WO2023091915A1 - Système de transduction chimique dans un capteur physiologique d'analyte enzymatique optique - Google Patents

Système de transduction chimique dans un capteur physiologique d'analyte enzymatique optique Download PDF

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WO2023091915A1
WO2023091915A1 PCT/US2022/079904 US2022079904W WO2023091915A1 WO 2023091915 A1 WO2023091915 A1 WO 2023091915A1 US 2022079904 W US2022079904 W US 2022079904W WO 2023091915 A1 WO2023091915 A1 WO 2023091915A1
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layer
solution
oxygen
enzymatic
enzymatic hydrogel
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PCT/US2022/079904
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English (en)
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Troy M. Bremer
Xuejun Yu
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Metronom Health, Inc.
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Publication of WO2023091915A1 publication Critical patent/WO2023091915A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/145—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue
    • A61B5/14532—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue for measuring glucose, e.g. by tissue impedance measurement
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/145—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue
    • A61B5/1455—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue using optical sensors, e.g. spectral photometrical oximeters
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N31/00—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods
    • G01N31/22—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods using chemical indicators
    • G01N31/223—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods using chemical indicators for investigating presence of specific gases or aerosols
    • G01N31/225—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods using chemical indicators for investigating presence of specific gases or aerosols for oxygen, e.g. including dissolved oxygen

Definitions

  • the disclosed and described technology relates generally to opto-enzymatic analyte physiological sensors such as may be used in an implantable sensor as part of a continuous analyte monitoring system, including a continuous glucose monitoring (CGM) system.
  • CGM continuous glucose monitoring
  • Diabetes is a disease of insufficient blood glucose regulation.
  • the body's beta cells monitor glucose and deliver just the right amount of insulin on, for example, a minute-by-minute basis for tissues in the body to uptake the right amount of glucose, keeping blood glucose at healthy levels.
  • this regulation primarily fails due to insufficient insulin production and secretion and/or a lack of normal sensitivity to insulin by the tissues of the body.
  • Glucose sensors can be used to monitor glucose levels in diabetic patients allowing proper dosing of diabetic treatments, including, for example, insulin.
  • analyte tracking and monitoring enable improved monitoring, diagnosis, and treatment of diseases, including diabetes.
  • Existing methods to measure, monitor, and track analyte levels may include sampling a bodily fluid, preparing the sample for measurement, and estimating the analyte level in the sample. For example, a diabetic may prick a finger to obtain a blood sample to measure glucose in a glucose monitoring unit.
  • Such existing methods may be painful, unpleasant or inconvenient for the patient, resulting in lower compliance with physician orders to, for example, take glucose readings at certain times each day or based on patient activity.
  • effective monitoring, diagnosis, and treatment may benefit from fusing multiple sensor readings that measure different aspects of a patient's state.
  • Readings from one or more analyte sensors, as well as other bio sensor systems and/or activity sensors may be combined with past readings to determine results that characterize a patient's state, and may be used to monitor, diagnose, and treat a patient. For example, an alarm may be triggered if a patient's glucose level exceeds a threshold.
  • Analyte sensors such as glucose sensors, can produce a digital electronic signal that depends on the concentration of a specific chemical or set of chemicals (analyte) in bodily fluid or tissue.
  • the sensor usually includes two main components, (1) a chemical or biological part that reacts or complexes with the analyte in question to form new chemical or biological products or changes in energy that can be detected by means of the second component and (2) a transducer.
  • the first component (chemical or biological) can be said to act as a receptor/indicator for the analyte.
  • the second component a variety of transduction methods can be used including, electrochemical and optical. After transduction, the signal is usually converted to an electronic digital signal that corresponds to a concentration of a particular analyte.
  • Example analytes that can be measured using the embodiments disclosed and described herein include, and are not limited to, glucose, galactose, fructose, lactose, peroxide, cholesterol, amino acids, alcohol, lactic acid, ketone, and mixtures of the foregoing.
  • analyte sensors that do not require unpleasant blood draws or sample preparation if measurements are to be taken multiple times each day; are sufficiently selective, sensitive, and provide repeatable and reproducible measurements; and have stable chemistry that provides consistent results.
  • a multilayered laminate for an opto- enzymatic analyte sensor comprising an oxygen sensing matrix layer, an optional barrier layer, an enzymatic hydrogel matrix layer, and an optional membrane.
  • the oxygen sensing matrix layer preferably: comprises a metalloporphyrin dye and a thermoplastic polymer; comprises at least a first surface adjacent to at least one optical waveguide and emits an optical emission in response to optical excitation via the waveguide wherein the optical characteristics of the optical emission are dependent on the partial pressure of oxygen in the oxygen sensing matrix; and/or comprises at least a second surface opposite the first surface and adjacent to the enzymatic hydrogel matrix layer either directly or through the optional barrier layer.
  • the optional barrier layer when present, is preferably: made from a solution comprising a first hydrogel monomer, a first urethane monomer, and a first crosslinker; and/or is disposed between and directly adhered to the oxygen sensing matrix layer and the enzymatic hydrogel layer and permits transfer of oxygen to occur readily between the oxygen sensing matrix and the enzymatic hydrogel layer such that changes in the partial pressure of oxygen in the enzymatic hydrogel layer are rapidly reflected in the partial pressure of oxygen in the oxygen sensing matrix. It also serves to limit or even block the entry of lipids into the oxygen sensing matrix.
  • the enzymatic hydrogel matrix layer is preferably made from a solution comprising at least one enzyme which catalyzes a chemical reaction consuming at least one target analyte and oxygen, at least one water soluble polymer or monomer and a multifunctional crosslinker; comprises at least a lower surface directly adjacent to the oxygen sensing matrix layer either directly or through the optional barrier layer and an upper surface optionally covered by the optional membrane layer; and/or each said enzymatic hydrogel matrix layer has at least one region permitting the entry of oxygen and a target analyte into the enzymatic hydrogel matrix layer.
  • the optional membrane layer when present, is preferably made from a solution comprising a second hydrogel monomer, a second urethane monomer, and a second crosslinker; and/or allows for a more consistent flow of oxygen into the enzymatic hydrogel layer immediately below and substantially blocks or reduces the quantity of lipids from outside the device, including from the blood, from entering lower layers of the laminate.
  • a method of making a laminate which includes: depositing an oxygen sensing matrix solution in at least two separate locations on a substrate and allowing a solvent in the solution to evaporate to form an oxygen sensing matrix layer; optionally depositing a barrier layer solution on top of each oxygen sensing matrix layer and allowing it to cure to form a barrier layer; depositing an enzymatic hydrogel matrix solution over at least one barrier layer, or over at least one oxygen sensing matrix layer if there is no barrier layer, and allowing it to cure to form at least one enzymatic hydrogel matrix layer; and optionally depositing a membrane layer solution on top of each enzymatic hydrogel matrix layer and allowing it to cure to form at least one membrane layer.
  • the oxygen sensing matrix solution is deposited in at least three separate locations, and the same enzymatic hydrogel matrix layer is placed over at least one of the three separate locations either directly or over a barrier layer covering the oxygen sensing matrix layer and allowed to cure.
  • a second enzymatic hydrogel layer comprising a different enzyme than the enzymatic hydrogel layer is placed over at least one of the three separate locations either directly or over a barrier layer covering the oxygen sensing matrix layer and allowed to cure.
  • the at least one oxygen sensing matrix include one or more of the following: the oxygen sensing matrix is formed through a crosslinking reaction in the presence of a crosslinker; the oxygen sensing matrix is formed by evaporation of solvent from a polymer-containing solution; and/or the metalloporphyrin dye is selected from the group consisting of IrOEP (iridium octaethylporphyrin), PdOEP (palladium octaethylporphyrin), Pttfpp (platinum tetrakis-(2,3,4,5,6-pentafluorophenyl)-porphyrin), Irtfpp (iridium tetrakis-(2,3,4,5,6-pentafluorophenyl)-porphyrin), PtOEP (platinum octaethylporphyrin), and combinations thereof.
  • IrOEP iridium octaethyl
  • the thermoplastic polymer in the OSP layer has a glass transition temperature lower than that of the substrate it lies upon, and is heated to at least its glass transition temperature, but below the glass transition temperature of the substrate, for a period of time following or accompanying heating to remove the solvent from the OSP layer.
  • At least a portion of the OSP layer is plasma treated prior to formation or placement of the barrier layer and/or at least a portion of the barrier layer is plasma treated prior to the formation or deposition of the enzymatic hydrogel matrix.
  • the enzyme includes one or more enzymes selected from glucose oxidase, catalase, lactase oxidase, 3- hydroxybutyrate dehydrogenase, NADH oxidase, diaphorase, other enzymes that react with an analyte and consume oxygen in the process, and combinations of the foregoing.
  • the multilayer laminate is made with biocompatible materials that allow for implantation and temporary residence in a human or other mammalian body.
  • the phosphorescent signal generated in the laminate and measured by the sensor allows for quantitative analysis of the target analyte, and the system may be directed toward detecting different concentration ranges and/or different analytes in the same sensor by use of different enzymatic hydrogel matrix materials in the same laminate or sensor.
  • a sensor can measure two or three different target ranges of glucose, glucose and lactate, glucose and ketone, two different ranges of glucose and lactate, and two different ranges of glucose and ketone.
  • the laminate and the sensor are stable for at least fourteen (14) days of use, fifteen (15) or more days of use, up to 2 weeks of use, and/or for two to three weeks of use.
  • the barrier layer substantially or completely blocks lipids, including those from the fluid containing the analyte being tested, from entering the oxygen sensing matrix layer through the barrier layer.
  • an oxygen mediated opto- enzymatic analyte transducer comprising an oxygen transport membrane, an optical oxygen restrictive laminate, at least one oxygen sensing matrix for measuring a target analyte (also called a target analyte dependent oxygen sensing matrix), at least one enzymatic hydrogel matrix, and at least one reference oxygen sensing matrix that serves as a reference (also called reference oxygen sensing matrix).
  • the oxygen transport membrane has one or more of the following characteristics: it is in contact with a fluid, such as interstitial fluid, comprising one or more target analytes and oxygen, it restricts the transport rates of said one or more target analytes through the oxygen transport membrane relative to the oxygen transport rate though the oxygen transport membrane; and/or the oxygen transport membrane comprises one or more polymer layers.
  • the oxygen transport membrane lies directly upon the membrane or the enzymatic hydrogel layer, and contains at least one port or hole to allow transportation of one or more analytes of interest into the sensor.
  • the optical oxygen restrictive laminate has one or more of the following characteristics: it comprises at least one surface in contact with said fluid comprising one or more target analytes and oxygen and/or it comprises at least two polymer laminate layers contacting one or more optical waveguides transmitting light.
  • the first polymer laminate layer of the oxygen restrictive laminate has one or more of the following characteristics: it is in contact with the fluid comprising one or more target analytes and oxygen; it has a lower index of refraction than said optical waveguide material, it is biocompatible for implantation into an animal; it restricts the oxygen transport rate relative to the oxygen transport rate though the oxygen transport layer; and/or it restricts the transport rates of said one or more target analytes through said oxygen transport membrane relative to the oxygen transport rate though the oxygen transport membrane.
  • the second polymer laminate layer of the optical oxygen restrictive laminate has one or more of the following characteristics: it adheres to the first polymer laminate layer of the oxygen restrictive laminate; it has a lower index of refraction than said optical waveguide material has a lower index of refraction than the waveguide material; it is optionally in contact with said fluid comprising one or more target analytes and oxygen; it is biocompatible for implantation into an animal; and/or it optionally adheres to a third polymer laminate layer of the oxygen restrictive laminate or to the oxygen transport membrane.
  • the oxygen sensing matrix includes one or more of the following characteristics: each oxygen sensing matrix emits an optical emission in response to optical excitation wherein the optical characteristics of the optical emission are dependent on the partial pressure of oxygen in the oxygen sensing matrix; the oxygen sensing matrix does not consume a substantial amount of oxygen; it comprises at least one surface adjacent to the optical oxygen restrictive laminate; the oxygen sensing matrix comprises at least a second surface adjacent to the at least one optical waveguide within the optical oxygen restrictive laminate; the target analyte dependent oxygen sensing matrix comprises at least one surface adhered to an enzymatic hydrogel matrix either directly or through a barrier layer between the oxygen sensing matrix and enzymatic hydrogel matrix; and/or the target analyte dependent oxygen sensing matrix is contacted on all surfaces by the oxygen restrictive laminate, an enzymatic hydrogel matrix and/or a barrier layer.
  • the at least one enzymatic hydrogel matrix includes one or more of the following: each enzymatic hydrogel matrix catalyzes a chemical reaction consuming at least one target analyte and oxygen; each enzymatic hydrogel matrix in a given sensor may have the same or different target analyte; the enzymatic hydrogel matrix comprises at least a first surface bounded by the oxygen transport layer; the enzymatic hydrogel matrix comprises least a second surface adhered or bonded either directly or indirectly (such as through a barrier layer) to a target analyte dependent oxygen sensing matrix such that changes in the partial pressure of oxygen in the enzymatic hydrogel matrix are rapidly reflected in the partial pressure of oxygen in the oxygen sensing matrix; each enzymatic hydrogel matrix has a target analyte entrance region that is not bounded by either the oxygen transport membrane or the oxygen restrictive matrix; each enzymatic hydrogel matrix is permissive to target analyte transport relative to the target analyt
  • the at least one reference oxygen sensing matrix includes one or more of the following characteristics: each reference oxygen sensing matrix emits an optical emission in response to optical excitation, wherein the optical characteristics of the optical emission are dependent on the partial pressure of oxygen in the reference oxygen sensing matrix; each reference oxygen sensing matrix is substantially not oxygen consuming; each reference oxygen sensing matrix comprises at least one surface adjacent to the optical oxygen restrictive laminate; each reference oxygen sensing matrix comprises at least a second surface adjacent to the optical oxygen restrictive laminate interrogated by light transmitted through the optical oxygen restrictive laminate; each reference oxygen sensing matrix is bound on all surfaces by the oxygen restrictive laminate and the oxygen transport membrane; and/or each reference oxygen sensing matrix is in contact with an enzymatic hydrogel matrix.
  • FIG. 1 is a schematic cross-section of a portion of a layered optical sensor according to implementations disclosed herein.
  • This disclosure relates to temporarily implantable optical enzymatic physiological sensors, more specifically, it relates to improvements to layered analyte sensors including, but not limited to, those described in U.S. Patent No. 7,146,203, U.S. Patent Publication No. 2019/0021672, and U.S. Patent Publication No. 2020/0008719, the disclosures of each of which references are hereby incorporated by reference in their entireties.
  • the disclosed sensors may include the presence of improved bonding or adherence between at least two layers of the sensor, either directly or through an intermediate layer which provides improved function of the sensor.
  • the enzymatic hydrogel there is bonding or adherence of the enzymatic hydrogel to the target analyte dependent oxygen sensing polymer matrix such that changes in the partial pressure of oxygen in the enzymatic hydrogel matrix are rapidly reflected in the partial pressure of oxygen in the oxygen sensing matrix.
  • the sensors provide an implantable, wearable, disposable and ideally low-cost way to continuously sense one or more analytes in the body, such as glucose.
  • the sensor is preferably relatively small in size, in low- cost to manufacture, has a short response time when exposed to target analyte(s), and made with biocompatible materials.
  • the disclosed sensor includes a phosphorescent oxygen sensitive polymer (OSP) and an enzymatic hydrogel layer together with one or more membranes or layers which provide functions such as protection or adhesion to continuously detect one or more target analytes including, but not limited to, glucose, lactate, and ketone (e.g. beta-hydroxybutyrate (BHB)).
  • OSP phosphorescent oxygen sensitive polymer
  • ketone e.g. beta-hydroxybutyrate (BHB)
  • BHB beta-hydroxybutyrate
  • the layers comprising the sensing chemistry are permanently embedded within a laminate that is placed in the underneath the skin in contact with interstitial fluid.
  • the multilayered laminate immobilizes the sensing chemistry so that it remains embedded within the sensor during use.
  • the stability and functionality of the sensor can be changed by the combination of different chemistry formulations.
  • FIG. 1 shows a schematic of the chemical sensing laminate portion of the sensor device according to certain implementations.
  • Laminate portions according to the disclosure herein and as illustrated in FIG. 1 form a portion of a larger sensor system as discussed and illustrated in U.S. Patent No. 7,146,203, U.S. Patent Publication No. 2019/0021672, and U.S. Patent Publication No. 2020/0008719.
  • Figures 20A-20E and 38-40 (among others) and their associated text in US 2020/0008719 provides additional background and context for the improvements to the chemical sensing laminate disclosed herein.
  • the first (bottom) layer 10 contains one or more depositions of oxygen sensing polymer (OSP) material and is in contact with one or more waveguides 50 immediately below the laminate.
  • OSP oxygen sensing polymer
  • an enzymatic hydrogel layer 30 which contains a polymer matrix including at least one enzyme which reacts with a target analyte such as glucose, lactate or ketone and consumes oxygen in the process.
  • the enzymatic hydrogel and OSP layers may be separated by an optional barrier layer 20 which is highly permeable to oxygen and water, and may also serve to protect the OSP layer from exposure to lipids (the presence of which can negatively affect performance) and enhance the adhesion between the OSP and enzymatic hydrogel layers.
  • an optional membrane 40 may be placed on the top of the enzymatic hydrogel layer to provide protection and help control the flow of oxygen and analytes into and across the area of the enzymatic hydrogel layer.
  • the oxygen and analyte molecules flow from one or more open ports in the sensor onto the membrane 40 (or the enzymatic hydrogel layer 30 in the absence of optional membrane 40)
  • the first (bottom) layer of the chemical sensing laminate portion comprises the oxygen sensing polymer (OSP).
  • the OSP layer also referred to herein as the oxygen sensing layer, lies immediately above the waveguides and has one or more surfaces directly in contact with the optical waveguides.
  • the waveguides permit the passage of light both into and away from the region of the OSP layer.
  • the OSP preferably has a lower index of refraction than the waveguide material and is optically coupled well with the waveguide material.
  • the OSP layer contains a phosphorescent dye, generally a metalloporphyrin, which is excited by green light and emits red light that is delivered and carried away by one or more waveguides.
  • the emission and phosphorescence lifetime of the dye is dependent on the oxygen concentration and therefore allows the OSP to function as an oxygen sensor.
  • the OSP layer does not have direct surface contact with the interstitial fluid or fluid containing the target analyte(s) and oxygen.
  • the OSP layer in preferred implementations is very stable with no substantial changes in performance or capability over at least 10 days, at least 14 days, at least 15 days, at least 21 days or more, either with or without the enzymatic hydrogel.
  • the OSP layer may be made with either of two category approaches.
  • One category approach is to build up the OSP polymer from monomers, dyes, and initiators.
  • the initiator generates free radicals and radical covalent bond all monomers and dyes into polymer matrix.
  • the polymer matrix may contain zero or more thermoplastic polymers to enhance the dye performance.
  • the second category approach is to embed or bind the dye (e.g. metalloporphyrin), including covalently, in a polymer matrix. Based on this second approach, it was discovered that in some implementations the OSP sensitivity/stability is related to one or more factors, such as the polymer material, polymer molecular weight, dye material, dye concentration and/or dispensing solvent.
  • the polymer material and the molecular weight can lead to different oxygen sensitivity from the OSP; for example, mixing poly(styrene-co- acrylonitrile) (PSAN) polymer can drag the OSP oxygen sensitivity range from 0%-5% to 15%-21%.
  • PSAN poly(styrene-co- acrylonitrile)
  • Different dye material has a different solubility in solvents. In some implementations, preferred solvents dissolve the dye fully without precipitation and form dimers.
  • the OSP layer solution containing the solvent is dispensed, all of the solvent should be removed from the OSP layer, which for many solvents requires heating or baking to achieve full solvent removal. Solvent left in the OSP layer is detrimental to the stability of the OSP layer performance. And each solvent has different vapor pressure, so the baking process needs to be optimized based on the solvent.
  • the OSP layer lies under a highly oxygen- permeable barrier layer 20. Since a wide range of polymers can be dissolved in lipid solutions, the barrier should be able to essentially block transmission of lipid into the OSP layer, but still permit water to come across to the OSP and also allow for transmission of oxygen into and out of the OSP.
  • the barrier layer can be made of any suitable material that meets these requirements, including but not limited to fluorinated ethylene propylene (FEP), polyurethane and/or polyester, and should also adhere or bond well to both the OSP and the enzymatic hydrogel layer which is in direct contact with the barrier on the other side from the OSP either directly or by the use of an adhesive material.
  • FEP fluorinated ethylene propylene
  • the barrier should be very oxygen permeable, but care should be taken with materials that have very high oxygen permeability to ensure that flux of oxygen does not cause crosstalk signals between neighboring channels in the sensor.
  • the OSP acts to detect the target analyte (e.g. glucose, lactate, and ketone) indirectly via a recognition event, whereby the enzymes in the enzymatic hydrogel layer react with target analyte (e.g. glucose, lactate or ketone) and in the process consume oxygen.
  • target analyte e.g. glucose, lactate or ketone
  • oxygen is highly permeable through the layers, a change in oxygen concentration in the enzymatic hydrogel due to oxygen consumption by the reaction with analyte leads to a change in oxygen concentration the OSP layer.
  • the change in OSP oxygen is then detected by the dye, because the presence of oxygen quenches the phosphorescence of the dye.
  • the waveguides detect the changes in fluorescence and the signal is converted to the unit of target analyte (e.g. mg/dL for glucose), and this allows for continuous measurement of analyte concentrations.
  • the reference OSP must be isolated from the enzymatic hydrogel material (and any barrier between the enzymatic hydrogel layer and the OSP) and is preferably sealed under a separate oxygen permeable and lipid impermeable barrier, which is preferably the same material as any barrier 40 between the OSP and enzymatic hydrogel. This allows for a separate measurement of OSP oxygen concentration independent of target analyte. Ultimately, the data from both the enzyme-adjacent OSP and the reference OSP will be used together to establish the final sensor measurement of analyte concentration.
  • the enzymatic hydrogel layer 30 is formed by using a crosslinker to covalently bind an enzyme to a polymer thereby immobilizing the enzyme in the polymer layer within the sensor laminate.
  • the enzyme used in a given enzymatic hydrogel should target a specific analyte of interest found in the interstitial fluid under the skin such as glucose, glucose, ketones (e.g. beta-hydroxybutyrate (BHB)), galactose, lactose, peroxide, cholesterol, amino acids, fructose, alcohol, lactic acid, and the like.
  • the enzymatic hydrogel layer should be permeable by the analyte, and when the enzyme reacts with the target analyte it should consume oxygen according to a known stoichiometric ratio. Because the enzymatic hydrogel and OSP layers allow for communication of oxygen between them (either directly or through a barrier layer) the oxygen consumed in the enzyme reaction will be sensed by the OSP layer allowing for the indirect measurement of the analyte concentration via the change in oxygen concentration in the OSP.
  • Suitable enzymes include, but are not limited to, glucose oxidase, catalase, lactate oxidase, 3 -hydroxybutyrate dehydrogenase, and diaphorase.
  • Sensors which measure more than one analyte have a similar structure to sensors that measure one analyte (See e.g. US 2020/0008719) with some minor modifications.
  • each different analyte should have its own separate enzymatic hydrogel that is reactive with that analyte.
  • each of the different types of enzymatic hydrogels should be in fluid contact (directly or through a barrier) with its own separate OSP portion (and separate associated barrier layer between the OSP and enzymatic hydrogel, if present) to permit the sensor to distinguish which enzymatic hydrogel corresponding to which analyte is consuming the oxygen, thereby allowing for calculation of concentration of two or more different analytes simultaneously with a single sensor.
  • the laminate forming the chemical portion of the sensor is made by building up layer upon layer from the bottom up by casting/pouring a liquid layer that is then cured or dried and/or by placing a pre-formed membrane or thin sheet of material upon the lower layer (with or without adhesive), care needs to be taken to ensure that there will be no significant chemical crossover between the portions noted above that should remain separate in order to accurately interpret the output of the sensor and calculate concentrations of multiple analytes.
  • the barrier layer 20 when present, can serve similar functions such as allowing for ease and consistency in oxygen movement as well as substantially blocking lipids from entering the OSP layer.
  • the enzyme hydrogel reacts with analyte while consuming oxygen that is present in the OSP. The change in oxygen concentration is detected optically and as a result allows for the continuous optical measurement of analyte concentrations.
  • the membrane (and/or the barrier 20, if present) is made from a polyester polyurethane.
  • This material has excellent resistance to lipids and is permeable to oxygen, glucose and other analytes of interest.
  • the thickness and consistency of thickness of the membrane is important. Too thin of a membrane does not permit the membrane to serve as an effective barrier and too thick of a membrane can cause a concentration gradient to form in the membrane that can reduce the accuracy of the measurements.
  • the membrane and/or barrier has a thickness of about 10 microns to about 50 microns, including about 10 microns to about 25 microns, about 15 microns to about 25 microns.
  • the useful thickness for a membrane or barrier will also vary with the chemistry of the material which affects how easily things like analyte and oxygen can move through the material, and can be determined at least in part by application of Fick’s law of diffusion.
  • the chemical portion of the sensor illustrated in FIG. 1 is generally prepared by depositing the material to form each layer directly on the layer beneath it in the finished sensor structure or on a substrate which is then placed upon the layer directly beneath it in the finished structure.
  • FIG. 1 is schematic and should not be taken as requiring that the various layers 10, 20, 30 and 40 are uniform and unbroken across the entire surface area of the laminate sensor or that the illustrated thicknesses are indicative of the actual relative thicknesses in a given sensor implementation; in some implementations, such as when more than one analyte is being measured by the sensor (i.e.
  • the laminate can be formed by building up the layers one on top of the other in a series of wells that create separation between different channels, each of which can be interrogated by its own separate waveguide to allow for each channel to be accurately measured without interference from other channels which may be associated with enzymatic hydrogels having different chemistry or concentrations that can consume oxygen at a different rate.
  • the OSP formulation comprises, or in some implementations consists of or consists essentially of, a metalloporphyrin dye, polystyrene, and solvent.
  • the OSP layer should be a hydrophobic layer.
  • the polystyrene forms the oxygen permeable polymer matrix to encapsulate the metalloporphyrin dye.
  • the metalloporphyrin dye is the signal transducer that shows a change in phosphorescence lifetime as a functional as oxygen concentration.
  • the solvent is a liquid media to dissolve both metalloporphyrin dye and polystyrene.
  • the solvent used in this OSP formulation should be able to fully dissolve both the dye and polystyrene, minimize dimerization of the dye, and be able to fully evaporate within 8-12 hours or overnight in a 50°C oven without causing the dissolution of the sensor waveguide material upon which the solution to form the layer is deposited (such as NOA63 from Norland Products, Inc., Jamesburg, New Jersey, USA). A summary of the components and their function is given below.
  • the metalloporphyrin dyes suitable for use in the OSP formulation include but are not limited to IrOEP (iridium octaethylporphyrin), PdOEP (palladium octaethylporphyrin), Pttfpp (platinum tetrakis-(2,3,4,5,6-pentafluorophenyl)-porphyrin), Irtfpp (iridium tetrakis-(2,3,4,5,6-pentafluorophenyl)-porphyrin), PtOEP (platinum octaethylporphyrin), or a mixture of different dyes.
  • IrOEP iridium octaethylporphyrin
  • PdOEP palladium octaethylporphyrin
  • Pttfpp platinum tetrakis-(2,3,4,5,6-pentaflu
  • iridium-containing metalloporphyrins have been found to demonstrate sensitivity between 0% oxygen and 5% oxygen, including 0% to 1%, 0% to 2%, 0% to 3%, and 0% to 4% which is a wider range of oxygen concentrations as compared to other metals such as platinum, palladium and copper.
  • a metalloporphyrin includes at least one functional group that is available for covalent bonding with the thermoplastic polymer forming the OSP matrix, either to the polymer directly or by using a crosslinker with suitable chemistry for the groups being joined.
  • Suitable functional terminal or pendant groups include, but are not limited to, N-methylacrylate.
  • N-methyl acrylate terminated iridium tetrakis- (2,3,4,5,6-pentafluorophenyl)-porphyrin (Irtfpp-N-MA) may be used.
  • Functional groups are present on the TFPP to be covalently linked to a polymer, such as a polystyrene or fluorinated polystyrene, with a bivalent -SH linker.
  • a polymer such as a polystyrene or fluorinated polystyrene
  • the reaction can be performed by mixing the materials at about 60°C in DMF. Bonding the metalloporphyrin to the polymer matrix of the OSP allows for the metalloporphyrin to resist being leached out of the OSP layer by any lipids that may reach the OSP layer. Therefore, in some implementations, covalently bonding metalloporphyrin to the thermoplastic polymer can allow for the elimination of a barrier layer without a degradation in performance and stability that could result when the barrier layer is absent and some lipids are present in the OSP.
  • the molecular weight (Mw) of the polystyrene in this OSP formulation may be about 1000 to 40,000 Daltons, about 1000 to 6000 Daltons, about 1000 to 4000 Daltons, about 6000 to 10,000 Daltons, about 10,000 to 40,000 Daltons, and values within and bordering such ranges, including but not limited to about 1000 Daltons, about 2000 Daltons, about 4000 Daltons, about 6000 Daltons, about 10,000 Daltons, and about 40,000 Daltons, although materials having molecular weights outside of these ranges can also be acceptable.
  • the weight ratio of dye over polystyrene can be 0.5% to 5%, including 0.5%, 1%, 2%, 3%, and 5%, although combinations at other ratios can also be used.
  • the solvents used this OSP formulation include, but are not limited to, xylene, toluene, acetone, ethyl acetate, THF, dichloromethane, chloroform, chlorobenzene, or a mixture thereof. Other solvents meeting the criteria noted herein may also be used.
  • the OSP may be prepared by mixing the components in a suitable vessel at any suitable temperature, including but not limited to about 2°C to 50°C and about 15°C to 30°C, including 2-8°C, 20 °C, 37°C, and 50°C, limited of course by the volatility or boiling point of the solvent and at dry or humid conditions.
  • a suitable temperature including but not limited to about 2°C to 50°C and about 15°C to 30°C, including 2-8°C, 20 °C, 37°C, and 50°C, limited of course by the volatility or boiling point of the solvent and at dry or humid conditions.
  • the temperature used for solvent evaporation includes, but is not limited to 20°C to 100°C, including 20°C, 37°C, 50°C, 80°C, and 100°C, and for a period of about 20 minutes to 48 hours, including about 20 minutes to 24 hours, about 1 hour to about 24 hours, about 24 hours to about 48 hours, and including 20 mins, 1 hour, 2 hours, 5 hours, 10 hours, and 24 hours, or longer until the solvent has fully evaporated.
  • the OSP layer is placed in the oven at a first temperature that is high enough to drive off the solvent but less than the glass transition temperature (Tg) of the polymer forming the matrix for a period of about 20 minutes to 1-3 hours until the solvent is evaporated.
  • Tg glass transition temperature
  • the oven temperature is then raised to at or above the Tg of the polymer (but below the Tg of any materials forming the sensor well or other materials containing and providing shape to the matrix) and the OSP layer is left at the temperature at or above the Tg of the polymer for about 12 hours to about 48 hours.
  • the polymer of the OSP layer By allowing the polymer of the OSP layer to sit at or above its Tg, it will reflow and enter a lower or minimum energy state that can increase its stability over time as compared to a layer that was not heat treated in this manner, especially in the presence of water.
  • the thickness of the OSP layer needs to be enough to fully and evenly cover where the waveguide interfaces with the OSP layer.
  • the thickness of the OSP layer is from about 1 mil to 20 mil, including about 1 mil to about 10 mil, about 2 mil, about 3 mil, about 5 mil, and about 10 mil.
  • the OSP formulation comprises (or in some implementations consists of or consists essentially of) the following:
  • the OSP was formulated by mixing the dye and polymer with the solvent in the amounts above at room temperature until the solids appeared to be fully dissolved.
  • a liner was placed on the sensor surface/waveguide material and then OSP wells were laser cut through the liner and into the waveguide material.
  • the OSP solution was dispensed into the sensor wells, overfilling above the liner, to fill into the bottom of the well and coat the waveguide interface.
  • the OSP coated card is then baked for 1-48 hours at 35-50 deg C, after which the liner is removed, leaving a patterned of filled OSP wells. .
  • the sensor is then baked at 50°C until the remaining solvent has evaporated.
  • the senor with the OSP layer may be placed in an oven at 35-45°C for 1-2 hours to remove the solvent, after which the liner is removed and then the OSP layer placed back in the oven and reheated to 50°C, which is at or above the Tg of the polystyrene but below the 55°C Tg of the substrate forming the sensor well, and allowed to reflow and eliminate any remaining gas bubbles from the OSP layer for a period of about 24-48 hours.
  • the oxygen sensing polymer matrix was fabricated using the same method above, except using toluene as the solvent instead of chloroform.
  • OSP layer may be used.
  • the oxygen sensing matrix is made by combining 1.7 mL of filtered 3-(3-methacryloxy-2-hydroxypropoxy)propylbis(trimethylsiloxy)methylsilane, 1.0 mL of N,N-Dimethylacrylamide, 10 mg Diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide, 28 pL of Diethylene glycol dimethacrylate and 45 mg of Pttfpp-N-MA, which is Platinum (II) 5- [(4-aminopropylmethacrylamide)-2,3,5,6 tetrafluorophenyl]-10,15,20-tris(pentafluorophenyl) porphyrin.
  • the resulting mixture was mixed for 5 min at 2000 rpm.
  • the oxygen sensing matrix solution was dispensed into the sensor well and then placed in a closed chamber and degassed with nitrogen for 3 min to 0.1% O2.
  • the sensing matrix solution was cured under a UV lamp for 120 s.
  • the oxygen sensing matrix is constructed from 1.7 mL of Filtered 3-(3-methacryloxy-2-hydroxypropoxy)propylbis(trimethylsiloxy)methylsilane, 1.0 mL of N,N-Dimethylacrylamide, 10 mg Diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide, 28 pL of Diethylene glycol dimethacrylate and 45 mg of Pttfpp-N-MA, which is Platinum (II) 5- [(4-aminopropylmethacrylamide)-2,3,5,6 tetrafluorophenyl]-10,15,20-tris(pentafluorophenyl) porphyrin.
  • 3% of a propylene imine tri -functional polyaziridine, trimethylolpropane tris(2 -methyl- 1 -aziridine propionate) was added by volume.
  • the solution was mixed for 5 min at 2000 rpm.
  • the oxygen sensing matrix solution was dispensed into the sensor, and then placed in a closed chamber and degassed with nitrogen for 3 min to 0.1% 02.
  • the sensing matrix solution was cured with UV for 120 s.
  • the degassed oxygen sensing matrix was cured with UV for 60 s, and/or a 5% to 10% solution of trimethylolpropane tris(2 -methyl- 1 -aziridine propionate) by volume was added in lieu of a 3% solution.
  • the propylene imine tri -functional polyaziridine, trimethylolpropane tris(2 -methyl- 1 -aziridine propionate) and is commercially available as PZ-28 from Poly Aziridine LLC (Palm Beach, FL, USA) and from other manufacturers such as PFAZ-322 (Sybron Chemicals, Birmingham, NJ, USA) or CX-100 (Zeneca, Waalwijk, Netherlands).
  • polyaziridine and polyepoxide could be used to crosslink carboxy, amide, hydroxyl and amine groups. It has been found that polyaziridines and the polyepoxides, if used separately or in combination with each other or with other crosslinkers, may enhance the protective properties of protein coatings, especially the water resistance of the films can easily be increased by the use of these reagents. (See European Patent Application EP0969056A1, published Jan. 5, 2000).
  • the enzymatic hydrogel is a polymer layer or matrix on top of OSP laminate layer either directly on top or in some implementations it is separated from the OSP layer by a barrier layer.
  • This layer is to provide an immobilized enzymatic hydrogel containing at least one enzyme that can react with target analytes (including glucose, lactate, and ketone) and in the process consumes oxygen.
  • the enzymatic hydrogel layer may be made by mixing enzyme, polymer and crosslinker and allowing them to react. Other components may be included in the reaction mixture in some implementations.
  • suitable enzymes include, but are not limited to glucose oxidase, catalase, lactate oxidase, 3 -hydroxybutyrate dehydrogenase, and diaphorase.
  • polymers include, but are not limited to:
  • More than one type of enzymatic hydrogel may be included in a device.
  • a device having three channels for measurement and one channel as a reference may have a first channel comprising a first enzymatic hydrogel that measures a first analyte, a second channel comprising a second enzymatic hydrogel that is tuned to measure a first concentration range of a second analyte, and a third channel comprising a third enzymatic hydrogel that is tuned to measure a second concentration range of the second analyte.
  • such a device may include three measurement channels each containing an enzymatic hydrogel tuned to measure different concentration ranges of the same analyte.
  • Example reactions appear below with additional details. These examples are provided as examples of particular implementations of the enzymatic hydrogels disclosed and claimed herein. They are meant to be illustrative and for purposes of example only, and should not be considered restrictive or limiting. Modifications and alterations to the materials, methods, conditions and the like can be made as will be understood by those skilled in the art. Additionally, other enzymes, polymers, and crosslinkers (including bi- tri- and tetra-functional crosslinkers) may also be used and that those others and the ones listed above and elsewhere herein may also be used in combinations other than shown and discussed below.
  • a multienzyme system it may be desirable to control the molar ratio between two enzymes for loading into a hydrogel, and the ratio is dependent at least in part on the enzyme Michaelis-Menten parameter.
  • the degree of crosslinking needed for enzyme stabilization for each enzyme may differ.
  • An enzyme may be pre-treated before including in the hydrogel with the other enzyme(s). The pretreatment may be used to define the number of residues on the enzyme that will be crosslinked by blocking or by labeling them using reactions in solution, prior to mixing with the hydrogel pre-cursors.
  • Traut's reagent (2-iminothiolane) reacts with primary amines from the enzyme ( — NH2) to introduce sulfhydryl ( — SH) groups while maintaining charge properties similar to the original amino group.
  • sulfhydryl groups can be specifically targeted for reaction in a variety of useful labeling, crosslinking and immobilization procedures.
  • the pH of this reaction should be between pH 6-8.
  • thiolene polymerization conditions are typically chosen to minimize side reactions and stabilize the enzyme.
  • Sulfhydryl-reactive chemical groups include haloacetyls, maleimides, aziridines, acryloyls, arylating agents, vinyl sulfones, pyridyl disulfides, TNB-thiols (including DTNB 5,5-dithio-bis-(2-nitrobenzoic acid)) and disulfide reducing agents. Most of these groups conjugate to sulfhydryls by either alkylation (usually the formation of a thioether bond) or disulfide exchange (formation of a disulfide bond).
  • An aziridine reactive group is a three-membered ring composed of one nitrogen and two carbon atoms.
  • the highly hindered nature of this heterocyclic ring gives it strong reactivity toward nucleophiles.
  • Sulfhydryls will react with aziridine-containing reagents in a ring-opening process, forming thioether bonds.
  • the simplest aziridine compound, ethylenimine, can be used to transform available sulfhydryl groups into amines.
  • the oxidase enzyme such as GOX, CAT, lactase, etc.
  • Traut’ s reagent to generate -SH reactive groups. These groups are then reacted with an aziridine or an aziridine derivative to create an immobilized enzyme network.
  • This crosslinked enzyme network may be modified by adding an appropriate hygroscopic monomer or polymer such as PAA to the network to modify transport through the network and to the crosslinked enzyme.
  • the pretreatment may be used to inhibit the degree of crosslinking for an enzyme that is achieved in a subsequent general reaction that immobilizes the enzyme into a hydrogel.
  • thiol-functionalized macromers can react with each other to form disulfide linkages, making them inaccessible for subsequent reaction with alkenes.
  • the extent of the nucleophilic functional groups (e.g., sulfhydryls) introduced onto the lysine (Lys) residues of enzymes (such as Lactate oxidase) can be controlled by the availability of an initiator, such as 2-iminothiolane (Trauf s reagent).
  • the sulfhydryl modified molecule can be used to initiate a further reaction so that the functional group is inaccessible in further reactions.
  • the pretreatment may be used to create functional groups for secondary reactions to further modify the enzyme.
  • Thiols on macromers can react with various functional groups that are present on biologies (i.e., off-target reactions leading to oxidation of cysteine residues on proteins).
  • the sulfhydryl modified molecule can be used to initiate a further reaction to add a crosslinker for subsequent reactions.
  • the extent of the nucleophilic functional groups (e.g., sulfhydryls) introduced onto the lysine (Lys) residues of enzymes can be controlled by the availability of an initiator, such as 2-iminothiolane (Trauf s reagent).
  • the number of lysine residues that are converted into thiol functional groups may be set by the molar ratio of the primary amines (e.g., Lys residues on lactate oxidase) to 2-iminothiolane (Trauf s reagent).
  • the pretreatment may be used to create functional groups for secondary reactions to modify the enzyme further.
  • the SH group on macromer can have secondary reactions with PEG-maleimide, as the diagram shown above.
  • the pretreated enzyme e.g. lactate oxidase
  • the pretreated enzymes can be linked to a bifunctional PEG-maleimide crosslinker, as shown in the diagram below.
  • the PEG-maleimide crosslinker can block the SH group on enzymes for further loading onto hydrogel precursors. Only the rest 50% of primary amine on the surface of enzyme is accessible for additional loading onto hydrogel precursors, such as polyacrylic acid (PAA).
  • PAA polyacrylic acid
  • the free primary amine can further react with one arm of a trifunctional aziridine crosslinker, with one arm of a trifunctional epoxide crosslinker, or with one arm of a tetrafunctional epoxide crosslinker.
  • the enzyme macromers are loaded with polyfunctional aziridine or epoxide.
  • the nonreact aziridine or epoxide groups will further link the enzyme to the hydrogel precursors (e.g. PAA, polymethacrylate (PMA), or Poly(acrylamide/acrylic acid)) to form the hydrogel.
  • the structure of trifunctional and tetrafunctional crosslinkers can also provide flexibility of the hydrogel matrix.
  • the pretreated enzyme e.g.
  • lactate oxidase may have 100% of the primary amine converted into thiol functional group, and 0% of primary amine are still displaying on the enzyme surface.
  • the pretreated enzymes may can further link to a bifunctional PEG-maleimide crosslinker, as shown in the diagram above. In this case, these specific enzyme macromers cannot incorporate into a subsequent reaction with an aziridine or epoxide crosslinker with a monomer such as PAA.
  • the pretreated enzyme may have 50% of the primary amine converted into the thiol functional group.
  • the other 50% of primary amines are still displaying on the enzyme surface.
  • the pretreated enzymes can be linked to a bifunctional PEG-maleimide crosslinker, as shown in the diagram above.
  • the PEG-maleimide crosslinker can block the SH group on enzymes for further loading onto hydrogel precursors.
  • the remaining 50% of primary amine on the surface on the enzyme can react further with a monofunctional PEG-epoxide crosslinker, so no free primary amine can bind to an aziridine group or an epoxide group.
  • These specific enzyme macromers cannot incorporate into a subsequent reaction with an aziridine or epoxide crosslinker with a monomer such as PAA.
  • the pretreated enzyme e.g. lactate oxidase
  • the other 80% of primary amine is still displaying on the enzyme surface.
  • the pretreated enzymes can be linked to a bifunctional PEG crosslinker with one side react group as a maleimide and the other side react group as a primary amine.
  • the bifunctional PEG crosslinker can extent the SH group to a primary amine group with a PEG spacer arm linker. After the pretreated enzyme is linked with the bifunctional PEG, only the PEG amine on the surface of enzyme is accessible for additional loading onto hydrogel precursors, such as PAA.
  • the PEG amine can further react with one arm of a trifunctional aziridine crosslinker, with one arm of a trifunctional epoxide crosslinker, or with one arm of a tetrafunctional epoxide crosslinker.
  • the enzyme macromers are loaded with polyfunctional aziridine or epoxide.
  • the non-reacted aziridine or epoxide groups will further link the enzyme to the hydrogel precursors (e.g. PAA, PMA, or poly(acrylamide/acrylic acid)) to form the hydrogel.
  • the structure of trifunctional and tetrafunctional crosslinkers can also provide flexibility of the hydrogel matrix.
  • the enzymatic hydrogel matrix is a polymer layer on top of OSP laminate layer. This layer is to provide an immobilized enzymatic hydrogel that can react with target analytes (including glucose, lactate, and ketone) and in the process the enzyme consumes oxygen.
  • the enzymatic hydrogel formulation comprises (or in some implementations consists of or consists essentially of) enzymes, buffer, salt, enzyme substrate molecules, water soluble polymers and multifunctional crosslinkers.
  • the enzyme comprises glucose oxidase (GOX) and may also include another enzyme such as catalase.
  • the sensitivity of the hydrogel should ideally cover most of the range of concentrations that can be reasonably expected to occur and which provide useful information regarding health.
  • the GOX hydrogel according to certain implementations shows glucose sensitivity between 20mg/dl glucose to 400mg/dl glucose and the lactate hydrogel according to certain implementations shows lactate sensitivity between 1 mmol/L to 20 mmol/L.
  • the enzymes used in this enzymatic hydrogel include, but are not limited to glucose oxidase, lactate oxidase, catalase, 3 -Hydroxybutyrate dehydrogenase, NADH oxidase, and diaphorase. Two or more enzymes may be used in the enzymatic hydrogel, and the combination of the enzymes must consume oxygen while reacting to the target analyte.
  • the water soluble polymer(s) used in forming the hydrogel matrix include, but are not limited to Poly(vinyl alcohol), Poly(allylamine) Poly(vinylamine), Chitosan, Polyacrylamide, Poly(N-vinylpyrrolidone), Poly(acrylic acid), Poly(2-vinylpyridine), Polyurethane, a block copolymer, and a mixture of multiple polymers.
  • the water-soluble polymer solution should be pH adjustable by mixing with salt buffer such as PBS, NaOH, and HC1.
  • the pH for the enzymatic hydrogel solution can be any pH between about pH 3 and pH 10, including between about pH 5 and pH 8, and about pH 4, about pH 5, about pH 6, about pH 7, about pH 8, and about pH 9.
  • the enzyme hydrogel solution can be mixed with substrates (i.e. glucose for a glucose sensor, lactate for a lactate sensor) to enhance the enzymatic activity while the enzyme is undergoing covalent bonding with the multifunctional crosslinker.
  • substrates i.e. glucose for a glucose sensor, lactate for a lactate sensor
  • the crosslinker(s) used in this hydrogel formulation can be a bifunctional crosslinker, a trifunctional crosslinker, a tetrafunctional crosslinker, or a mixture of two or more such crosslinkers.
  • bifunctional crosslinkers include, but are not limited to Ethylene glycol diglycidyl ether, glutaraldehyde, Tetra(ethylene glycol) diacrylate, N,N’-(methylenedi-p-phenylene)bis(aziridine-l-carboxamide) and PEG-(nitrophenyl carbonate).
  • trifunctional crosslinkers include, but are not limited to Glycidyl Glycerol -Ether, Trimethylolpropane tris(2-methyl-l -aziridine propionate), Pentaerythritol Tris (3-(l-Aziridinyl) Propionate, and TSAT (tris-(succinimidyl)aminotriacetate).
  • tetrafunctional crosslinkers include but are not limited to 4-Arm PEG-Acrylate, Pentaerythritol tetraacrylate, and 4-Arm PEG-Succinimidyl Glutarate.
  • the enzymatic hydrogel can be dispensed at a wide range of temperatures including but not limited to 2°C to 50°C and about 15°C to 30°C, including 2-8°C, 20 °C, 37°C, and 50°C, and the enzymatic hydrogel dispensing condition can be dry, humid, or contain chemical vapor.
  • the enzymatic hydrogel is allowed to cure under certain conditions to let the crosslinker covalently bond with the enzyme and the polymer.
  • the curing temperature used includes, but is not limited to 20°C to 100°C, including 20°C, 37°C, 50°C, 80°C, and 100°C, for a period of about 20 minutes to 72 hours, including 20 mins, 1 hour, 2 hours, 5 hours, 10 hours, 24 hours, 48 hours, and 72 hours, or until the material is cured.
  • the desired thickness of enzymatic hydrogel can be about 0.5 to 5 mil, including about 0.5-lmil, about 1- 2mil, and about 2-4mil.
  • the ratio of the polymer in hydrogel solution can be 1% wt/vol to 20% wt/vol, including 1% wt/vol to 10 wt/vol, 10% wt/vol to 20% wt/vol, 1% wt/vol to 5% wt/vol, 1% wt/vol, 2% wt/vol, 3% wt/vol, 4% wt/vol, 5% wt/vol, 10% wt/vol, 15% wt/vol and 20%wt/vol.
  • the ratio of crosslinker to hydrogel solution can be from 1 :99 to 1 :5, including 1 :99 to 1 :75, 1 :75 to 1 :50, 1 :50 to 1 :25, 1 :25 to 1 :5, 1 :90, 1 :80, 1 :70, 1 :60, 1 :50, 1 :40, 1 :30, 1 :20, 1 : 10, and 1 :5.
  • the enzyme can be present at 10 g/ml to 500 g/ml, including 10 g/ml to 100 g/ml, 10 g/ml to 250 g/ml, 100 g/ml to 500 g/ml, 10 g/ml to 50 g/ml, and 10 g/ml to 20 g/ml.
  • the enzymatic hydrogel matrix is fabricated using an enzymatic hydrogel solution that is crosslinked. Specifically, glucose oxidase enzyme was crosslinked with a hygroscopic polymer, polyacrylic acid, using a trifunctional epoxide crosslinker, glycidyl glycerol-ether, polyfunctional, to immobilize the glucose oxidase enzyme in a hydrophilic matrix (see example reaction 1 above).
  • the enzymatic hydrogel solution was made as follows, polyacrylic acid (PAA, MW 40000, 0, 2 g) was added to phosphate buffered saline (PBS) (pH 7.0, 50mM, 100 mL) and stirred for 16 h at room temperature. The pH of the final solution was adjusted by adding NaOH to be about pH 8. In a separate container, 0.5 g glucose oxidase (GOX)was added to 5.0 g of pH7.0 PBS. The solution was mixed with a speedmixer at 140 rpm for 60 sec. Polyacrylic acid solution (5.0g) was added to the enzymatic hydrogel (GOX) solution and mixed using a speedmixer set at 1500 rpm for 700s.
  • PAA polyacrylic acid
  • PBS phosphate buffered saline
  • GOX glucose oxidase
  • the enzymatic hydrogel solution was deposited in a thin layer on an oxygen sensing polymer matrix and cured in a high humidity environment (>80%) at room temperature for 20 hours.
  • This same formulation could also be deposited on a barrier layer (which covers the OSP) instead of directly upon the OSP prior to curing.
  • the enzymatic hydrogel matrix was fabricated using an enzymatic hydrogel solution that is crosslinked. Specifically, glucose oxidase enzyme was crosslinked with a hygroscopic polymer, poly(methacrylic acid), using a bifunctional araziridine crosslinker, N,N’-(methylenedi-p-phenylene)bis(aziridine-l-carboxamide), to immobilize the glucose oxidase enzyme in a hydrophilic matrix (see example reaction 2).
  • the enzymatic hydrogel solution was made as follows. Poly(methacrylic acid) (PMA, MW 40000, 0, 5 g) was added to phosphate buffered saline (PBS) (pH 7.4, 50mM, 90 mL) and stirred for 16 h at room temperature. The pH of the final solution was adjusted to pH 8 by adding NaOH. In a separate container, 10 g glucose oxidase (GOX)was added to 1.0 g of pH7.0 PBS. The solution was mixed with a speedmixer at 1400 rpm for 90 sec.
  • PMA Poly(methacrylic acid)
  • PBS phosphate buffered saline
  • pH 7.4, 50mM phosphate buffered saline
  • GOX glucose oxidase
  • Poly(methacrylic acid) (5.0g) was added to the enzymatic hydrogel (GOX) solution and mixed using a speed mixer set at 1400 rpm for 20s.
  • a crosslinker, N,N’-(methylenedi-p- phenylene)bis(aziridine-l -carboxamide) (0.5g) was added into the GOX/PMA solution and mixed with a speedmixer set at 1400 rpm for 300s.
  • the enzymatic hydrogel solution was deposited in a thin layer on an oxygen sensing polymer matrix and cured in a high humidity environment (>80%) at room temperature for 12 hours. This same formulation could also be deposited on a barrier layer instead of directly upon the OSP and then allowed to cure.
  • the enzymatic hydrogel matrix was fabricated using an enzymatic hydrogel solution that is crosslinked. Specifically, glucose oxidase enzyme was crosslinked with a hygroscopic polymer, poly(methacrylic acid), using a 4-Arm PEG- Epoxide crosslinker to immobilize the glucose oxidase enzyme in a hydrophilic matrix (see example reaction 3).
  • the enzymatic hydrogel solution was made as follows. Polyacrylic acid (PAA, MW 5000, 0, 1 g) was added to phosphate buffered saline (PBS) (pH 7.0, 50mM, 90 mL) and stirred for 18 h at room temperature.
  • PBS phosphate buffered saline
  • the pH of the final solution was adjusted to pH 8.5 by adding NaOH.
  • 50 g glucose oxidase (GOX) was added to 5.0 g of pH7.0 PBS.
  • the solution was mixed with a speedmixer at 1500 rpm for 90sec.
  • Polyacrylic acid (5.0g) was added to the enzymatic hydrogel (GOX) solution and mixed using a speed mixer set at 1500 rpm for 60s.
  • a crosslinker, 4-Arm PEG-Epoxide (1g) was added into the GOX/PAA solution and mixed with a speedmixer set at 1400 rpm for 60s.
  • the enzymatic hydrogel solution was deposited in a thin layer on an oxygen sensing polymer matrix and cured in a high humidity environment (>80%) at room temperature for 12 hours. This same formulation could also be deposited on a barrier layer instead of directly upon the OSP prior to curing.
  • the enzymatic hydrogel matrix is fabricated using an enzymatic hydrogel solution that is crosslinked.
  • Glucose oxidase enzyme was crosslinked with a hygroscopic polymer, polyacrylic acid, using an araziridine crosslinker, Trimethylolpropane tris(2 -methyl- 1 -aziridine propionate), to immobilize the glucose oxidase enzyme in a hydrophilic matrix (see example reaction 4).
  • the enzymatic hydrogel solution was made as follows. Polyacrylic acid (PAA, MW 40, 0, 10 g) was added to phosphate buffered saline (PBS) (pH 7.0, 50mM, 90 mL) and stirred for 16 h at room temperature. The pH of the resulting solution was adjusted to pH 5. In a separate container, 0.50 g glucose oxidase (GOX)was added to 5.0 g of pH7.0 PBS. The solution was mixed with a speedmixer at 150 rpm for 20 sec. Polyacrylic acid solution (0.9g) was added to the enzymatic hydrogel (GOX) solution and mixed using a speed mixer set at 140 rpm for 20s.
  • PAA Polyacrylic acid
  • PBS phosphate buffered saline
  • GOX glucose oxidase
  • a crosslinker Trimethylolpropane tris(2-methyl-l-aziridine propionate) (0.1g) was added into the GOX/PAA solution and mixed with a speedmixer set at 1500 rpm for 200s.
  • the enzymatic hydrogel solution was deposited in a thin layer on an oxygen sensing polymer matrix and cured in a high humidity environment (>80%) at room temperature for 16 hours. This same formulation could also be deposited on a barrier layer instead of directly upon the OSP and then cured.
  • the enzymatic hydrogel matrix is fabricated using an enzymatic hydrogel solution that is crosslinked.
  • glucose oxidase enzyme was crosslinked with a hygroscopic polymer, Poly(HEMA), using a bifunctional acrylic crosslinker, Tetra(ethylene glycol) diacrylate (TetEGDA), to immobilize the glucose oxidase enzyme in a hydrophilic matrix (see example reaction 5).
  • the enzymatic hydrogel solution was made as follows. Poly (HEMA) was added to phosphate buffered saline (PBS) (pH 7.0, 50mM, 90 mL) and stirred for 16 h at room temperature. The pH of the final solution was adjusted to pH 7. In a separate container, 80 g glucose oxidase (GOX) was added to 1.0 g of pH7.0 PBS. The solution was mixed with a speedmixer at 500 rpm for 240 sec. Poly (HEMA) solution (10.0g) was added to the enzymatic hydrogel (GOX) solution and mixed using a speed mixer set at 1400 rpm for 90s.
  • PBS phosphate buffered saline
  • GOX glucose oxidase
  • the reaction was started by adding a water soluble, biocompatible initiator.
  • ammonium persulfate (APS). 0.1% wt/vol was added to the GOX/Poly(HEMA)/TetEGDA mixed solution.
  • the enzymatic hydrogel solution was deposited in a thin layer on an oxygen sensing polymer matrix and cured in a high humidity environment (>85%) at 37°C for 18 hours. In another implementation, this same formulation could be deposited upon a barrier layer instead of directly upon the OSP layer before curing.
  • the enzymatic hydrogel matrix was fabricated using an enzymatic hydrogel solution that is crosslinked. Specifically, glucose oxidase (GOX) enzyme and Catalase (CAT) were crosslinked with a hygroscopic polymer, poly (acrylamide-co-acrylic acid), using a trifunctional aziridine crosslinker, trimethylolpropane tris(2-methyl-l-aziridine propionate), to immobilize the GOX/CAT enzyme in a hydrophilic matrix.
  • GOX glucose oxidase
  • CAT Catalase
  • the enzymatic hydrogel solution was made as follows. Poly (acrylamide- co-acrylic acid) (PAAA, MW 520,000, 5 g) was added to phosphate buffered saline (PBS) (pH 7.4, 80mM, 45 mL) and stirred for 16 h at room temperature. The pH of the solution was adjusted to pH 5. In a separate container, 90 g Glucose oxidase (GOX) and 10 g Catalase (CAT) were added to 1.0 g of pH7.4 PBS. The solution was mixed with a speedmixer at 2000 rpm for 60 sec.
  • PBS phosphate buffered saline
  • CAT Catalase
  • Poly (acrylamide-co-acrylic acid) solution (1 mL) was added to the enzymatic hydrogel (GOX/CAT) solution and mixed using a speed mixer set at 2000 rpm for 60s.
  • a crosslinker, Trimethylolpropane tris(2 -methyl- 1 -aziridine propionate) (0.2 mL), was added into the GOX/CAT/PAAA solution and mixed with a speedmixer set at 2000 rpm for 60s.
  • the enzymatic hydrogel solution was deposited in a thin layer on an oxygen sensing polymer matrix and cured in a high humidity environment (>80%) at room temperature for 48 hours. In an alternative implementation, the solution is deposited on a barrier layer covering the OSP prior to curing.
  • the enzymatic hydrogel matrix is fabricated using an enzymatic hydrogel solution that is crosslinked.
  • glucose oxidase enzyme and catalase enzyme (GOX/CAT) were crosslinked with a hygroscopic polymer, polyacrylic acid, using a trifunctional epoxide crosslinker, glycidyl glycerol-ether, polyfunctional, to immobilize the GOX/CAT enzyme in a hydrophilic matrix (see example reaction 1).
  • the enzymatic hydrogel solution was made as follows, polyacrylic acid (PAA, MW 40000, 0.2 g) was added to phosphate buffered saline (PBS) (pH 7.0, 50mM, 100 mL) and stirred for 16 h at room temperature. The pH of the resulting solution was adjusted to pH 8. In a separate container, 50 g glucose oxidase (GOX) and 9 g Catalase (CAT) were added to 1.1 g of pH7.0 PBS. The solution was mixed with a speedmixer at 1500 rpm for 6 seconds.
  • PBS phosphate buffered saline
  • CAT 9 g Catalase
  • Polyacrylic acid solution (5.0g) was added to the enzymatic hydrogel (GOX/CAT) solution and mixed using a speed mixer set at 1400 rpm for 600s.
  • a crosslinker, glycidyl glycerol-ether, polyfunctional (0.15 g) was added into the GOX/CAT/PAA solution and mixed with a speedmixer set at 1500 rpm for 60s.
  • the enzymatic hydrogel solution was deposited in a thin layer on an oxygen sensing polymer matrix and cured in a high humidity environment (>80%) at 37°C for 6 hours. In other implementations, this solution is deposited on a barrier layer covering the OSP (instead of directly on the OSP) and then cured.
  • the enzymatic hydrogel matrix is fabricated using an enzymatic hydrogel solution that is crosslinked.
  • glucose oxidase enzyme and catalase enzyme (GOX/CAT) were crosslinked with a hygroscopic polymer, poly(methacrylic acid), using a bifunctional araziridine crosslinker, N,N’-(methylenedi-p- phenylene)bis(aziridine-l -carboxamide), to immobilize the GOX/CAT enzyme in a hydrophilic matrix (see example reaction 2).
  • the enzymatic hydrogel solution was made as follows. Poly(methacrylic acid) (PMA, MW 40000, 0, 5 g) was added to phosphate buffered saline (PBS) (pH 7.4, 50mM, 90 mL) and stirred for 18 h at room temperature. The pH of the solution was then adjusted to pH 6. In a separate container, 50 g glucose oxidase (GOX) and 6 g Catalase (CAT) were added to 0.9 g of pH7.0 PBS. The solution was mixed with a speedmixer at 1400 rpm for 90sec.
  • PMA Poly(methacrylic acid)
  • PBS phosphate buffered saline
  • CAT 6 g Catalase
  • Poly(methacrylic acid) (1.0g) was added to the enzymatic hydrogel (GOX/CAT) solution and mixed using a speed mixer set at 1400 rpm for 20s.
  • a crosslinker, N,N’-(methylenedi-p- phenylene)bis(aziridine-l -carboxamide) (0.5g) was added into the GOX/CAT/PMA solution and mixed with a speedmixer set at 1500 rpm for 300s.
  • the enzymatic hydrogel solution was deposited in a thin layer on an oxygen sensing polymer matrix and cured in a high humidity environment (>80%) at room temperature for 12 hours. In other implementations, this solution is deposited on a barrier layer covering the OSP and then cured.
  • the enzymatic hydrogel matrix is fabricated using an enzymatic hydrogel solution that is crosslinked.
  • glucose oxidase enzyme and catalase enzyme (GOX/CAT) were crosslinked with a hygroscopic polymer, poly(methacrylic acid), using a 4-Arm PEG-Epoxide crosslinker to immobilize the GOX/CAT enzyme in a hydrophilic matrix (see example reaction 3).
  • the enzymatic hydrogel solution was made as follows. Polyacrylic acid (PAA, MW 5000, 0, 1 g) was added to phosphate buffered saline (PBS) (pH 7.0, 50mM, 90 mL) and stirred for 16 h at room temperature. The pH of the resulting solution was adjusted to pH 8. In a separate container, 45 g glucose oxidase (GOX) and 15 g Catalase (CAT) were added to 5.0 g of pH7.0 PBS. The solution was mixed with a speedmixer at 1400 rpm for 70sec.
  • PBS phosphate buffered saline
  • CAT Catalase
  • Polyacrylic acid (5.0g) was added to the enzymatic hydrogel (GOX/CAT) solution and mixed using a speed mixer set at 1400 rpm for 90s.
  • a crosslinker, 4-Arm PEG-Epoxide (1.1g) was added into the GOX/CAT/PAA solution and mixed with a speedmixer set at 1400 rpm for 90s.
  • the enzymatic hydrogel solution was deposited in a thin layer on an oxygen sensing polymer matrix and cured in a high humidity environment (>80%) at 4°C - 8°C for 22 hours. In other implementations, this solution is deposited on a barrier layer covering the OSP.
  • the enzymatic hydrogel matrix is fabricated using an enzymatic hydrogel solution that is crosslinked.
  • glucose oxidase enzyme and catalase enzyme (GOX/CAT) were crosslinked with a hygroscopic polymer, polyacrylic acid, using an araziridine crosslinker, Trimethylolpropane tris(2-methyl-l -aziridine propionate), to immobilize the GOX/CAT enzyme in a hydrophilic matrix (see example reaction 4).
  • the enzymatic hydrogel solution was made as follows. Polyacrylic acid (PAA, MW 40, 0, 10 g) was added to phosphate buffered saline (PBS) (pH 7.0, 50mM, 90 mL) and stirred for 16 h at room temperature. The pH of the resulting solution was adjusted to pH 5.5. In a separate container, 50 g glucose oxidase (GOX) and 15 g Catalase (CAT) were added to 1.0 g of pH7.0 PBS. The solution was mixed with a speedmixer at 160 rpm for 60 sec.
  • PBS phosphate buffered saline
  • CAT Catalase
  • Polyacrylic acid solution (1.0g) was added to the enzymatic hydrogel (GOX/CAT) solution and mixed using a speed mixer set at 160 rpm for 30s.
  • a crosslinker Trimethylolpropane tris(2 -methyl- 1 -aziridine propionate) (0.1g), was added into the GOX/CAT/PAA solution and mixed with a speedmixer set at 1500 rpm for 240s.
  • the enzymatic hydrogel solution was deposited in a thin layer on an oxygen sensing polymer matrix and cured in a high humidity environment (>80%) at room temperature for 18 hours. In other implementations, this solution is deposited on a barrier layer covering the OSP prior to curing.
  • the enzymatic hydrogel matrix is fabricated using an enzymatic hydrogel solution that is crosslinked.
  • glucose oxidase enzyme and catalase enzyme (GOX/CAT) were crosslinked with a hygroscopic polymer, Poly(HEMA), using a bifunctional acrylic crosslinker, tetra(ethylene glycol) diacrylate (TetEGDA), to immobilize the GOX/CAT enzyme in a hydrophilic matrix (see example reaction 5).
  • the enzymatic hydrogel solution was made as follows. Poly (HEMA) was added to phosphate buffered saline (PBS) (pH 7.0, 50mM, 90 mL) and stirred for 16 h at room temperature. The pH of the final solution was adjusted to pH 7. In a separate container, 65 g glucose oxidase (GOX) and 9g Catalase (CAT) were added to 0.9 g of pH 7.0 PBS. The solution was mixed with a speedmixer at 1500 rpm for 240 sec. Poly (HEMA) solution (10.0g) was added to the enzymatic hydrogel (GOX/CAT) solution and mixed using a speed mixer set at 1400 rpm for 90s.
  • PBS phosphate buffered saline
  • CAT 9g Catalase
  • a crosslinker tetra(ethylene glycol) diacrylate (0.12g) was added into the GOX/CAT/Poly(HEMA) solution and mixed with a speedmixer set at 1400 rpm for 60s.
  • the reaction will start by adding a water soluble, biocompatible initiator.
  • a water soluble, biocompatible initiator In this specific example, we use ammonium persulfate (APS). 0.12% wt/vol APS is added to the GOX/CAT/Poly(HEMA)/TetEGDA mixed solution.
  • the enzymatic hydrogel solution was deposited in a thin layer on an oxygen sensing polymer matrix and cured in a high humidity environment (>80%) at 37°C for 24 hours. In other implementations, this solution is deposited on a barrier layer covering the OSP before curing.
  • the enzymatic hydrogel matrix is fabricated using an enzymatic hydrogel solution that is crosslinked.
  • lactate oxidase enzyme was crosslinked with a hygroscopic polymer, polyacrylic acid, using a trifunctional epoxide crosslinker, glycidyl glycerol-ether, polyfunctional, to immobilize the lactate oxidase enzyme in a hydrophilic matrix (see example reaction 1).
  • the enzymatic hydrogel solution was made as follows. Polyacrylic acid (PAA, MW 100,000, 2 g) was added to phosphate buffered saline (PBS) (pH 7.4, 80mM, 10 mL) and stirred for 16 h at room temperature. The pH of the resulting solution was adjusted to pH 7.8. In a separate container, 10 g lactate oxidase (LOX)was added to 1.0 g of pH 7.4 PBS. The solution was mixed with a speedmixer at 1200 rpm for 50 sec. Polyacrylic acid solution (0.5 g) was added to the enzymatic hydrogel (LOX) solution and mixed using a speed mixer set at 1400 rpm for 30s.
  • PAA Polyacrylic acid
  • LOX lactate oxidase
  • the enzymatic hydrogel solution was deposited in a thin layer on an oxygen sensing polymer matrix and cured in a high humidity environment (>80%) at room temperature for 20 hours. In other implementations, this solution is deposited on a barrier layer covering the OSP before being allowed to cure.
  • the enzymatic hydrogel matrix is fabricated using an enzymatic hydrogel solution that is crosslinked.
  • lactate oxidase enzyme was crosslinked with a hygroscopic polymer, poly(methacrylic acid), using a bifunctional araziridine crosslinker, N,N’-(methylenedi-p-phenylene)bis(aziridine-l-carboxamide), to immobilize the lactate oxidase enzyme in a hydrophilic matrix (see example reaction 2).
  • the enzymatic hydrogel solution was made as follows. Poly(methacrylic acid) (PMA, MW 9500, 1.5 g) was added to phosphate buffered saline (PBS) (pH 7.4, 60mM, 10 mL) and stirred for 16 h at room temperature. The pH of the final solution was adjusted to pH 6. In a separate container, 20 g lactate oxidase (LOX)was added to 1.0 g of pH7.0 PBS. The solution was mixed with a speedmixer at 1400 rpm for 80sec. Poly(methacrylic acid) solution (0.5g) was added to the enzymatic hydrogel (LOX) solution and mixed using a speed mixer set at 1400 rpm for 80s.
  • PMA Poly(methacrylic acid)
  • PBS phosphate buffered saline
  • LOX lactate oxidase
  • a crosslinker N,N’-(methylenedi-p-phenylene)bis(aziridine- 1 -carboxamide) (0.5g), was added into the LOX/PMA solution and mixed with a speedmixer set at 1400 rpm for 200s.
  • the enzymatic hydrogel solution was deposited in a thin layer on an oxygen sensing polymer matrix and cured in a high humidity environment (>80%) at 37 °C for 2 hours.
  • the solution is deposited on a barrier layer covering the OSP prior to curing.
  • the enzymatic hydrogel matrix is fabricated using an enzymatic hydrogel solution that is crosslinked. Specifically, lactate oxidase enzyme was crosslinked with a hygroscopic polymer, Polyacrylic acid, using a 4-Arm PEG-Epoxide crosslinker to immobilize the glucose oxidase enzyme in a hydrophilic matrix (see example reaction 3).
  • the enzymatic hydrogel solution was made as follows. Polyacrylic acid (PAA, MW 100,000, 2.5 g) was added to phosphate buffered saline (PBS) (pH 7.0, 50mM, 10 mL) and stirred for 16 h at room temperature. The pH of the final solution was adjusted by adding NaOH to a final pH of 7.8. In a separate container, 30 g lactate oxidase (LOX) was added to 1.0 g of pH7.0 PBS. The solution was mixed with a speedmixer at 1400 rpm for 80sec. Polyacrylic acid (0.5 g) was added to the enzymatic hydrogel (LOX) solution and mixed using a speed mixer set at 1400 rpm for 60s.
  • PAA Polyacrylic acid
  • LOX lactate oxidase
  • the enzymatic hydrogel solution was deposited in a thin layer on an oxygen sensing polymer matrix and cured in a high humidity environment (>80%) at 4 °C for 20 hours. In another implementation, the solution is deposited on a barrier layer covering the OSP prior to curing.
  • the enzymatic hydrogel matrix is fabricated using an enzymatic hydrogel solution that is crosslinked.
  • lactate oxidase enzyme was crosslinked with a hygroscopic polymer, polyacrylic acid, using an araziridine crosslinker, Trimethylolpropane tris(2 -methyl- 1 -aziridine propionate), to immobilize the glucose oxidase enzyme in a hydrophilic matrix (see example reaction 4).
  • the enzymatic hydrogel solution was made as follows. Polyacrylic acid (PAA, MW 40,000, 3 g) was added to phosphate buffered saline (PBS) (pH 7.0, 50mM, 10 mL) and stirred for 16 h at room temperature. The pH of the resulting solution was adjusted to pH 5. In a separate container, 40g lactate oxidase (LOX) was added to 1.0 g of pH7.0 PBS. The solution was mixed with a speedmixer at 1200 rpm for 60 sec. Polyacrylic acid solution (1.0g) was added to the enzymatic hydrogel (LOX) solution and mixed using a speedmixer set at 1400 rpm for 30s.
  • PAA Polyacrylic acid
  • LOX lactate oxidase
  • the enzymatic hydrogel solution was deposited in a thin layer on an oxygen sensing polymer matrix and cured in a high humidity environment (>80%) at room temperature for 16 hours. In an alternative implementation, the solution is deposited on a barrier layer covering the OSP prior to curing.
  • the enzymatic hydrogel matrix is fabricated using an enzymatic hydrogel solution that is crosslinked.
  • lactate oxidase enzyme was crosslinked with a hygroscopic polymer, Poly(HEMA), using a bifunctional acrylic crosslinker, tetra(ethylene glycol) diacrylate (TetEGDA), immobilize the lactate oxidase enzyme in a hydrophilic matrix (see example reaction 5).
  • the enzymatic hydrogel solution was made as follows. Poly (HEMA) was added to phosphate buffered saline (PBS) (pH 7.0, 50mM, 90 mL) and stirred for 16 h at room temperature. The pH of the resulting solution was adjusted to pH 7. In a separate container, 70 g lactate oxidase (LOX) was added to 1.0 g of pH7.0 PBS. The solution was mixed with a speedmixer at 500 rpm for 200 sec. Poly (HEMA) solution (1.0g) was added to the enzymatic hydrogel (LOX) solution and mixed using a speed mixer set at 1400 rpm for 60s.
  • PBS phosphate buffered saline
  • LOX lactate oxidase
  • the reaction was started by adding a water soluble, biocompatible thermo/photo initiator.
  • ammonium persulfate (APS) 0.1% wt/vol was added to the LOX/Poly(HEMA)/TetEGDA mixed solution.
  • the enzymatic hydrogel solution was deposited in a thin layer on an oxygen sensing polymer matrix and cured in a high humidity environment (>80%) at room temperature for 20 hours. In an alternative implementation, the solution is deposited on a barrier layer covering the OSP prior to curing.
  • the enzymatic hydrogel matrix is fabricated using an enzymatic hydrogel solution that is crosslinked.
  • lactate oxidase enzyme and catalase enzyme LOX/CAT
  • LOX/CAT lactate oxidase enzyme and catalase enzyme
  • the enzymatic hydrogel solution was made as follows. Polyacrylic acid (PAA, MW 100,000, 2 g) was added to phosphate buffered saline (PBS) (pH 7.4, 80mM, 10 mL) and stirred for 16 h at room temperature. The pH of the solution was adjusted to pH 7.8. In a separate container, 40 g lactate oxidase (LOX) and 4 g catalase (CAT) were added to 1.0 g of pH7.4 PBS. The solution was mixed with a speedmixer at 1200 rpm for 50 sec.
  • PBS phosphate buffered saline
  • CAT catalase
  • Polyacrylic acid solution (0.5 g) was added to the enzymatic hydrogel (LOX/CAT) solution and mixed using a speed mixer set at 1400 rpm for 30s.
  • the enzymatic hydrogel solution was deposited in a thin layer on an oxygen sensing polymer matrix and cured in a high humidity environment (>80%) at room temperature for 20 hours. In an alternative implementation, the solution is deposited on a barrier layer covering the OSP prior to curing.
  • the enzymatic hydrogel matrix is fabricated using an enzymatic hydrogel solution that is crosslinked.
  • lactate oxidase enzyme and catalase enzyme (LOX/CAT) were crosslinked with a hygroscopic polymer, poly(methacrylic acid), using a bifunctional araziridine crosslinker, N,N’-(methylenedi-p- phenylene)bis(aziridine-l -carboxamide), to immobilize the LOX/CAT enzyme in a hydrophilic matrix (see example reaction 2).
  • the enzymatic hydrogel solution was made as follows. Poly(methacrylic acid) (PMA, MW 9500, 1.5 g) was added to phosphate buffered saline (PBS) (pH 7.4, 60mM, 10 mL) and stirred for 16 h at room temperature. The pH of the resulting solution was adjusted to pH 6. In a separate container, 40 g lactate oxidase (LOX) and 4 g catalase (CAT) were added to 1.0 g of pH7.0 PBS. The solution was mixed with a speedmixer at 1400 rpm for 80sec.
  • PMA Poly(methacrylic acid)
  • PBS phosphate buffered saline
  • CAT catalase
  • Poly(methacrylic acid) solution (0.5g) was added to the enzymatic hydrogel (LOX/CAT) solution and mixed using a speed mixer set at 1400 rpm for 80s.
  • a crosslinker N,N’- (methylenedi-p-phenylene)bis(aziridine-l -carboxamide) (0.5g), was added into the LOX/CAT/PMA solution and mixed with a speedmixer set at 1400 rpm for 200s.
  • the enzymatic hydrogel solution was deposited in a thin layer on an oxygen sensing polymer matrix and cured in a high humidity environment (>80%) at 37 °C for 2 hours. In alternative implementations, the solution is deposited on a barrier layer covering the OSP before curing.
  • the enzymatic hydrogel matrix is fabricated using an enzymatic hydrogel solution that is crosslinked.
  • lactate oxidase enzyme and catalase enzyme LOX/CAT
  • LOX/CAT lactate oxidase enzyme and catalase enzyme
  • the enzymatic hydrogel solution was made as follows. Polyacrylic acid (PAA, MW 100,000, 2.5 g) was added to phosphate buffered saline (PBS) (pH 7.0, 50mM, 10 mL) and stirred for 16 h at room temperature. The pH of the final solution was adjusted to pH 7.8. In a separate container, 30 g lactate oxidase (LOX) and 3 g Catalase (CAT) were added to 1.0 g of pH 7.0 PBS. The solution was mixed with a speedmixer at 1400 rpm for 80 sec.
  • PBS phosphate buffered saline
  • CAT 3 g Catalase
  • Polyacrylic acid 0.5 g was added to the enzymatic hydrogel (LOX/CAT) solution and mixed using a speed mixer set at 1400 rpm for 60s.
  • a crosslinker, 4-Arm PEG-Epoxide (0.2g) was added into the LOX/CAT/PAA solution and mixed with a speedmixer set at 1400 rpm for 60s.
  • the enzymatic hydrogel solution was deposited in a thin layer on an oxygen sensing polymer matrix and cured in a high humidity environment (>80%) at 4 C for 20 hours. In an alternative implementation, the solution is deposited on a barrier layer covering the OSP prior to curing.
  • the enzymatic hydrogel matrix is fabricated using an enzymatic hydrogel solution that is crosslinked.
  • lactate oxidase enzyme and catalase enzyme LOX/CAT
  • LOX/CAT lactate oxidase enzyme and catalase enzyme
  • the enzymatic hydrogel solution was made as follows. Polyacrylic acid (PAA, MW 40,000, 3 g) was added to phosphate buffered saline (PBS) (pH 7.0, 50mM, 10 mL) and stirred for 16 h at room temperature. The pH of the final solution may need to be adjusted by adding NaOH. The final pH should between pH 5-pH 8. In this example, we adjust the pH to 5. In a separate container, 40g lactate oxidase (LOX) and 8 g catalase were added to 1.0 g of pH7.0 PBS. The solution was mixed with a speedmixer at 1200 rpm for 60 sec.
  • PBS phosphate buffered saline
  • LOX lactate oxidase
  • 8 g catalase were added to 1.0 g of pH7.0 PBS. The solution was mixed with a speedmixer at 1200 rpm for 60 sec.
  • Polyacrylic acid solution (1.0g) was added to the enzymatic hydrogel (LOX/CAT) solution and mixed using a speedmixer set at 1400 rpm for 30s.
  • a crosslinker, trimethylolpropane tris(2 -methyl- 1 -aziridine propionate) (0.4g) was added into the LOX/CAT/PAA solution and mixed with a speedmixer set at 1400 rpm for 120s.
  • the enzymatic hydrogel solution was deposited in a thin layer on an oxygen sensing polymer matrix and cured in a high humidity environment (>80%) at room temperature for 16 hours. In alternative implementations, the solution is deposited on a barrier layer covering the OSP prior to curing.
  • the enzymatic hydrogel matrix is fabricated using an enzymatic hydrogel solution that is crosslinked.
  • lactate oxidase enzyme and catalase enzyme (LOX/CAT) were crosslinked with a hygroscopic polymer, Poly(HEMA), using a bifunctional acrylic crosslinker, tetra(ethylene glycol) diacrylate (TetEGDA), immobilize the lactate oxidase enzyme in a hydrophilic matrix (see example reaction 5).
  • the enzymatic hydrogel solution was made as follows. Poly (HEMA) was added to phosphate buffered saline (PBS) (pH 7.0, 50mM, 90 mL) and stirred for 16 h at room temperature. The pH of the resulting solution was adjusted to pH 7. In a separate container, 70 g lactate oxidase (LOX) and 7 g catalase (CAT) were added to 1.0 g of pH7.0 PBS. The solution was mixed with a speedmixer at 500 rpm for 200 sec. Poly (HEMA) solution (1.0g) was added to the enzymatic hydrogel (LOX/CAT) solution and mixed using a speed mixer set at 1400 rpm for 60s.
  • PBS phosphate buffered saline
  • CAT catalase
  • the reaction will start by adding a water soluble, biocompatible thermo/photo initiator.
  • a water soluble, biocompatible thermo/photo initiator In this specific example, we use ammonium persulfate (APS). 0.1% wt/vol APS is added to the LOX/CAT/Poly(HEMA)/TetEGDA mixed solution.
  • the enzymatic hydrogel solution was deposited in a thin layer on an oxygen sensing polymer matrix and cured in a high humidity environment (>80%) at room temperature for 20 hours. In an alternative implementation, the solution is deposited on a barrier layer covering the OSP prior to curing.
  • the enzymatic hydrogel matrix is fabricated using an enzymatic hydrogel solution that is crosslinked.
  • 3 -Hydroxybutyrate dehydrogenase (HBDH) enzyme and NADH oxidase (NO) were crosslinked with a hygroscopic polymer, Poly (acrylamide-co-acrylic acid), using a trifunctional aziridine crosslinker, Trimethylolpropane tris(2-methyl-l-aziridine propionate), to immobilize the HBDH/NO enzyme in a hydrophilic matrix.
  • HBDH 3 -Hydroxybutyrate dehydrogenase
  • NO NADH oxidase
  • the enzymatic hydrogel solution was made as follows.
  • Poly (acrylamide- co-acrylic acid) PAAA, MW 100,000, 2 g was added to phosphate buffered saline (PBS) (pH 7.4, 80mM, 10 mL) and stirred for 16 h at room temperature. The pH of the solution was adjusted to pH 8. In a separate container, 40 g 3 -Hydroxybutyrate dehydrogenase (HBDH) and 10 g NADH oxidase (NO) were added to 1.0 g of pH7.4 PBS. The solution was mixed with a speedmixer at 1200 rpm for 50 sec.
  • HBDH -Hydroxybutyrate dehydrogenase
  • NO NADH oxidase
  • Poly (acrylamide-co-acrylic acid) solution (0.5 g) was added to the enzymatic hydrogel (HBDH/NO) solution and mixed using a speedmixer set at 1400 rpm for 30s.
  • a crosslinker, Trimethylolpropane tris(2-methyl-l -aziridine propionate) (0.1g) was added into the HBDH/NO/PAAA solution and mixed with a speedmixer set at 1400 rpm for 70s.
  • the enzymatic hydrogel solution was deposited in a thin layer on an oxygen sensing polymer matrix and cured in a high humidity environment (>80%) at room temperature for 48 hours. In an alternative implementation, the solution is deposited on a barrier layer covering the OSP prior to curing.
  • Membrane is the optional layer of the laminate that lies on top of the enzymatic hydrogel layer, and this layer, when present, can control the diffusion rate of the sensor target analyte as well as block lipids from entering the laminate. It is sometimes referred to as an oxygen transport layer or laminate.
  • the same types of materials used in the membrane may also be used as the optional barrier layer between the enzymatic hydrogel layer and OSP.
  • the membrane layer is directly exposed to the fluid containing oxygen and the target analyte(s) and preferably adheres well to the enzymatic hydrogel layer, is water permeable and has fast response to changes in the analyte.
  • the thickness of the membrane and/or barrier should be consistent and even across the entire layer.
  • the membrane or barrier may comprise a thin polymer sheet that is purchased or made separately (e.g. FEP membrane may be purchased in suitable thicknesses such as 15 or 25 microns) and placed upon the laminate stack in the desired location.
  • the membrane or barrier layer is created by casting or depositing a liquid formulation into a well in the sensor body.
  • the membrane or barrier formulation comprises (or in some implementations consists of or consists essentially of) one or more hydrogel polymer backbone monomers, urethane monomers, and/or ester monomers, crosslinker and an initiator.
  • Suitable hydrogel backbone monomers include but are not limited to dimethylacrylamide, 2-Hydroxyethyl acrylate, HEMA, diurethane dimethacrylate, and silane monomers with multiple functional groups.
  • the crosslinker can be a bifunctional, trifunctional, a tetrafunctional crosslinker, or a mixture thereof.
  • the amount of crosslinker used will vary depending upon its functionality and can be readily calculated by those skilled in the art. Examples include but are not limited to bisphenol dimethacrylate, butanediol dimethacrylate, poly(ethylene glycol) dimethacrylate, di(ethylene glycol) dimethacrylate, ethylene glycol dimethacrylate.
  • the ratio of the total monomers (same or different) and initiator can vary widely due to the different possible chemistries and reactivities, and include ratios of about 1 : 100 to 1 : 1 by weight, including about 1 :90, 1 :80, 1 :70, 1 :60, 1 :50, 1 :30, 1 :20, 1 : 10, 1:9, 1 :8, 1 :7, 1 :6, 1 :5, 1 :4, 1 :3, 1 :2, and 1 : 1 by weight.
  • Any suitable type of initiator may be used in this formulation, including but not limited to thermal initiators and a UV initiators.
  • the membrane can be dispensed on top of dry enzymatic hydrogel or wet enzymatic hydrogel, and the barrier can be dispensed upon the OSP.
  • the membrane or barrier can be dispensed at a wide range of temperatures including but not limited to 2°C to 50°C and about 15°C to 30°C, including 2-8°C, 20 °C, 37°C, and 50°C, and the conditions can be dry, humid, or contain chemical vapor.
  • the curing temperature used includes, but is not limited to 20°C to 100°C, including 20°C, 37°C, 50°C, 80°C, and 100°C, for a period including but not limited to 1 minute to 72 hours, including 1 min, 2 mins, 3 mins, 4 mins, 10 mins, 20 mins, 1 hour, 2 hours, 5 hours, 10 hours, 24 hours, 48 hours, and 72 hours.
  • At least the surface of the OSP layer to which the barrier layer will be attached is subjected to plasma treatment prior to deposition of the barrier layer and/or at least the surface of the barrier layer to which the enzymatic hydrogel will be attached is subjected to plasma treatment prior to the deposition of the enzymatic hydrogel layer.
  • the plasma treatment may enhance adherence between the layers and/or reduce negative effects at the interfaces between layers.
  • the plasma treatment may be done using a gas that is about 95:5 (Argon: Oxygen) to 70:30 (Argon: Oxygen) including about 90: 10 (Argon: Oxygen) to 80:20 (Argon: Oxygen), and 85: 15 (Argon: Oxygen) to 80:20 (Argon: Oxygen).
  • the gas for the plasma treatment is 80:20 (Argon: Oxygen) at a flow rate of at least 5cc/min, including lOcc/min and 15cc/min for more than one minute, including 1 to 5 minutes and 1 to 20 minutes.
  • a surface energy of >55 dyne was achieved on a PVDF.
  • the parameters for plasma treatment may be adjusted by those skilled in the art to achieve a similar change in surface function as measured in dynes in other systems.
  • membrane or barrier formulation the components used are as listed in this Table below.
  • the materials and methods described herein may be used in a physiological sensor including, but not limited to, an optical enzymatic analyte sensor capable of measuring the concentration of glucose, lactic acid or other physiological compounds of interest in a human or other mammal, such as those described in U.S. Patent No. 7,146,203 entitled IMPLANTABLE BIOSENSOR AND METHODS OF USE THEREOF, issued December 5, 2006; U.S. Patent Publication No. 2019/0021672 entitled SYSTEMS AND METHODS FOR CONTINUOUS HEALTH MONITORING USING AN OPTO-ENZYMATIC ANALYTE SENSOR, published January 24, 2019; and U.S. Patent Publication No.
  • Conditional language such as “can,” “could,” “might,” or “may,” unless specifically stated otherwise, or otherwise understood within the context as used, is generally intended to convey that certain implementations include, while other implementations do not include, certain features, chemicals, elements, and/or steps. Thus, such conditional language is not generally intended to imply that such features, chemicals, elements, and/or steps are in any way required for one or more implementations or that one or more implementations necessarily include logic for deciding, with or without user input or prompting, whether these features, elements, and/or steps are included or are to be performed in any particular embodiment.
  • the term ‘including’ should be read to mean ‘including, without limitation,’ ‘including but not limited to,’ or the like;
  • the term ‘comprising’ as used herein is synonymous with ‘including,’ ‘containing,’ or ‘characterized by,’ and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps; “comprising” is intended to also include the narrower terms “consisting of’ and “consisting essentially of,” the latter term meaning that the scope is limited to the recited elements or steps and any others that do not materially affect the basic and novel characteristics of what is already recited;
  • the term ‘having’ should be interpreted as ‘having at least;’ the term ‘includes’ should be interpreted as ‘includes but is not limited to;’ the term ‘example’ is used to provide exemplary instances of the item in discussion, not an exhaustive or limiting list thereof; adjectives such as ‘known’, ‘normal’, ‘ standard’ , and terms of similar meaning should not be construed
  • a group of items linked with the conjunction “and” should not be read as requiring that each and every one of those items be present in the grouping, but rather should be read as ‘and/or’ unless expressly stated otherwise.
  • a group of items linked with the conjunction ‘or’ should not be read as requiring mutual exclusivity among that group, but rather should be read as ‘and/or’ unless expressly stated otherwise.

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Abstract

Les capteurs physiologiques d'analyte opto-enzymatique divulgués présentent une liaison ou une adhérence améliorée entre au moins deux couches du capteur, soit directement, soit par l'intermédiaire d'une couche intermédiaire, ce qui assure une fonction améliorée du capteur. Dans certains modes de réalisation, il existe une liaison ou une adhérence de l'hydrogel enzymatique à la matrice polymère de détection d'oxygène dépendante de l'analyte cible, soit directement, soit par l'intermédiaire d'une couche barrière intermédiaire, de telle sorte que des variations de la pression partielle d'oxygène dans la matrice d'hydrogel enzymatique sont rapidement réfléchies dans la pression partielle d'oxygène dans la matrice de détection d'oxygène. Les capteurs fournissent un moyen implantable, à porter sur soi, jetable et idéalement à faible coût permettant de détecter en continu un ou plusieurs analytes dans le corps, tels que le glucose.
PCT/US2022/079904 2021-11-16 2022-11-15 Système de transduction chimique dans un capteur physiologique d'analyte enzymatique optique WO2023091915A1 (fr)

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