Oxidase-based sensors and methods of using

Abstract

Oxidase-based sensors and methods of using the sensors are provided.

Claims

1. A sensor for detecting an analyte, comprising one or more analyte sensing populations comprising: (a) one or more polymers, wherein the one or more polymers is formed from one or more monomers, one or more comonomers and one or more crosslinkers; wherein the one or more monomers are selected from the group consisting of: 2-hydroxyethyl methacrylate (HEMA) and hydroxypropyl methacrylate (HPMA); the one or more comonomers are selected from the group consisting of: HPMA and n-hexylacrylate (nHA); and the one or more crosslinkers is ethylene glycol dimethacrylate (EGDMA); wherein the one or more comonomers are is different from the one or more monomers; (b) one or more oxidases; and (c) one or more oxygen sensitive dyes.

2. The sensor of claim 1, wherein the analyte is lactate and the one or more oxidases is lactate oxidase.

3. The sensor of claim 1, wherein the analyte is glucose and the one or more oxidases is glucose oxidase.

4. The sensor of claim 1, further comprising an oxygen reference population comprising an oxygen reference dye.

5. A method for detecting an analyte in a subject, comprising placing a sensor of claim 1 in a subject, wherein the sensor generates detectable luminescent signal.

6. The sensor of claim 1, further comprising an additional analyte sensing population.

7. The sensor of claim 6, wherein the additional analyte sensing population detects the analyte at a different oxygen concentration.

8. The sensor of claim 4, wherein the oxygen reference population is between 0.1 mm and 5 mm from the one or more analyte sensing populations.

9. The sensor of claim 1, wherein the analyte is alcohol and the one or more oxidases is alcohol oxidase.

10. The sensor of claim 1 wherein the one or more polymers are formed from HEMA monomer, HPMA comonomer, and EGDMA crosslinker.

11. The sensor of claim 10, wherein: the HEMA monomer is 40 wt % to 80 wt % of the one or more polymers; the HPMA comonomer is 15 wt % to 30 wt % of the one or more polymers; and the EGDMA is 4 wt % to 20 wt % of the one or more polymers.

12. The sensor of claim 10, wherein: the HEMA monomer is about 63 wt % of the one or more polymers; the HPMA comonomer is about 27 wt % of the one or more polymers; and the EGDMA is about 10 wt % of the one or more polymers.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1A shows a schematic of general sensing mechanism of the oxidase-based sensors described herein. FIG. 1B shows a schematic of general sensing mechanism of the lactate oxidase-based sensors described herein. FIG. 1C shows a schematic of general sensing mechanism of the glucose oxidase-based sensors described herein.

(2) FIG. 2 illustrates the phosphorescence signal vs. lactate concentration of sensors including methacrylate monomers, acrylate comonomers, and methacrylate crosslinkers (closed squares) as compared to that of sensors including mixed methacrylate and methacrylamide monomers, comonomers, and crosslinkers (open circles). These measurements were taken at 2% oxygen.

(3) FIG. 3 shows the performance of the glucose oxidase containing sensor (red line) in pigs, as compared to glucose levels (black line) and oxygen levels (blue line).

(4) FIG. 4 shows the performance of lactate sensors with sensing population 1 at 21% oxygen and sensing population 2 at 2% oxygen. Sensing population 1 includes mixed methacrylate and methacrylamide monomers, comonomers, and crosslinkers. Sensing population 2 includes methacrylate monomers, acrylate comonomers, and methacrylate crosslinkers.

(5) FIG. 5 illustrates alcohol oxidase-based sensors tested at 5% oxygen. Sensors show a response when exposed to increasing concentrations of ethanol.

DETAILED DESCRIPTION

(6) Oxidase-based sensors designed to measure analytes, such as lactate and glucose at physiological oxygen concentrations are described herein. Additionally, sensors containing distinct sensing populations measuring different concentration ranges are described.

(7) Sensors described herein include one or more polymers, one or more oxidases, and one or more oxygen sensitive dyes. Exemplary oxidases include but are not limited to naturally occurring oxidases, genetically engineered oxidases, monooxygenases, glucose oxidase, lactate oxidase, pyruvate oxidase, alcohol oxidase, bilirubin oxidase, and histamine oxidase. Additionally, the oxidase-based sensors may further include one or more oxygen sensitive reference dye. As described herein, the measurement of the analyte by the described sensors does not require implanted electronics.

(8) An embodiment relates to a sensor including two or more sensing populations. The two or more sensing populations may measure the same analyte, or the two or more sensing populations may measure different analytes. In an embodiment, one lactate sensing population measures lactate at a first percentage of oxygen, and a second sensing population measures lactate at a second percentage of oxygen. In a further aspect of the embodiment, additional sensing populations that measure lactate at different percentages of oxygen are also contemplated. Each sensing population includes one or more polymers, one or more lactate oxidases, and one or more oxygen sensitive dyes. This embodiment is non-limiting; although described for lactate, other oxidases, such as glucose oxidase, and their corresponding analytes are contemplated.

(9) Measurement of an Analyte Using Oxidase-Based Sensors Described Herein

(10) Without being bound by a particular mechanism, it is believed that in the sensors described herein as the analyte is enzymatically converted, oxygen is consumed by the enzyme. The sensors measure the amount of oxygen, and the depletion of oxygen is directly related to the analyte concentration for a given oxygen concentration, that is, the concentration of oxygen and the analyte are inversely proportional. The particular analyte will be specific for the corresponding oxidase in the sensor; for example, lactate is the analyte for the lactate oxidase, and glucose is the analyte for the glucose oxidase. In an exemplary embodiment, without being bound by a particular mechanism, it is believed that in the exemplary sensors described herein, as the analyte is enzymatically converted, oxygen is consumed by the enzyme (FIG. 1A). The sensors measure the amount of oxygen, and the depletion of oxygen is directly related to the analyte concentration for a given oxygen concentration. In an exemplary embodiment, without being bound by a particular mechanism, it is believed that in the exemplary lactate sensors described herein, as the lactate is enzymatically converted, oxygen is consumed by the enzyme (FIG. 1B). The sensors measure the amount of oxygen, and the depletion of oxygen is directly related to the lactate concentration for a given oxygen concentration. In an exemplary embodiment, without being bound by a particular mechanism, it is believed that in the exemplary glucose sensors described herein, as the glucose is enzymatically converted, oxygen is consumed by the enzyme (FIG. 1C). The sensors measure the amount of oxygen, and the depletion of oxygen is directly related to the glucose concentration for a given oxygen concentration.

(11) After initial sensor injection, measurements are collected non-invasively through luminescent near infrared (NIR) signals with a specially designed optical reader. In an embodiment, the optical reader is located outside of the body. These continuous analyte sensors have the potential to transform the field of analyte monitoring, such as lactate and glucose monitoring, by providing non-invasive, real-time, continuous analyte measurements in a user-friendly, cost-effective format.

(12) Polymers

(13) Sensors described herein may comprise several types of polymers. Each sensing population comprises one or more polymers, as described below. In addition, the sensors may further comprise one or more additional scaffold polymers. The scaffold polymers may form the scaffold of the sensor. The one or more sensing populations may be included in the scaffold. The one or more sensing populations may be covalently or non-covalently bound to the scaffold.

(14) Polymers Useful in Oxidase-Based Sensors Functioning at a First Concentration of Oxygen

(15) In an aspect, oxidase-based sensors may be useful in detecting a particular analyte at a first concentration of oxygen. The first concentration of oxygen may be a concentration of oxygen that is found in the tissue under normal conditions. The first concentration of oxygen may be about 6% or less, about 5% or less, about 3% or less, about 2% or less, or about 1% or less.

(16) In an exemplary embodiment, FIG. 2 illustrates the phosphorescence signal vs. lactate concentration of sensors including methacrylate monomers, acrylate comonomers, and methacrylate crosslinkers (closed squares) as compared to that of sensors including mixed methacrylate and methacrylamide monomers, comonomers, and crosslinkers (open circles). These measurements were taken at 2% oxygen. As shown, sensors including methacrylate monomers, acrylate comonomers, and methacrylate crosslinkers (closed squares) respond to increasing lactate in a manner that can be correlated to lactate concentration (at 2% oxygen). Sensors including mixed methacrylates and methacrylamides monomers, comonomers, and crosslinkers (open circles) did not show a correlation to the lactate concentrations tested (at 2% oxygen).

(17) In an aspect, as illustrated in FIG. 2, the one or more polymers may be formed from one or more methacrylate or acrylate monomers, one or more methacrylate or acrylate comonomers, and one or more methacrylate or acrylate crosslinkers.

(18) In an embodiment, the methacrylate or acrylate monomers and comonomers may be selected from the group consisting of: 3-chloro-2-hydroxypropyl methacrylate (3C2HPMA), 2-hydroxyethylmethacrylate (HEMA), pentafluorobenzyl methacrylate (PFBMA), butylmethacrylate (BMAcrylate), hydroxypropyl methacrylate (HPMA), methyl methacrylate (MMA), n-hexylacrylate (nHA), 2-methacryloyloxyethyl phosphorylcholine (MPC), hexyl methacrylate (HexMA), 2,2,3,3,4,4,4-heptafluorobutyl methacrylate (HFMA), and polyethylene glycol methacrylate (Mn=500) (PEGMA 500). In an embodiment, the monomer and the comonomer are not the same. In an aspect, the methacrylate or acrylate monomers and comonomers may be selected from the group consisting of: HEMA, HPMA, and nHA. In an aspect, the methacrylate or acrylate monomers and comonomers may be selected from the group consisting of: HPMA and nHA.

(19) In an embodiment, the methacrylate or acrylate crosslinker may be selected from the group consisting of: bisphenol A glycerolate diacrylate (BPADA), ethylene glycol dimethacrylate (EGDMA), 1,6-hexanediol diacrylate (HDDA), neopentyl glycol diacrylate (NPDA), pentaerythritol triacrylate (PEA3), pentaerythritol tetraacrylate (PEA4), poly(etheylene glycol) diacrylate (Mn=700) (PEGDA 700), diurethane dimethacrylate (UDMA), di(trimethylolpropane) tetra-acrylate (DTMPTA) and tetraethylene glycol dimethacrylate (TEGDMA). In an embodiment, the crosslinker may be selected from the group consisting of: bisphenol A glycerolate diacrylate (BPADA), ethylene glycol dimethacrylate (EGDMA), 1,6-hexanediol diacrylate (HDDA), neopentyl glycol diacrylate (NPDA), pentaerythritol triacrylate (PEA3), pentaerythritol tetraacrylate (PEA4), poly(etheylene glycol) diacrylate (Mn=700) (PEGDA 700), and diurethane dimethacrylate (UDMA). In an aspect, the methacrylate or acrylate crosslinkers may be EGDMA.

(20) In one embodiment, the one or more polymers may be formed from one or more methacrylamide or acrylamide monomers, one or more methacrylamide or acrylamide comonomers, and one or more methacrylamide or acrylamide crosslinkers. In an embodiment, the methacrylamide or acrylamide monomers, comonomers, and crosslinkers may be selected from the group consisting of: poly(ethylene glycol) diacrylamide (Mn=3700) (PEGDAAm (3700)), N-Isopropylacrylamide (NIPAAm), N-(2-hydroxyethyl) methacrylamide (HEMAM), dimethacrylamide (DMA), and N,N′-methylenebis(acrylamide) (Bis). In an aspect, the methacrylamide or acrylamide monomer or comonomer may be DMA.

(21) The polymers of the present invention may be described by the weight percentage of up to three primary components (monomer, comonomer, and crosslinker) in the precursor solution. Prior to polymerization, these components may comprise about 10-90% volume of the precursor solution. In one embodiment, these components may comprise about 30-80% volume of the precursor solution. In one embodiment, these components may comprise about 50-70% volume of the precursor solution. In one embodiment, these components may comprise about 70% volume of the precursor solution. The remaining volumetric components may be sensing elements, dyes, co-solvents, crosslinkers that incorporate into the polymer.

(22) The polymers of the present invention may be described by the weight percentage of up to three primary components (monomer, comonomer, and crosslinker) in the precursor solution.

(23) Prior to polymerization, these components may comprise about 10-90% w/w of the precursor solution. In one embodiment, these components may comprise about 30-80% w/w of the precursor solution. In one embodiment, these components may comprise about 50-70% w/w of the precursor solution. In one embodiment, these components may comprise about 70% w/w of the precursor solution. The remaining volumetric components may be sensing elements, dyes, co-solvents, crosslinkers that incorporate into the polymer.

(24) In particular embodiments, the weight percentage of methacrylate or acrylate monomer as compared to the other primary comonomers and crosslinkers in the precursor solution (Table 2) may be: about 40 to 100% w/w. In an embodiment, weight percentage of methacrylate or acrylate monomer (Table 2) may be: about 60 to 80% w/w. In an embodiment, weight percentage of methacrylate or acrylate monomer (Table 2) may be: about 60 to 75% w/w.

(25) In particular embodiments, the weight percentage of methacrylate or acrylate comonomer as compared to the other primary monomers and crosslinkers in the precursor solution (Table 2) may be: 0 to 50% w/w. In an embodiment, weight percentage of methacrylate or acrylate comonomer (Table 2) may be: 5 to 30% w/w. In an embodiment, weight percentage of methacrylate or acrylate comonomer (Table 2) may be: 15 to 30% w/w.

(26) In particular embodiments, the weight percentage of methacrylate or acrylate crosslinker as compared to the other primary monomers and comonomers in the precursor solution (Table 2) may be: 0 to 25% w/w. In an embodiment, weight percentage of methacrylate or acrylate crosslinker (Table 2) may be: 4 to 20% w/w. In an embodiment, weight percentage of methacrylate or acrylate crosslinker (Table 2) may be: 5 to 17% w/w.

(27) In particular embodiments, the weight percentage of methacrylamide or acrylamide monomer as compared to the other primary comonomers and crosslinkers in the precursor solution (Table 3) may be: 85 to 100% w/w. In an embodiment, weight percentage of methacrylamide or acrylamide monomer (Table 3) may be: 85 to 95% w/w. In an embodiment, weight percentage of methacrylamide or acrylamide monomer (Table 3) may be: 90 to 95% w/w.

(28) In particular embodiments, the weight percentage of methacrylamide or acrylamide crosslinker as compared to the other primary monomers and comonomers in the precursor solution (Table 3) may be: 0 to 15% w/w. In an embodiment, weight percentage of methacrylamide or acrylamide crosslinker (Table 3) may be: 4 to 12% w/w. In an embodiment, weight percentage of methacrylamide or acrylamide crosslinker (Table 3) may be: 5 to 10% w/w.

(29) Analyte Sensing Protein

(30) The sensor includes an analyte sensing protein. In an embodiment, the analyte sensing protein may be an oxidase. In an aspect, analyte sensing proteins may include but are not limited to naturally occurring oxidases, genetically engineered oxidases, monooxygenases, glucose oxidase, lactate oxidase, pyruvate oxidase, alcohol oxidase, bilirubin oxidase, and histamine oxidase.

(31) Exemplary lactate oxidases include, but are not limited to, lactate 2-monooxygenase and lactate monooxygenase. Lactate 2-monooxygenase may be derived from different species including, but not limited to, Aerococcus viridans, Pediococcus species, and native microorganism. Glucose oxidase, also known as GOx or notatin, may be derived from different species including, but not limited to, Penicillium notatum, and Aspergillus niger. As shown in FIG. 1B, lactate oxidases consume oxygen and convert lactate to either pyruvate and hydrogen peroxide or acetate, carbon dioxide, and water. Similarly, glucose oxidases consume oxygen and convert glucose to hydrogen peroxide and D-glucono-δ-lactone. The reduction of oxygen in the vicinity of the enzyme can be measured by using an oxygen-sensitive dye, such as a porphyrin dye. These dye molecules are quenched in the presence of oxygen, so the reduction of oxygen by the action of oxidases causes an increase in luminescence and phosphorescent lifetime. Luminescence and phosphorescent lifetimes from the oxygen-sensitive dyes is thus proportional to the concentration of the analyte in the sensor.

(32) In an embodiment, the one or more oxidases may be commercially available or produced by a user. The oxidase may be naturally occurring, may be recombinant, may contain mutations, or may have post transcriptional modifications such as glycosylation, or the like.

(33) In an embodiment, the oxidase may be a monomer, dimer, trimer, or tetramer.

(34) The lactate oxidase can be engineered from different species that include but are not limited to Aerococcus viridans and Pediococcus species. The glucose oxidase can be engineered from different species that include but are not limited to Penicillium notatum, and Aspergillus niger.

(35) In an embodiment, the oxidase may be physically entrapped or chemically bound within the sensor. In an embodiment, the oxidase may be attached to the polymer, such through a covalent or non-covalent linkage. In an embodiment, the oxidase may not be chemically conjugated to the polymer. In another embodiment, the oxidase may be attached to the surface of the sensor, such as via covalent or non-covalent linkages. In yet another embodiment, oxidase may be present within the sensor through more than one of the above means, e.g., oxidase may be attached to the polymer via a covalent linkage and physically entrapped within the sensor. In an embodiment, the oxidase may be on the surface of the sensor and also within the sensor. In an embodiment, the sensor may be covered by an exterior coating.

(36) Oxygen Sensitive Dye

(37) Sensors described herein also include an oxygen sensitive dye. In an embodiment, the oxygen sensitive dye may be a porphyrin dye. The oxygen sensitive dye may be a NIR porphyrin molecule.

(38) In an embodiment, the oxidase and the oxygen sensitive dye are co-located in the sensor.

(39) In an embodiment, the oxygen sensitive dye may be selected from one described in U.S. Pat. No. 9,375,494, which is hereby incorporated by reference herein.

(40) In an embodiment, the oxygen sensitive dye may be covalently attached to the polymer. In an embodiment, the oxygen sensitive dye may be covalently attached to the oxidase. In an embodiment, the oxygen sensitive dye may be non-covalently bound to the polymer.

(41) Sensor Design

(42) In an embodiment, the sensor may be 1-10 mm in length. The sensor may be 0.25-1 mm in diameter. In an embodiment, the sensor may be rod-shaped, spherical, block-like, cube-like, disk-shaped, cylindrical, oval, round, random or non-random configurations of fibers and the like. In an embodiment, the sensor may be a microsphere or a nanosphere.

(43) In an embodiment, one sensor may include two or more sensing populations. These two or more sensing populations may be in distinct portions of the sensor. In an aspect, each of the two or more sensing populations may detect different analytes. In an aspect, each of the two or more sensing populations may detect different concentrations of the same analyte. In an aspect, a first sensing population of a sensor may measure an analyte at a first concentration of oxygen, and a second sensing population of the sensor may measure the analyte at a second concentration of oxygen. In an embodiment, the second concentration of oxygen may be higher than the first concentration of oxygen. In an embodiment, at least one of the concentrations of oxygen may be a physiological concentration of oxygen.

(44) In an embodiment, one or more of the sensing populations may be microspheres, nanospheres, microparticles, nanoparticles, and the like. In an embodiment, the scaffold of the sensor may include a polymer that be different from, or the same as, the polymer in a sensing population.

(45) In an embodiment, the sensor may include distinct layers where the oxidase is physically entrapped or chemically bound to or within specific layers of the sensor. In a further embodiment, the sensor may include additional layers; the additional layers may provide other features such as mechanical strength, elasticity, conductivity or other properties. The additional layers may detect different analytes or different concentrations of the same analyte. The additional layers may include a reference dye.

(46) In certain embodiments, the sensor includes additional moieties (e.g., non-sensing or additional sensing moieties different from the sensing moieties), for example reference (or calibration) moieties. Reference or calibration moieties include, but are not limited to, dyes, fluorescent particles, lanthanides, nanoparticles, microspheres, quantum dots or other additives or elements of the implant whose signal does not change due to the presences of the analyte (e.g., glucose). See, e.g., Chaudhary et al. (2009) Biotechnology and Bioengineering 104(6):1075-1085, which is hereby incorporated herein by reference in its entirety. Fluctuations in the reference (calibration) signal(s) can be used to correct or calibrate the sensing signal(s).

(47) Oxygen Reference Dye

(48) In an embodiment, the oxidase-based sensor may also include an additional oxygen-sensitive dye that serves as a reference for the amount of locally present oxygen.

(49) In an embodiment, the oxygen reference dye may be a porphyrin dye. The oxygen reference dye may be a NIR porphyrin ring molecule. In an embodiment, the oxygen reference dye may include the same type of chemistry as the oxygen-sensitive dye. The oxygen reference dye may be selected from one described in U.S. Pat. No. 9,375,494, which is hereby incorporated by reference herein.

(50) The oxygen reference dye may be covalently or non-covalently attached to a polymer. The polymer and the one or more oxygen reference dyes may form an oxygen reference population. The polymer of the oxygen reference population may be the same or different from the polymer of the scaffold. In an embodiment, one or more of the oxygen reference dye populations may be microspheres, nanospheres, microparticles, nanoparticles, and the like.

(51) In an embodiment, the one or more analyte sensing populations is adjacent to the oxygen reference population with no space between. In an embodiment, there may be a spacer between the analyte sensing portion and the oxygen reference population. The spacer may include the same or different polymer materials as the analyte and oxygen sensing populations. In an aspect, the spacer may separate the analyte sensing populations and oxygen reference population between 0.1 and 5 mm. In an embodiment, the spacer may be between 0.5 and 2 mm. In an embodiment, the spacer may be greater than 0.2 mm. In an embodiment, the spacer may be greater than 0.5 mm.

(52) In an embodiment, multiple sensors containing the same or different sensing populations may be implanted near each other. For example, one or more sensors containing only the first sensing population may be implanted near one or more sensors containing only the second sensing population. For example, one or more sensors containing only the oxygen reference population may be implanted near the one or more sensors containing only the first sensing population and/or the second sensing population. In an aspect, one sensor may include multiple sensing populations. For example, one or more sensors containing the first sensing population and the second sensing population may be implanted near one or more sensors containing a third sensing population. For example, one or more sensors containing both the first sensing population and the second sensing population may be implanted near one or more sensors containing one or more oxygen reference populations. In an aspect, a sensor may include one or more sensing populations and one or more reference populations. The sensors may be implanted in a particular design, such as a ring, or another geometry.

(53) Properties

(54) In addition, the scaffold of the invention may be constructed such that it has conduits, pores or pockets that are hollow or filled with degradable, angiogenic, or other substances (e.g. stem cells). Once in the body, the biodegradation of the material filling the conduits, pores or pockets, creates space for tissue, including capillaries to integrate with the material. The degradable material that initially fills the conduits, pores or pockets may enhance vessel growth or tissue growth within the scaffold. This architecture promotes new vessel formation and maintains healthy viable tissue within and around the implant.

(55) Methods of Using Oxidase-Based Sensors

(56) Oxidase-based sensors as described herein are useful in the monitoring of a number of conditions. The oxidase-based sensors may be placed subcutaneously, surrounding tissue of muscle, subcutaneous fat, dermis, in muscle, in skin, in the limbs, sternum, neck, ear, brain, or other locations.

(57) In an embodiment, the lactate sensors described herein may be useful in monitoring trauma, sepsis, exercise physiology/performance optimization, skin grafts, wound healing, shock, and other disease states as described in Andersen et al. (2013) Mayo Clin Proc 88 (10): 1127-1140, which is hereby incorporated herein by reference in its entirety.

EXAMPLES

Example 1

Lactate Sensor

(58) Sensor Fabrication: Formulations with monomer mixtures for each sensor type are listed in Table 1 and apply to FIGS. 2 and 4. FIG. 2 contains formulations 2 (open circles) and 3 (closed squares). FIG. 4 contains formulations 1 (open circles) and 3 (closed squares). All formulations contained 0.56 mM 2-aminoethylmethacrylate in distilled water, 19 mM of Irgacure 651, and enzymatic components were dissolved in 20 mM PBS such that the PBS volume was 18% of the total mixture volume. The mixture was then polymerized. After polymerization, the polymeric material was rinsed with distilled water and placed in 20 ml of PBS with 0.7 mM N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and 1.8 mM N-hydroxysulfosuccinimide (NHS) for at least 8 hours except for Formulation 2. The material was then removed, rinsed three times with PBS, and cut into sensor rods with dimensions of 5 mm×0.5 mm×0.5 mm.

(59) Sensor Performance Testing: Sensors were placed in a customized test fixture with controllable oxygen levels. All sensors were tested in either 500 or 800 ml of PBS and allowed to equilibrate to either 2 or 21% oxygen at 37° C. Pumps were used to dispense lactate at stepwise increases in concentration (Table 1). At each lactate concentration, the sensor phosphorescence signal was equilibrated. Response curves were generated by averaging the phosphorescence signal of the last 2 minutes of each step prior to increasing lactate.

(60) TABLE-US-00001 TABLE 1 Lactate sensor formulations depicted in FIGS. 2 and 4. Monomer/ Total Monomer/ Comonomer/ Enzymatic Volume Comonomer/ Crosslinker Components Oxygen Lactate Formulation (uL) Crosslinker (M) Cosolvents Dye (w/v %) Conc. (%) Conc. (mM) 1 500 HEMA/ 3.0/ 1.4M 1 mM 2.1% LOx.sup.4 21 0, 0.8, 1.6, 3.2, DMA/ 0.9/ DMSO.sup.1 BMAP.sup.3 0.3% 6.4, 8 EGDMA 0.3 2.9M EG.sup.2 in DMSO catalase.sup.5 2 500 HEMA/ 3.0/ 3.7M 1 mM 2.1% LOx.sup.6 2 0, 1, 2, 4, 8, 10 DMA/ 0.9/ DMSO BMAP EGDMA 0.3 in DMSO 3 125 HEMA/ 2.6/ 2.7M NMP 1 mM 2.1% LOx.sup.7 2 0, 0.8, 1.6, 3.2, nHA/ 0.8/ BMAP 0.3% 6.4, 8 EGDMA 0.3 in NMP catalase.sup.8 .sup.1Dimethysulfoxide .sup.2Ethylene glycol .sup.3Pd-BMAP-AEME-4 (U.S. Pat. No. 9,375,494) .sup.4from Aerococcus viridans .sup.5from bovine liver .sup.6from Aerococcus viridans .sup.7from Aerococcus viridans .sup.8from bovine liver

Example 2

Lactate Sensors

(61) TABLE-US-00002 TABLE 2 Methacrylate and acrylate-based oxidase-based sensor compositions (w/w % of monomer and/or polymer content of major components) Wt % Wt % Wt % Monomer Comonomer Crosslinker Monomer Comonomer Crosslinker 3-chloro-2- tetraethylene glycol 98.2 1.8 hydroxypropyl dimethacrylate methacrylate 2-hydroxyethyl tetraethylene glycol 98 2 methacrylate dimethacrylate 2-hydroxyethyl ethylene glycol 95.1 4.9 methacrylate dimethacrylate hydroxypropyl ethylene glycol 95 5 methacrylate dimethacrylate 2-hydroxyethyl pentaerythritol 94.4 5.6 methacrylate tetraacrylate 2-hydroxyethyl pentaerythritol 94.4 5.6 methacrylate triacrylate pentafluorobenzyl ethylene glycol 92.3 7.7 methacrylate dimethacrylate 2-hydroxyethyl 1,6-hexanediol 92.1 7.9 methacrylate diacrylate 2-hydroxyethyl ethylene glycol 90.2 9.8 methacrylate dimethacrylate hydroxypropyl ethylene glycol 90 10 methacrylate dimethacrylate 2-hydroxyethyl diurethane 89.8 10.2 methacrylate dimethacrylate 2-hydroxyethyl di(trimethylolpropane) 89.6 10.4 methacrylate tetra-acrylate 2-hydroxyethyl pentaerythritol 88.9 11.1 methacrylate triacrylate hydroxypropyl pentaerythritol 88.7 11.3 methacrylate triacrylate 2-hydroxyethyl diurethane 79.6 20.4 methacrylate dimethacrylate 2-hydroxyethyl 2-methacryloyloxyethyl ethylene glycol 87.6 0.5 11.9 methacrylate phosphorylcholine dimethacrylate 2-hydroxyethyl hydroxypropyl ethylene glycol 85.7 9.4 4.9 methacrylate methacrylate dimethacrylate 2-hydroxyethyl hexyl methacrylate ethylene glycol 82.6 7.4 10 methacrylate dimethacrylate 2-hydroxyethyl butylmethacrylate ethylene glycol 82.3 7.7 10 methacrylate dimethacrylate 2-hydroxyethyl methyl methacrylate ethylene glycol 82 8 9.9 methacrylate dimethacrylate 2-hydroxyethyl hydroxypropyl ethylene glycol 81.3 8.9 9.8 methacrylate methacrylate dimethacrylate 2-hydroxyethyl 2,2,3,3,4,4,4- ethylene glycol 79.2 11.3 9.6 methacrylate heptafluorobutyl dimethacrylate methacrylate 2-hydroxyethyl n-hexyl acrylate ethylene glycol 78.7 16.3 5.1 methacrylate dimethacrylate 2-hydroxyethyl n-hexyl acrylate ethylene glycol 78.5 13 8.5 methacrylate dimethacrylate 2-hydroxyethyl n-hexyl acrylate ethylene glycol 78.5 11.5 10 methacrylate dimethacrylate 2-hydroxyethyl butylmethacrylate ethylene glycol 78.4 16.5 5.1 methacrylate dimethacrylate 2-hydroxyethyl n-hexyl acrylate ethylene glycol 77.9 7.2 15 methacrylate dimethacrylate 2-hydroxyethyl hydroxypropyl ethylene glycol 76.8 8.4 14.8 methacrylate methacrylate dimethacrylate 2-hydroxyethyl hydroxypropyl ethylene glycol 76.3 18.8 4.9 methacrylate methacrylate dimethacrylate 2-hydroxyethyl hexyl methacrylate ethylene glycol 74.8 15 10.2 methacrylate dimethacrylate 2-hydroxyethyl n-hexyl acrylate ethylene glycol 74.5 15.4 10.1 methacrylate dimethacrylate 2-hydroxyethyl butylmethacrylate ethylene glycol 74.3 15.6 10.1 methacrylate dimethacrylate 2-hydroxyethyl methyl methacrylate ethylene glycol 73.7 16.2 10 methacrylate dimethacrylate 2-hydroxyethyl hydroxypropyl ethylene glycol 72.4 17.8 9.8 methacrylate methacrylate dimethacrylate 2-hydroxyethyl polyethylene glycol ethylene glycol 71.5 18.7 9.7 methacrylate methacrylate (Mn = 500) dimethacrylate 2-hydroxyethyl 3-chloro-2- ethylene glycol 70.5 19.9 9.6 methacrylate hydroxypropyl dimethacrylate methacrylate 2-hydroxyethyl n-hexyl acrylate ethylene glycol 70 24.8 5.2 methacrylate dimethacrylate 2-hydroxyethyl 2,2,3,3,4,4,4- ethylene glycol 68.7 22 9.3 methacrylate heptafluorobutyl dimethacrylate methacrylate hydroxypropyl 2-hydroxyethyl ethylene glycol 67.3 22.8 9.9 methacrylate methacrylate dimethacrylate 2-hydroxyethyl n-hexyl acrylate ethylene glycol 66.2 23.5 10.3 methacrylate dimethacrylate 2-hydroxyethyl n-hexyl acrylate tetraethylene glycol 65.9 23.4 10.7 methacrylate dimethacrylate 2-hydroxyethyl n-hexyl acrylate poly(ethylene glycol) 65.6 23.3 11.1 methacrylate diacrylate (Mn = 700) 2-hydroxyethyl methyl methacrylate ethylene glycol 65.2 24.6 10.1 methacrylate dimethacrylate 2-hydroxyethyl hydroxypropyl ethylene glycol 64 27 9 methacrylate methacrylate dimethacrylate 2-hydroxyethyl hydroxypropyl ethylene glycol 63.4 26.7 9.9 methacrylate methacrylate dimethacrylate 2-hydroxyethyl n-hexyl acrylate poly(ethylene glycol) 61.6 21.9 16.5 methacrylate diacrylate (Mn = 700) 2-hydroxyethyl n-hexyl acrylate poly(ethylene glycol) 57.6 20.4 21.9 methacrylate diacrylate (Mn = 700) 2-hydroxyethyl methyl methacrylate ethylene glycol 47.6 42 10.4 methacrylate dimethacrylate 2-hydroxyethyl hydroxypropyl ethylene glycol 45.4 44.7 9.9 methacrylate methacrylate dimethacrylate

(62) TABLE-US-00003 TABLE 3 Methacrylamide and acrylamide-based oxidase-based sensor compositions (w/w % of monomer and/or polymer content of major components) Wt % Wt % Monomer Crosslinker Monomer Crosslinker poly(ethylene glycol) 100 diacrylamide (Mn = 3700) N-Isopropylacrylamide N,N′-methylenebis(acrylamide) 95.2 4.8 N-(2-hydroxyethyl)methacrylamide N,N′-methylenebis(acrylamide) 88.9 11.1

Example 3

Glucose Sensors

(63) Sensors of Table 4 were be fabricated as follows: 0.56 mM (3-aminopropyl)methacrylamide, 15 mM of 2,2′-azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride, and enzymatic components were dissolved in 10 mM PBS such that the PBS volume was 21% of the total mixture volume. All components were mixed, molded, and polymerized. Glucose sensors were then placed in 20 ml of PBS with 0.7 mM N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and 1.8 mM N-hydroxysulfosuccinimide (NHS) for at least 8 hours. Oxygen sensors were placed in 20 ml of PBS for at least 1 hour. After soaking in PBS, sensors were placed in distilled water for at least 15 minutes then removed and dried under vacuum. After drying, sensors were ethylene oxide sterilized for implantation.

(64) TABLE-US-00004 TABLE 4 Oxygen and glucose sensor formulations depicted in FIG. 3. Total Monomer/ Monomer/ Enzymatic Volume Comonomer/ Comonomer/ Components Sensor (uL) Crosslinker Crosslinker (M) Cosolvents Dye (w/v %) Glucose 125 HEMA/ 3.0/ 0.9M DMSO 1 mM BMAP 3.6% glucose Sensor nHA/ 0.5/ 3.2M EG in DMSO oxidase.sup.9/0.6% EGDMA 0.3 catalase.sup.10 Oxygen 125 HEMA/ 3.0/ 1.5 M DMSO 1 mM BMAP N/A Sensor nHA/ 0.5/ 3.2M EG in DMSO EGDMA 0.3

(65) Sensors were injected subcutaneously into a female Sinclair mini-pig with an 18 gauge needle attached to a custom built trocar-like injection device. Sensor signal was monitored throughout the experiment using a custom handheld optical reader system. Under anesthesia, a dextrose infusion was administered to the animal to achieve a glucose challenge. Blood glucose measurements were recorded using a commercially available handheld glucometer to track increases and decreases in blood glucose values. FIG. 3 shows the blood glucose measurements and phosphorescent lifetime signals of glucose and oxygen sensors implanted for 28 days (n=1 each).

Example 4

Alcohol Sensors

(66) Alcohol sensors were made by dissolving enzymatic components (e.g., alcohol oxidase) and 2,2-dimethoxy-1,2-diphenylethan-1-one into one or more solvents. This mixture was combined 1:1 (v/v) with a solution containing 90% HEMA and 10% ethylene glycol dimethacrylate (wt %). The mixture was then polymerized. After polymerization, the polymeric material was rinsed with distilled water and cut into sensor rods with dimensions of 5 mm×0.5 mm×0.5 mm and stored in PBS until tested. Sensor Performance Testing: Sensors were placed in a customized test fixture with controllable oxygen levels. All sensors were tested in either 500 or 800 mL of PBS and allowed to equilibrate to 5% oxygen at 37° C. Pumps were used to dispense ethanol at stepwise increases in concentration. At each ethanol concentration, the sensor phosphorescence signal was equilibrated. Response curves were generated by averaging the phosphorescence signal of the last 2 minutes of each step prior to increasing ethanol. An increase in sensor signal with increasing concentrations of ethanol exhibits a clear sensitivity to the analyte added to the test vessel. FIG. 5 illustrates alcohol oxidase-based sensors tested at 5% oxygen. Sensors show a response when exposed to increasing concentrations of ethanol.

(67) While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed in practicing the disclosure. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.

(68) All patents, patent applications and publications mentioned herein are hereby incorporated by reference in their entirety.

(69) Although disclosure has been provided in some detail by way of illustration and example for the purposes of clarity of understanding, it will be apparent to those skilled in the art that various changes and modifications can be practiced without departing from the spirit or scope of the disclosure. Accordingly, the foregoing descriptions and examples should not be construed as limiting.