two different membrane polymers or a mixture of two different membrane polymers, where one membrane polymer covers the other active region. Such a multicomponent membrane may be deposited by dip coating techniques in non-limiting examples.

[0065] Other suitable mass transfer-restricting films can be deposited by in situ polymerization, such as in situ photopolymerization, as will be discussed in more detail below.

[0066] Electron transfer agents may be present in any of the active regions disclosed herein. A suitable electron transfer agent can facilitate the transfer of electrons to adjacent working electrodes after one or more test substances have undergone an enzymatic redox reaction within the corresponding active region, thereby generating an electron flow indicating the presence of a particular test substance. The amount of current generated is proportional to the amount of test substance present. Depending on the sensor configuration used, electron transfer agents in active regions responding to different test substances may be the same or different. For example, when two different active regions are located on the same working electrode, the electron transfer agents in each active region may be different (e.g., chemically different so that the electron transfer agents exhibit different redox potentials). When multiple working electrodes are present, since each working electrode may be detected separately, the electron transfer agents in each active region may be the same or different.

[0067] Suitable electron transfer agents may include electroreducible and electrooxidizable ions, complexes, or molecules (e.g., quinones) having redox potentials several hundred millivolts higher or lower than the redox potential of a standard calomel electrode (SCE). According to some embodiments, suitable electron transfer agents may include low-potential osmium complexes, such as those described in U.S. Patents 6,134,461 and 6,605,200, which are incorporated herein by reference in their entirety. Further examples of suitable electron transfer agents include those described in U.S. Patents 6,736,957, 7,501,053, and 7,754,093, the respective disclosures of which are incorporated herein by reference in their entirety. Other suitable electron transfer agents may include metal compounds or complexes of ruthenium, osmium, iron (e.g., polyvinylferrocene or hexacyanoferrates), or cobalt (e.g., their metallocene compounds). Suitable ligands for metal complexes may include, for example, bipyridine, biimidazole, phenanthroline, or pyridyl(imidazole) bidentate or more. Other suitable bidentate ligands may include, for example, amino acids, oxalic acid, acetylacetone, diaminoalkanes, or o-diaminoarenes. Any combination of monodentate, bidentate, tridentate, tetradentate ligands, or ligands with a higher number of dentates may be present in the metal complex to achieve a complete coordination sphere.

[0068] An active region suitable for detecting any of the test substances disclosed herein may comprise a polymer to which an electron transfer agent is covalently bonded. Any of the electron transfer agents disclosed herein may comprise functional groups suitable for promoting covalent bonding to the polymer within the active region. Examples of suitable polymer-bonded electron transfer agents may include those described in U.S. Patents 8,444,834, 8,268,143, and 6,605,201, the disclosures of which are incorporated herein by reference in their entirety. Suitable polymers to be included in the active region may include, but are not limited to, polyvinylpyridine (e.g., poly(4-vinylpyridine)), polyvinylimidazole (e.g., poly(1-vinylimidazole)), or any copolymer thereof. Exemplary copolymers that may be suitable to be included in the active region include, for example, those containing monomer units such as styrene, acrylamide, methacrylamide, or acrylonitrile. When two or more different active regions are present, the polymers within each active region may be the same or different.

[0069] The covalent bonding of the electron transfer agent to the polymer within the active region may be carried out by polymerizing monomer units having the covalently bonded electron transfer agent, or the electron transfer agent may react separately with the polymer after the polymer has already been synthesized. A bifunctional spacer can covalently bond the electron transfer agent to the polymer within the active region, with the first functional group reacting with the polymer (e.g., a functional group capable of quaternizing pyridine nitrogen atoms or imidazole nitrogen atoms), and the second functional group reacting with the electron transfer agent (e.g., a functional group that reacts with ligands that coordinate to metal ions).

[0070] Similarly, one or more enzymes within an active region may be covalently bonded to the polymer containing the active region. When an enzyme system comprising multiple enzymes is present in a given active region, in some embodiments all of the multiple enzymes may be covalently bonded to the polymer, and in other embodiments only a portion of the multiple enzymes may be covalently bonded to the polymer. For example, one or more enzymes comprising an enzyme system may be covalently bonded to the polymer so that a non-covalently bonded enzyme is physically incorporated into the polymer, and at least one enzyme may be non-covalently bonded to the polymer. Covalent bonding of enzymes(s) to a polymer in a given active region may occur via a crosslinking agent introduced with a suitable crosslinking agent. Crosslinking agents suitable for reaction with free amino groups in enzymes (e.g., free side-chain amines in lysine) may include, for example, polyethylene glycol diglycidyl ether (PEGDGE) or other polyepoxides, cyanuryl chloride, N-hydroxysuccinimide, imide esters, epichlorohydrin, or derivatized variants thereof. Crosslinking agents suitable for reaction with free carboxylic acid groups in enzymes may include, for example, carbodiimide. Enzyme crosslinking to polymers is generally intermolecular, but in some embodiments it can be intramolecular. In certain embodiments, all of the enzyme within a given active region can be covalently bonded to the polymer.

[0071] Electron transfer agents and / or enzymes may be bound to the polymer in the active region by means other than covalent bonding. In some embodiments, electron transfer agents and / or enzymes may be bound to the polymer ionically or coordinationally. For example, a charged polymer may be ionically bound to an electron transfer agent or enzyme that is conversely charged. In yet another embodiment, electron transfer agents and / or enzymes may be physically incorporated into the polymer without being bound to it. Physically incorporated electron transfer agents and / or enzymes may still interact appropriately with the fluid and facilitate the detection of the substance without substantially leaching out of the active region.

[0072] The polymer within the active region(s) contains NAD that is not covalently bonded to the polymer. +Alternatively, another cofactor may be selected to restrict its outward diffusion. Restricting the outward diffusion of the cofactor can promote a moderate sensor lifetime (several days to several weeks), while still allowing sufficient inward diffusion of the test substance to facilitate detection.

[0073] The active regions (multiple) in the test substance sensors disclosed herein have a size of approximately 0.01 mm. 2 From approximately 1 mm 2 Or approximately 0.05 mm 2 From approximately 0.3 mm 2 The active region may comprise one or more individual spots (e.g., one to about 20 spots, or even more individual spots), but larger or smaller individual spots within the active region are also contemplated herein. When two different active regions are present, the number and / or size of individual spots may be the same or different for each type of active region.

[0074] Furthermore, it should be understood that the sensitivity (output current) of the substance sensor to a given substance or combination of substances can be changed by altering the coverage (area or size) of the active region, the area ratio of the active regions relative to each other, and the identity, thickness, and / or composition of the mass transfer limiting film covering the active region. These parameter changes can be readily made by those skilled in the art, provided they are given the benefit of the disclosures herein.

[0075] In more specific embodiments, the substance sensor of this disclosure may comprise a sensor tail configured to be inserted into tissue. Suitable tissues are not considered particularly limiting and are described in more detail above. Similarly, considerations for deploying the sensor tail to a specific location within a given tissue, such as the dermis of the skin, are described above.

[0076] In certain embodiments of this disclosure, a mass transfer restriction film covering one or more active regions may comprise a crosslinked polyvinylpyridine homopolymer or copolymer. The composition of the mass transfer restriction film may be the same as or different from that of mass transfer restriction films covering different types of active regions. When the film composition differs at two different locations, the film may comprise a bilayer film or a homogeneous mixture of two different film polymers, one of which may be a crosslinked polyvinylpyridine or polyvinylimidazole homopolymer or copolymer. Suitable techniques for depositing a mass transfer restriction film on an active region(s) may include, for example, spray coating, painting, inkjet printing, screen printing, stenciling, roller coating, dip coating, and any combination thereof. Dip coating techniques may be particularly desirable for polyvinylpyridine and polyvinylimidazole polymers and copolymers.

[0077] Other suitable mass transfer limiting films can be polymerized in situ on the surface of one or more working electrodes. Specific examples of mass transfer limiting films that can be formed in situ include photopolymerized (photocured) mass transfer limiting films. When multiple types of active regions are present, a photopolymerized mass transfer limiting film can be selectively formed on each type of active region. That is, a mass transfer limiting film having a first portion can cover a first type of active region, and a second portion having a different composition from the first portion can cover a second type of active region. The mass transfer limiting film may or may not be continuous between two or more active regions. More specifically, such a photopolymerized mass transfer limiting film can be formed by depositing one or more monomers on a second surface in contact with one or more active regions and photopolymerizing the one or more monomers. Suitable deposition techniques for depositing monomers include, for example, spraying, screen printing, or any combination thereof. Film chemicals that can be obtained through photopolymerization include, for example, acrylate polymers and copolymers, thiol-en copolymers, or any combination thereof. Polymerization to form both of these types of films can occur under conditions that do not interfere with the detected chemical in the active region. Further in-situ crosslinking can also occur on the surface of the working electrode(s) during the process of forming mass transfer limiting films in situ.

[0078] In non-limiting embodiments, a mass transfer limiting membrane suitable for incorporation in a substance sensor of this disclosure may comprise a polymer or copolymer comprising one or more acrylate monomers. The term “acrylate monomer” refers to acrylic acid, methacrylic acid, or derivatives thereof. A monomer that may be either an acrylate or a methacrylate is referred to herein as “(meth)acrylate monomer.” That is, the designation of a particular monomer as “(meth)acrylate” refers to both the particular acrylate monomer and the corresponding methacrylate monomer form. Suitable acrylate monomers that may be present in the mass transfer limiting membrane of the disclosure herein include, for example, 2-hydroxyethyl (meth)acrylate, poly(ethylene glycol)methyl ether (meth)acrylate, alkyl ether poly(ethylene glycol) (meth)acrylate, poly(ethylene glycol) (meth)acrylate, poly(propylene glycol) (meth)acrylate, alkyl ether poly(propylene glycol) (meth)acrylate, isobutyl (meth)acrylate, alkyl (meth)acrylate, 2-(trimethylsilyloxy)ethyl (meth)acrylate, 3-[tris(trimethylsiloxy)silyl]propyl (meth)acrylate, N,N-dimethylacrylamide, glycidyl (meth)acrylate, or any combination thereof. Glycidyl (meth)acrylate may be further functionalized in some cases. Crosslinkable acrylates, such as di(ethylene glycol)di(meth)acrylate, poly(ethylene glycol)di(meth)acrylate, trimethylolpropane triacrylate, or any combination thereof, may exist in combination with any of the aforementioned (meth)acrylate monomers. Any molecule containing two or more sufficiently reactive alkene groups may also function as a crosslinking agent when forming a mass transfer limiting film. The amount of crosslinkable (meth)acrylate may be selected to give a desired degree of crosslinking, for example, a weight percentage of crosslinkable (meth)acrylate up to about 1% by weight relative to the total mass of monomers.The selection of specific (meth)acrylate monomers, crosslinkable (meth)acrylates or similar polyene crosslinking agents, or any combination thereof, and the ratios between them, may be chosen to provide sufficient permeability to a given test substance. For example, two or more (meth)acrylate monomers having different levels of hydrophilicity and hydrophobicity may be selected, and their ratios may be modified to give specific membrane properties depending on the water content and the hydrophilicity or hydrophobicity of the test substance. Membranes containing glycidyl (meth)acrylate can also be crosslinked with diol or triol crosslinking agents, such as ethylene glycol, propylene glycol, butanediol, hexanediol, glycerol, poly(ethylene glycol), poly(propylene glycol), or any combination thereof.

[0079] In addition, (meth)acrylamide, alkyl or dialkyl(meth)acrylamide, (meth)acrylic acid, or alkyl(meth)acrylate esters can be copolymerized with any of the aforementioned (meth)acrylate monomers or crosslinkable acrylates. Other olefinic unsaturated monomers can also be copolymerized with any of the above (meth)acrylate monomers. Other monomers that can be copolymerized include alpha-olefins such as ethylene, propylene, butene, pentene, hexene, heptene, octene, nonene, decene, undecene, dodecene, or mixtures thereof. Alpha-olefins that are liquid at room temperature may be particularly desirable. Such comonomers may be included to facilitate further tuning of the physical properties of the resulting film. For example, one or more alpha-olefin monomers may be added to increase the hydrophobicity of the film.

[0080] Some acrylate monomers can undergo photopolymerization or thermal polymerization in the absence of an initiator. Other acrylate monomers can undergo photopolymerization in the presence of a suitable initiator that generates radical species when exposed to electromagnetic radiation such as ultraviolet light (e.g., from a mercury lamp). In non-limiting embodiments, suitable photoinitiators may include, for example, 2,2-dimethoxy-2-phenylacetophenone, camphorquinone, azo compounds, and peroxides (e.g., benzoyl peroxide with an N,N-dimethyltoluidine accelerator). Other suitable photoinitiators may include, but are not limited to, Norrish type I initiators (e.g., 2,2-dimethoxy-2-phenylacetophenone, hydroxyacetophenone, aminoacetophenone, and acetophenone phosphine oxide, etc.), Norrish type II initiators (e.g., benzophenone, benzyl formate, and thioxanthone, etc., possibly including amine synergistic accelerators), phenolglyoxalic acid methyl esters, alpha-aminoketones, benzoyl diphenylphosphine oxide, or any combination thereof. Typical examples include the CAROCUR, IRGACUR, and LUCIRIN photoinitiators from BASF. A suitable photoinitiator can be present in amounts up to approximately 1% by weight relative to the total mass of the monomer.

[0081] Thiol-ene copolymers can also be appropriately incorporated as mass transfer limiting membranes in the substance sensors disclosed herein. Advantageously, thiol-ene copolymers can be formed in air, in contrast to (meth)acrylate polymers, which may require the use of inert air for sufficient polymerization to occur. Thiol-ene polymers can be obtained by reacting a monomer containing two or more thiol groups with a monomer containing two or more non-conjugated alkene groups. Any of the above photoinitiators may be used as needed. Monomers having two thiol groups or two non-conjugated alkene groups may yield linear polymers. Monomers having three or more thiol groups or three or more non-conjugated alkene groups may yield varying degrees of crosslinking. Any type of non-crosslinkable monomer may be used, for example, to reduce the crosslinking density. Crosslinkable monomers, which may be essentially tripod- or tetrad-shaped, may have variable-length spacers between the central atom and the terminal thiol or terminal alkene groups. The length of the spacers and the amount of crosslinkable monomer may also affect the crosslinking density.

[0082] Particularly suitable monomers containing thiol groups may be quadrupedal in which each arm of the quadrupedal structure is terminated with a thiol group. Compounds 1 and 2 below are exemplary examples of monomers containing quadrupedal thiol groups that may be suitable for use in forming mass transfer limiting films by photopolymerization. [ka] The following compound 3 (dithiol) can be used to reduce the crosslink density by introducing a linear moiety into the thiol-ene polymer. Smaller dithiols, such as ethanedithiol and propanedithiol, can also be used to reduce the crosslink density, similar to the larger polyetherdithiol compound 3. Similarly, trithiols can be used to reduce the crosslink density. [ka]

[0083] Particularly suitable en monomers may be bilegged, tripod, or tetralegged, depending on the desired crosslinking density. Compounds 4 to 6 are exemplary examples of bilegged, tripod, and tetralegged en monomers, respectively, where A is a spacer group whose structure is not particularly limited and which may optionally contain one or more heteroatoms. Compound 4 Compound 5 Compound 6 Compounds 7 to 10 below are exemplary examples of specific en monomers that may be suitable for forming mass transfer-restricting films by in situ photopolymerization. [ka]

[0084] The photopolymerized mass transfer restriction film may be present in a sensor of a substance equipped with the carbon working electrode described above, or it may be present in a sensor of a substance equipped with a conventional carbon working electrode or any other type of working electrode. In either case, the photopolymerized mass transfer restriction film may be advantageous because it can be easily adapted to meet the specific needs of a particular application. Furthermore, the photopolymerized mass transfer restriction film may be obtained by depositing readily distributed monomers, which can be deposited at one or more specific locations by spraying or screen printing techniques. As a result of deposition specificity, photopolymerized mass transfer restriction films with different compositions may cover different types of active regions. Alternatively, the photopolymerized mass transfer restriction film may cover a first type of active region, while a second type of active region may be covered with a polyvinylpyridine or polyvinylimidazole polymer or copolymer deposited by dip coating.

[0085] Accordingly, some of the test substance sensors of this disclosure may comprise a first working electrode, one or more first active regions disposed on the first working electrode and responding to a first test substance, and a first photopolymerized material migration-restricting film formed directly on the first working electrode and covering at least one of the one or more first active regions. The first working electrode may be a first carbon electrode, such as the carbon electrode described in more detail above. Conventional carbon working electrodes may also be suitable. In non-limiting embodiments, the first active region may comprise a test substance-responsive enzyme or enzyme system. Any of the test substances discussed in more detail above may be assayed by the first active region.

[0086] The substance sensor may also comprise a second working electrode and one or more second active regions located on the second working electrode that respond to a second substance different from the first substance. The second working electrode may be a second carbon electrode, such as the carbon electrodes described in more detail above, or a conventional carbon working electrode may also be suitable. The second mass transfer limiting film may cover at least one or more second active regions and may have a different composition from the first photopolymerized mass transfer limiting film. The second mass transfer limiting film may contain a dip-coated polyvinylpyridine or polyvinylimidazole polymer or copolymer, and more preferably, the second photopolymerized mass transfer limiting film may have a different composition from the first mass transfer limiting film. The first and / or second photopolymerized mass transfer limiting films may be discontinuous so that the film does not extend between adjacent spots of a given active region or between two different active regions configured to detect different substances.

[0087] Accordingly, a method for forming a substance sensor having a photopolymerized mass transfer limiting film may include: providing a substance sensor having at least one first working electrode on which one or more first active regions that respond to a first substance are disposed; applying one or more first monomers on a first working electrode that is in contact with one or more first active regions; and in situ polymerization of one or more first monomers on the first working electrode by photopolymerization or the like to form a first mass transfer limiting film covering at least one or more first active regions. In a non-limiting embodiment, photopolymerization may include exposing one or more first monomers (e.g., a mixture of a first monomer and a second monomer) to ultraviolet light and optionally a photoinitiator. When the substance sensor further comprises a second working electrode, the method may further comprise applying one or more second monomers onto the second working electrode in contact with one or more second active regions, and polymerizing one or more second monomers onto the second working electrode by photopolymerization or the like to form a second mass transfer limiting film covering at least one or more second active regions. The second mass transfer limiting film may have the same composition as the first mass transfer limiting film or a different composition. In various embodiments, both the first and second mass transfer limiting films can be photopolymerized.

[0088] A sensor for a substance to be tested can be prepared as an assembly using an electrode material (e.g., carbon) having multiple active regions arranged on top of it. Polymerization can occur as described above when one or more first monomers are applied to the active regions, such as by syringe deposition of a solution containing one or more first monomers. In non-limiting examples, the solution containing one or more first monomers may be deposited at a volume of about 100 to 200 nL. 365 nm and 1 to 30 mW / cm² 2 After polymerization is carried out by irradiation with ultraviolet light for up to 5 minutes, the electrode material can be laser-cut, and in some cases the mass transfer limiting film can be cut, thereby isolating individual test substance sensors.

[0089] Embodiments disclosed herein include the following:

[0090] A. A substance sensor having a carbon working electrode. The substance sensor comprises a dielectric substrate, one or more openings penetrating the dielectric substrate and each filled with carbon conductor pillars extending between a first surface and a second surface of the dielectric substrate, a carbon conductor coating disposed on the first surface of the dielectric substrate in direct contact with each carbon conductor pillar, and a dielectric coating disposed on the carbon conductor coating, one or more active regions located on the second surface of the dielectric substrate, electrically conductive with each of the carbon conductor pillars and responding to the substance, and a mass transfer limiting film covering at least one or more active regions.

[0091] B. A method for manufacturing a substance sensor having a carbon working electrode. The method comprises: providing a dielectric substrate having one or more openings that penetrate the dielectric substrate and extend between a first surface and a second surface; filling one or more openings with a carbon conductor to form carbon conductor pillars therein; depositing a carbon conductor coating on the first surface of the dielectric substrate and bringing the carbon conductor coating into direct contact with each carbon conductor pillar; depositing a dielectric coating on the carbon conductor coating; forming one or more active regions on the second surface of the dielectric substrate that are electrically conductive with the carbon conductor pillars in one or more openings and respond to a substance; and depositing a mass transfer limiting film on at least one of the active regions.

[0092] C. A substance sensor having a mass transfer limiting film. The substance sensor comprises a first working electrode, one or more first active regions disposed on the first working electrode and responding to a first substance, and a first photopolymerized mass transfer limiting film formed directly on the first working electrode and covering at least one or more of the first active regions.

[0093] D. A method for manufacturing a substance sensor having a mass transfer limiting film. The method provides a substance sensor comprising at least one first working electrode on which one or more first active regions that respond to a first substance are disposed, the first working electrode comprising applying one or more first monomers to a first working electrode in contact with one or more first active regions, and in situ polymerization of one or more first monomers on the first working electrode to form a first mass transfer limiting film covering at least one or more first active regions.

[0094] Each of embodiments A through D may have one or more of the following additional elements in any combination.

[0095] Element 1: One or more openings are provided with multiple vias that penetrate the dielectric substrate.

[0096] Element 2: Each via has an active region directly positioned on a carbon conductor pillar placed inside.

[0097] Element 3: The substance sensor is positioned on a second surface of a dielectric substrate and further comprises a carbon conductor strip covering multiple vias, with one or more active regions positioned directly on the carbon conductor strip.

[0098] Element 4: One or more openings are provided with slots that penetrate the dielectric substrate.

[0099] Element 5: Multiple active regions are directly positioned on carbon conductor pillars placed within slots.

[0100] Element 6: One or more active regions comprise one or more test substance-responsive enzymes.

[0101] Element 7: The mass transfer limiting film is a photopolymerized mass transfer limiting film.

[0102] Element 8: The photopolymerization material migration-restricting film is selectively formed on one or more active regions.

[0103] Element 9: The mass transfer limiting membrane comprises an acrylate polymer or copolymer, a thiol-en copolymer, or any combination thereof.

[0104] Element 10: The carbon working electrode is positioned on the sensor tail, which is configured to be inserted into the tissue.

[0105] Element 11: The method further comprises depositing a carbon conductor strip covering a plurality of vias on a second surface of a dielectric substrate, wherein one or more active regions are directly located on the carbon conductor strip.

[0106] Element 12: The mass transfer limiting film is deposited by dip coating.

[0107] Element 13: The mass transfer limiting membrane is polymerized in situ on one or more active regions.

[0108] Element 14: The mass transfer limiting film is a photopolymerized mass transfer limiting film, formed by depositing one or more monomers on a second surface of a dielectric substrate that is in contact with one or more active regions, and then photopolymerizing the one or more monomers.

[0109] Element 15: The photopolymerization material migration-restricting film comprises an acrylate polymer or copolymer, a thiol-en copolymer, or any combination thereof.

[0110] Element 16: The first photopolymerization material migration-restricting film comprises an acrylate polymer or copolymer, a thiol-en copolymer, or any combination thereof.

[0111] Element 17: The test substance sensor further comprises a second working electrode, one or more second active regions disposed on the second working electrode and responding to a second test substance different from the first test substance, and a second mass transfer limiting film covering at least one of the second active regions and having a different composition from the first photopolymerized mass transfer limiting film.

[0112] Element 18: The second mass transfer limiting film is a second photopolymerized mass transfer limiting film having a different composition from the first photopolymerized mass transfer limiting film.

[0113] Element 19: The first photopolymerized material migration-restricting film is crosslinked.

[0114] Element 20: The first photopolymerized material migration limiting film is discontinuous.

[0115] Element 21: The first photopolymerization material migration-restricting film is positioned on substantially one or more active regions.

[0116] Element 22: One or more first active regions comprise one or more test substance-responsive enzymes.

[0117] Element 23: The first working electrode is positioned on a sensor tail configured to be inserted into tissue.

[0118] Element 24: The first mass transfer limiting film is a photopolymerized mass transfer limiting film formed by photopolymerizing one or more first monomers.

[0119] Element 25: Applying one or more first monomers comprises screen printing one or more first monomers, spraying one or more first monomers, or any combination thereof.

[0120] Element 26: The test substance sensor further comprises a second working electrode having one or more second active regions positioned on it, the one or more second active regions responding to a second test substance different from the first test substance, and the method further comprises applying one or more second monomers onto the second working electrode in contact with one or more second active regions, and in situ polymerizing one or more second monomers onto the second working electrode to form a second mass transfer limiting film having a different composition from the first mass transfer limiting film and covering at least one or more second active regions.

[0121] Element 27: The second mass transfer limiting film is a photopolymerized mass transfer limiting film formed by photopolymerizing one or more second monomers.

[0122] Element 28: The first mass transfer limiting membrane is discontinuous.

[0123] Element 29: The first mass transfer limiting membrane is selectively formed on one or more first active regions.

[0124] As a non-limiting example, exemplary combinations applicable to A include, but are not limited to, 1 and 2, 1 and 3, 1 to 3, 1 and 6, 1 and 7, 1 and 8, 1 and 9, 1 and 10, 4 and 5, 4 and 6, 4 to 6, 4 and 7, 4 and 5 and 7, 4 and 8, 4 and 5 and 8, 4 and 9, 4 and 5 and 9, 4 and 10, and 4 and 5 and 10. Exemplary combinations applicable to B include, but are not limited to, 1 and 2, 1 and 11, 1 and 2 and 11, 1 and 6, 1 and 7, 1 and 8, 1 and 9, 1 and 10, 4 and 5, 4 and 6, 4 to 6, 4 and 7, 4 and 5 and 7, 4 and 8, 4 and 5 and 8, 4 and 9, 4 and 5 and 9, 4 and 10, 4 and 5 and 10, 1 and 12, 1 and 2 and 12, 1 and 13, 1 and 2 and 13, 1 and 2 and 11 and 13, 4 and 12, 4 and 5 and 12, 4 and 13, 4 and 5 and 13, 1 and 13 and 14, 4 and 13 and 14, 1 and 13 to 15, and 4 and 13 to 15. Exemplary combinations applicable to C include, but are not limited to, 16 and 17, 16 to 18, 16 and 19, 16 and 20, 16 and 21, 16 and 22, 16 and 23, 17 and 18, 17 and 19, 17 and 20, 17 and 21, 17 and 22, 17 and 23, 20 and 21, 20 and 22, 20 and 23, 21 and 22, 21 and 23, and 22 and 23. Exemplary combinations applicable to D include, but are not limited to, 16 and 24, 16 and 24 and 25, 24 and 25, 16 and 24 and 26, 24 and 26, 24 and 26 and 27, 16 and 24 and 28, 24 and 28, 16 and 24 and 26 and 27, 24 and 29, 16 and 24 and 29, 26 and 27, 26 and 28, 26 to 28, 26 and 29, 26 and 27 and 29, and 28 and 29.

[0125] To facilitate a better understanding of the embodiments described herein, the following examples of various representative embodiments are given. The following examples should not be read to limit or define the scope of the invention.

[0126] Examples General procedure for sensor manufacturing. Apply the appropriate sensing layer chemicals first, about 0.05 to 0.3 mm thick. 2 The detection layer chemical was deposited on the working electrode (e.g., a carbon working electrode) as a group of dots or slots having an area of ​​. Next, approximately 100 to 200 nL of monomer solution containing monomers, a crosslinking agent, and an initiator was manually deposited over a working electrode area of ​​approximately 1.5 mm × 2.5 mm using a microsyringe. The resulting construct was then transferred into an argon-filled chamber containing a UV lamp emitting at 365 nm. The mass transfer limiting film was then subjected to a UV load of 1 to 30 mW / cm². 2 The sensors were photopolymerized under UV power for up to 5 minutes. Next, the individual sensors were isolated by laser cutting.

[0127] The active region for various test substances utilized a poly(vinylpyridine)-bonded transition metal complex having the structure shown in the following chemical formula 11 as an electron transfer agent. Further details regarding this transition metal complex and electron transfer using it are provided in the shared U.S. Patent No. 6,605,200, which is incorporated above by reference. The subscripts for each monomer indicate exemplary atomic ratios and do not indicate any particular monomer order. [ka]

[0128] Example 1: Glucose sensor covered with a photopolymerized film. The glucose-responsive active region was deposited using the formulation shown in Table 1 below and then mixed in 10 mM HEPES buffer (pH=8.05). [Table 1] A total of 12 nL of the formulations shown in Table 1 were deposited onto the carbon working electrode using a piezoelectric dispensing system, resulting in a layer of approximately 0.1 mm. 2 A glucose-responsive active region with a certain area was formed. Next, 200 nL volumes of 2-hydroxyethyl methacrylate (HEMA) and poly(propylene glycol) methacrylate (POMA) were deposited on the active region and polymerized with monomers in various molar ratios in the presence of 1 wt% of 2,2-dimethoxy-2-phenylacetophenone photoinitiator and 1 wt% of di(ethylene glycol) dimethacrylate crosslinking agent. Polymerization and crosslinking were carried out under argon for 5 minutes at a UV irradiation wavelength of 365 nm and 6 mW / cm². 2 The test was performed at the output. Next, the sensor was isolated by laser cutting the photopolymerized film to form a 0.7 mm wide tail. Figure 13 shows plots of the sensor response of several glucose sensors covered with mass transfer limiting films formed from HEMA:POMA in various ratios, after the addition of various amounts of 1 M glucose stock solution to achieve a range of glucose concentrations in PBS at 33°C. Figure 14 shows the corresponding plot of the sensor response as a function of glucose concentration. The test substance sensor with the HEMA:POMA film showed an average drift of less than 10% over 14 days, while the film with HEMA only drifted almost 100% (data not shown). HEMA:POMA ratios of 2:1 and 4:1 resulted in a good balance of signal, response linearity, and extended response stability.

[0129] Example 2: Ketone sensor coated with HEMA-POMA membrane. The ketone-responsive active region was deposited using the formulation shown in Table 2 below and then mixed in 10 mM MES buffer (pH=5.5). [Table 2-1] [Table 2-2] A total of 12 nL of the formulations shown in Table 2 were deposited onto the carbon working electrode using a piezoelectric dispensing system, resulting in a layer of approximately 0.1 mm.2 A ketone-responsive active region with a certain area was formed. Next, 200 nL volumes of 2-hydroxyethyl methacrylate (HEMA) and poly(propylene glycol) methacrylate (POMA) were deposited on the active region and polymerized with monomers in various molar ratios in the presence of 1 wt% of 2,2-dimethoxy-2-phenylacetophenone photoinitiator and 1 wt% of di(ethylene glycol) dimethacrylate crosslinking agent. Polymerization and crosslinking were carried out under argon for 5 minutes at a UV irradiation wavelength of 365 nm and 6 mW / cm². 2 The test was performed at the output. Next, the sensor was isolated by laser cutting the photopolymerized film to form a 0.7 mm wide tail. Then, a PVP-co-styrene layer was formed on the HEMA-POMA copolymer by dip coating the sensor tail. Figures 15A and 15B show plots of the sensor response of several ketone sensors covered with mass transfer limiting membranes formed from various ratios of HEMA:POMA after adding various amounts of 1 M ketone stock solution to achieve a range of ketone concentrations in PBS at 33°C. Figures 16A and 16B show corresponding plots of the sensor response as a function of ketone concentration. A HEMA:POMA ratio of 4:6 resulted in a good balance of signal and response linearity. Sensor performance comparable to the dip-coated PVP / PVP-co-styrene mass transfer limiting membrane control was achieved, which results in good performance when assaying ketones (US Patent Application No. 16 / 774,835 referenced above). The sensor response changed by approximately 2.4% over two weeks of measurement at this HEMA:POMA ratio (data not shown).

[0130] Example 3: Variation in crosslinking density in thiol-ene polymer systems. 2,2'-(ethane-1,2-diylbis(oxy))bis(ethane-1-thiol) (EDDEE) and 2-(5-mercapto-3-oxopentyl)-2-(((3-mercaptopropanoyl)oxy)methyl)propane-1,3-diylbis(3-mercaptopropanoate) (PETMP) were combined in various molar ratios and photopolymerized with tri(ethylene glycol) divinyl ether (TEGDVE) as shown in Table 3 below. The molar ratio of the two thiols to TEGDVE was maintained at 1:1 in all cases. Polymerization was carried out by exposing the monomer mixture to 365 nm UV light at 6 mW / cm². 2 This was performed by exposure to air at the specified power density for 5 minutes. Table 3 also summarizes the obtained polymer properties. [Table 3] Items 1 through 12 in Table 1 are ranked in approximate order from lowest to highest crosslink density. Crosslink density was evaluated qualitatively by the appearance of the polymer and semi-quantitatively by the amount of water absorbed by the polymer. Water content was measured by the ratio of the difference between the dry polymer mass and the wet polymer mass to the wet polymer mass after immersion of one polymer in water for 8 hours. As shown in Table 3, a higher amount of tetrafunctional thiol PETMP resulted in a higher crosslink density value. This example demonstrates that it is possible to adjust the permeability of thiol-ene polymer systems to account for the sizes of different test substances. For water-containing systems, a higher water content of the polymer generally results in a higher permeability value.

[0131] Example 4: Ketone sensor covered with a thiol-ene film. A ketone sensor was prepared as in Example 2 and covered with thiol-ene copolymers from items 6, 8, 11, and 12 of Table 3. Photopolymerization was carried out as in Example 3. Next, the sensor was isolated by laser cutting the photopolymerized film to form a 0.7 mm wide tail. Next, a PVP-co-styrene layer was formed on the thiol-ene copolymer by dip coating the sensor tail. Figure 17 shows plots of the sensor response of several ketone sensors covered with mass transfer limiting films formed from thiol-ene polymers in various ratios after the addition of various amounts of 1 M ketone stock solution to achieve a range of ketone concentrations in PBS at 33°C. Figure 18 shows the corresponding plots of the sensor response as a function of ketone concentration. In general, except for the film formed from item 6, which was an adhesive film, the sensor response increased as the crosslinking density decreased.

[0132] Unless otherwise indicated, all figures representing quantities, etc., in this specification and related claims should be understood to be modified in all cases by the term “approximately.” Therefore, unless otherwise indicated, the numerical parameters described herein and in the appended claims are approximations that may vary depending on the desired properties sought 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, not in any attempt to limit the application of the doctrine of equivalents to the scope of the claims.

[0133] One or more exemplary embodiments incorporating various features are presented herein. Not all features of a physical implementation are described or shown in this application for clarity. It is understood that in developing a physical implementation incorporating an embodiment of the present invention, a number of implementation-specific decisions will need to 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 may vary. While the developer's efforts may be time-consuming, such efforts are still routine for those skilled in the art and will benefit those who are interested in this disclosure.

[0134] While various systems, tools, and methods are described herein in terms of "equipping" various components or processes, systems, tools, and methods may also be "essentially made of" or "consist of" various components and processes.

[0135] As used herein, the phrase “at least one of” with the terms “and” or “or” that precede a set 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 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” 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, respectively.

[0136] Accordingly, the disclosed systems, tools, and methods are fully applicable to achieve the purposes and benefits mentioned, as well as those inherent to them. 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, no limitation is intended on the details of the configurations or designs shown herein, other than those described in the following claims. Accordingly, the specific exemplary embodiments disclosed above may be changed, combined, or modified, and it is obvious that all such variations will be considered within the scope of this disclosure. The systems, tools, and methods disclosed exemplary herein can be adequately implemented without any elements not specifically disclosed herein and / or any optional 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 "essentially consist of" or "consist of" various components and processes. All the numbers and scopes disclosed above may vary to some extent. Whenever a numerical range with lower and upper limits is disclosed, any number and any range that falls within that range are specifically disclosed. In particular, all ranges of values ​​disclosed herein (in the form of “about a to about b,” or equivalently “approximately a to b,” or equivalently “approximately a to b”) should be understood to describe 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. In addition, the indefinite article “a” or “an” used in the claims is defined herein to mean one or more 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 that may be incorporated herein by reference, the definition consistent with this specification should be adopted.

Claims

1. The first working electrode and Displaced on the first working electrode, and comprising one or more first active regions that respond to a first test substance, A first photopolymerization material migration limiting film is formed directly on the first working electrode and covers at least one of the first active regions, A sensor for a substance to be tested, comprising the first photopolymerized substance migration limiting film comprising a thiol-en copolymer.

2. The second working electrode, Displaced on the second working electrode, one or more second active regions that respond to a second test substance different from the first test substance, A second mass transfer limiting film covering at least one or more of the second active regions, having a different composition from the first photopolymerized mass transfer limiting film, The sensor for the substance to be tested according to claim 1, further comprising the above.

3. The sensor for testing a substance according to claim 2, wherein the second mass transfer limiting film is a second photopolymerized mass transfer limiting film having a different composition from the first photopolymerized mass transfer limiting film.

4. The first photopolymerized substance migration limiting film is crosslinked, as described in claim 1, for the substance sensor.

5. The sensor for a substance to be tested according to claim 1, wherein the first photopolymerized substance migration limiting film is discontinuously arranged on the first working electrode.

6. The sensor for a substance to be tested according to claim 5, wherein the first photopolymerized substance migration-restricting film is arranged substantially on one or more active regions.

7. The test substance sensor according to claim 1, wherein the one or more first active regions comprises one or more test substance-responsive enzymes.

8. The sensor for testing a substance according to claim 1, wherein the first working electrode is positioned on a sensor tail configured to be inserted into tissue.

9. The sensor for testing a substance according to claim 1, wherein the thiol-en copolymer is a reaction product of a) a monomer containing two or more alkene groups and b) a mixture of a monomer having two thiol groups and a monomer having three or more thiol groups.

10. The sensor for testing a substance according to claim 9, wherein the monomer containing two or more alkene groups includes a mixture of a monomer containing two or more non-conjugated alkene groups and a monomer having three or more non-conjugated alkene groups.

11. The sensor for testing a substance according to claim 9, wherein the monomer containing two or more alkene groups includes a monomer containing two non-conjugated alkene groups.

12. The thiol-ene copolymer is composed of ethanedithiol, propanedithiol, the following compounds 1, 2, 3, 7, 8, 9, 10, and mixtures thereof. 【Chemistry 1】 A sensor for a substance to be tested according to claim 1, which is a reaction product of one or more monomers selected from.

13. A method for preparing a sensor for a test substance, The substance sensor comprises at least a first working electrode, the first working electrode having one or more first active regions disposed on the first working electrode that respond to a first substance, The method involves applying one or more first monomers onto the first working electrode that is in contact with one or more first active regions, To form a first mass transfer limiting film covering at least one of the first active regions, the one or more first monomers are photopolymerized in situ on the first working electrode. A method comprising, wherein the first mass transfer limiting membrane comprises a thiol-en copolymer.

14. The method according to claim 13, wherein applying the one or more first monomers comprises screen printing the one or more first monomers, spraying the one or more first monomers, or any combination thereof.

15. The substance sensor further comprises a second working electrode, the second working electrode having one or more second active regions positioned on the second working electrode and responding to a second substance different from the first substance, The method involves applying one or more second monomers onto the second working electrode that is in contact with one or more second active regions, To form a second mass transfer limiting film that covers at least one or more second active regions and has a different composition from the first mass transfer limiting film, the one or more second monomers are photopolymerized in situ on the second working electrode, The method according to claim 13, further comprising:

16. The method according to claim 13, wherein the first mass transfer limiting film is discontinuously arranged on the first working electrode.

17. The method according to claim 13, wherein the first mass transfer limiting membrane is selectively formed on one or more first active regions.

18. The method according to claim 13, wherein the thiol-ene copolymer is a reaction product of a) a monomer containing two or more alkene groups and b) a mixture of a monomer having two thiol groups and a monomer having three or more thiol groups.

19. The method according to claim 18, wherein the monomer containing two or more alkene groups includes a mixture of a monomer containing two or more non-conjugated alkene groups and a monomer having three or more non-conjugated alkene groups.

20. The method according to claim 18, wherein the monomer containing two or more alkene groups includes a monomer containing two non-conjugated alkene groups.

21. The thiol-ene copolymer is comprised of ethanedithiol, propanedithiol, the following compounds 1, 2, 3, 7, 8, 9, 10, and mixtures thereof. 【Chemistry 2】 The method according to claim 13, wherein the reaction product is one or more monomers selected from.

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