Luminescent enzyme-based sensors

Abstract

This document describes medical systems for detecting biological analytes. For example, this document describes sensors for continuous monitoring of biological analytes such as glucose and / or lactate in aqueous solutions and body fluids (e.g., blood) based on fluorescent or luminescent signal readouts.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Application No. 63 / 406,567, filed September 14, 2022. The disclosure of the prior application is considered part of the disclosure of this application (and is incorporated by reference herein). [Technical Field]

[0002] This document relates to medical systems for sensing biological analytes using enzymes. For example, this document relates to sensors for continuous monitoring of glucose and / or lactate in aqueous solutions and body fluids based on fluorescent or luminescent signal readouts. [Background technology]

[0003] Monitoring biological analytes, such as pH, blood gases, electrolytes, and metabolites, is one of the primary means for assessing an individual's overall health and the state of their bodily functions, particularly in critical care settings. For example, dedicated analyzers are used in near-patient testing environments to provide continuous, real-time measurement and detection of blood analytes during critical care situations. Blood analyte measurements provide valuable information regarding an individual's oxygenation, gas exchange, acid-base homeostasis, and ventilation status. While various biological analyte sensor technologies have been developed, improvements in design, functionality, and accuracy are constantly being sought. Furthermore, enzymes and / or probes may lose functionality if inaccurate or incompatible solvents or membrane materials are used, or if interference barriers are improperly positioned. Currently, no sensors exist that can continuously monitor biological analytes, such as glucose and / or lactate, in blood during surgery or at the bedside in real time. Summary of the Invention

[0004] This document describes medical systems for sensing biological analytes using enzymes. For example, this document describes sensors for continuous monitoring of biological analytes such as glucose and / or lactate in aqueous solutions and body fluids based on luminescence signal readouts.

[0005] In one aspect, the present disclosure is directed to a blood parameter measurement device having a tubular housing defining an interior space configured to receive blood and a sensor connected to the tubular housing. The sensor can have (i) a first layer including an enzyme that produces hydrogen peroxide when reacting with at least one biological analyte in the blood, and (ii) a second layer having a substance that chemically reacts with hydrogen peroxide. The first layer is closer to the interior space than the second layer.

[0006] In some cases, the enzyme is selected from the group consisting of glucose oxidase (GOx), lactate oxidase (LOx), cholesterol oxidase (ChOx), galactose oxidase (GAOx), pyruvate oxidase (POx), xanthine oxidase (XAOx), monoamine oxidase A (MAO-Ax), monoamine oxidase B (MAO-Bx), D-amino acid oxidase (D-AAOx), L-amino acid oxidase (L-AAOx), lactose oxidase (LOx), and superoxide dismutase (SOD). In some cases, the enzyme is or includes glucose oxidase (GOx). In some cases, the enzyme is or includes lactate oxidase (LOx).

[0007] In some cases, the material chemically reactive with hydrogen peroxide includes a Europium(III)-tetracycline (EuTu) complex. In some cases, the first layer and the second layer are directly adjacent to one another.

[0008] In some cases, the sensor further comprises an intermediate layer between the first layer and the second layer. In some cases, the first layer is an annular layer defining an open space and substantially centered about the second layer. In some cases, the sensor further comprises a protective layer positioned between the first layer and the interior space.

[0009] In some cases, the sensor further comprises a reference dye. In some cases, the reference dye is selected from the group consisting of 9,10 di(phenylenthynyl)anthracene (DPEA), CD405M, 1-anilinonaphthalene-8-sulfonic acid, 8-benzyloxy-5,7-diphenylquinoline, 4-methylumbelliferyl acetate, octadecyl 7-hydroxycoumarine-3-carboxylate, perylene, tetracene, H9-40, pyranine, etyl eosin, coumarin 30, coumarin 153, and CF™ 405M. In some cases, the reference dye has an excitation maximum at 400±10 nm and an emission maximum at 450±10 nm. In some cases, the sensor further comprises a reference layer including the reference dye. The reference layer may be positioned farther from the interior space than the second layer.

[0010] The technology described herein can provide one or more advantages and / or benefits. For example, the technology facilitates real-time monitoring of blood parameters, which provides critical information necessary for target-directed perfusion during cardiopulmonary bypass surgery, and continuous analyte detection in blood via luminescence measurements. The technology also enables ongoing bedside monitoring of a patient's bodily fluids, including, but not limited to, blood. The described technology is an affordable system that uses relatively inexpensive components and can utilize inexpensive mass production and / or roll-to-roll manufacturing processes.

[0011] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the present invention, the preferred methods and materials are described herein. In case of conflict, the present specification, including definitions, will control. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0012] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description herein. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. [Brief explanation of the drawings]

[0013] [Figure 1] 1 is a schematic diagram of a patient undergoing open heart surgery while being supported using a heart-lung system and an extracorporeal circuit. [Figure 2] FIG. 1 is a perspective view of an exemplary blood parameter measurement system according to some embodiments. [Figure 3] 1 illustrates a schematic diagram of a multi-layer structure of an exemplary sensor according to some embodiments. [Figure 4] 1 illustrates a schematic diagram of a multi-layer structure of an exemplary sensor according to some embodiments. [Figure 5] 1A and 1B illustrate schematic diagrams of multi-layer structures of exemplary sections of a sensor according to some embodiments. [Figure 6] 1 illustrates a schematic diagram of a multi-layer structure of an exemplary sensor according to some embodiments. [Figure 7] 1 illustrates a schematic diagram of a multi-layer structure of an exemplary sensor according to some embodiments. [Figure 8] 1 illustrates a schematic diagram of a multi-layer structure of an exemplary sensor according to some embodiments. [Figure 9] 1 is a flowchart of an exemplary method for fabricating a multi-layer sensor according to some embodiments. [Figure 10] 1 is a flowchart of an exemplary method for fabricating a multi-layer sensor according to some embodiments. [Figure 11] 1 illustrates schematically the use of sensor measurements in a flow cell or flow-through format. [Figure 12] 1 illustrates a schematic diagram of a multi-layer structure of an exemplary sensor according to some embodiments. DETAILED DESCRIPTION OF THE INVENTION

[0014] Like reference numbers refer to corresponding parts throughout.

[0015] This document describes medical systems and devices for sensing and / or measuring biological analytes. For example, this document describes optical sensors, and in some cases enzyme-based sensors, for continuous monitoring of biological analytes, such as glucose and / or lactate, in aqueous solutions and bodily fluids (e.g., blood) based on fluorescent or luminescent signal readouts.

[0016] 1 , various types of medical procedures may be performed on the patient 10 while the patient 10 is connected to a heart-lung machine ("HLM") system 100. Before, during, and / or after such procedures, parameters of the patient's 10 blood may be measured to monitor the condition of the patient 10. As described further below, types of patient parameters that may be measured include glucose and / or lactate in the patient's 10 blood.

[0017] In this example, patient 10 is undergoing open-heart surgery, during which the patient's 10 heart 12 and lungs are intentionally temporarily shut down. Because the patient's 10 body has a continuing metabolic need to receive a supply of circulating oxygenated blood during the medical procedure, HLM system 100 performs such functions. That is, HLM system 100 is connected to patient 10 and performs the functions of the patient's 10 heart 12 and lungs to keep the patient 10 alive and healthy during the open-heart surgery. Types of procedures that can be performed on patient 10 in the illustrated manner include, but are not limited to, coronary artery bypass grafts, heart valve repair, heart valve replacement, heart transplants, lung transplants, ablation procedures, septal defect repair, congenital heart defect repair, aneurysm repair, pulmonary endarterectomy, pulmonary thrombectomy, etc.

[0018] In the illustrated example, the HLM system 100 includes components and subsystems such as the HLM 110, an extracorporeal circuit 120, one or more temperature control systems 130, a blood monitoring system 140 (e.g., a CDI® blood parameter monitoring system), a perfusion data management system 150, and a regional oximetry system 160. Some types of procedures using the HLM system 100 may not require all of the components and subsystems shown. Some types of procedures using the HLM system 100 may require additional components and / or subsystems not shown.

[0019] The extracorporeal circuit 120 is connected to the patient 10 and the HLM 110. Other systems, such as a temperature control system 130, a blood monitoring system 140, and a perfusion data management system 150, may also be configured to interface with the extracorporeal circuit 120. The extracorporeal circuit 120 is connected to the patient 10 at the patient's heart 12. Oxygen-depleted blood (venous blood) from the patient 10 is extracted from the patient 10 using a venous catheter 121 at the patient's heart 12. The blood circulates through the extracorporeal circuit 120 to receive oxygen and remove carbon dioxide. The oxygenated blood is then returned through the extracorporeal circuit 120 to the patient's heart 12 via an aortic cannula 129.

[0020] Briefly, the extracorporeal circuit 120 operates by withdrawing oxygen-depleted venous blood from the patient 10 via a venous catheter 121 and depositing the venous blood in a reservoir 123 via a venous tubing 122. Blood from the reservoir 123 is drawn therefrom by a pump 124. The illustrated embodiment includes a single-use centrifugal pump as the pump 124, although in some cases a peristaltic pump such as the HLM 110 is used instead. Pressure generated by the pump 124 advances the blood to an oxygenator 125. The now oxygen-enriched arterial blood exits the oxygenator 125 and passes through an arterial filter 126, which removes emboli, through arterial tubing 127, and via an aortic cannula 129.

[0021] During a surgical procedure using the HLM system 100, various vital signs of the patient 10 are measured and / or monitored. For example, the HLM system 100, as shown, includes a blood monitoring system 140. The blood monitoring system 140 uses one or more blood gas sensors (e.g., venous shunt sensors, arterial shunt sensors, H / S cuvettes, etc.) located at various locations along the extracorporeal circuit 120 (e.g., venous tubing 122, arterial tubing 127, etc.) to monitor the priming solution and the patient's arterial and / or venous extracorporeal blood during the surgical procedure. The blood monitoring system 140 may also use one or more other biological analyte (e.g., blood gas or blood metabolite) sensors, which may include an air detector, an air bubble sensor, an arterial optical fluorescence sensor, a venous optical fluorescence sensor, an arterial optical reflectance sensor, a venous optical reflectance sensor, a hematocrit level sensor, and / or a hemoglobin sensor. Monitored parameters may include, but are not limited to, pH, pCO2, pO2, K + , temperature, SO2, hematocrit, hemoglobin, base excess, bicarbonate, oxygen consumption, and oxygen delivery. Parameters monitored in the blood of patient 10 can also include other blood metabolites, such as glucose and / or lactate, in accordance with the disclosure provided herein. Additional non-limiting blood metabolites monitored in the blood of patient 10 can also include cholesterol, galactose, pyruvate, xanthine, amines (e.g., dopamine, norepinephrine, and / or serotonin), benzylamine, phenethylamine, D-amino acids, L-amino acids, lactose, creatine, insulin, heparin, and / or superoxide radical (O2 - Additionally, in some cases, the devices and systems described herein can be used to monitor substances such as cell culture solutions and organ preservation solutions (e.g., packed red blood cells, human albumin, succinylated gelatin, NaHCO3, NaCl, insulin, heparin Na, antibiotics, calcium gluconate, etc.).

[0022] 2, a blood parameter measurement system 200 (or simply "system 200"), particularly useful for surgical procedures and / or bedside monitoring of a patient, includes a shunt sensor 220 (also referred to herein as a "blood parameter measurement device 220" or simply "device 220") and an optical probe 240. Optical probe 240 is in wireless or wired communication with a control and monitoring device (not shown).

[0023] The blood parameter measuring device 220 and the optical probe 240 are removably coupleable together. In the coupled configuration, the optical probe 240 operates with the blood parameter measuring device 220 to measure various parameters of the bodily fluid within or flowing through the blood parameter measuring device 220.

[0024] In use, the blood parameter measuring device 220 is fluidly coupled to an extracorporeal source of bodily fluid (e.g., blood). The bodily fluid flows through the blood parameter measuring device 220. For example, in some cases, one end of the blood parameter measuring device 220 is connected to a first tube that supplies blood to the blood parameter measuring device 220, and the other end of the blood parameter measuring device 220 is connected to a second tube through which the blood flows away from the blood parameter measuring device 220. Thus, the bodily fluid (e.g., blood) flows through the blood parameter measuring device 220.

[0025] The blood parameter measuring device 220 includes a tubular housing 222 having mounted thereon one or more sensors 224. The tubular housing 222 defines an interior space configured to receive blood.

[0026] The one or more sensors 224 are specifically configured to respond to one or more particular parameters of a bodily fluid (e.g., blood, etc.). The one or more sensors 224 each comprise a multi-layer assembly that can be adhesively attached to the tubular housing 222 of the blood parameter measuring device 220. In some cases, the adhesive used to adhesively attach the one or more sensors 224 to the tubular housing 222 can be pressure-sensitive. The innermost layer of the multi-layer assembly can be in direct contact with the bodily fluid within or flowing through the interior space of the blood parameter measuring device 220. In the illustrated exemplary embodiment, a series of four sensors 224 are included as part of the blood parameter measuring device 220. For example, the sensors 224 can include an ion (potassium) sensor, a pH sensor, a carbon dioxide sensor, and an oxygen sensor. Additionally or optionally, the sensors 224 can also include one or more sensors for measuring additional biological analytes, such as metabolites, e.g., glucose and / or lactate, in accordance with the disclosure provided herein.

[0027] In some embodiments, each of the one or more sensors 224 can optionally include a fluorescent ionophore compound ("ionophore") containing a complexing moiety for binding ions and a fluorescent moiety. The compound has a wavelength of maximum absorbance of at least about 350 nm. Suitable fluorescent moieties preferably contain closely spaced nπ* and ππ* excited states. Suitable fluorescent moieties are preferably capable of ion-dependent out-of-plane puckering when coupled to an appropriate complexing moiety. The ππ* state of suitable fluorescent moieties is also preferably sufficiently high in energy that ion-dependent mixing dominates over nonradiative coupling to the ground state. Particularly preferred fluorescent moieties include coumarin moieties, although other aromatic carbonyl or nitroaromatic or N-heterocyclic moieties may also be used. Suitable ion-complexing moieties include cyclic "cage" moieties capable of binding ions. The cage may be capable of selective binding of ions. In some cases, preferred ion-complexing moieties include crypto and crown ether moieties.

[0028] In some embodiments, the ionophore is covalently attached to a suitable substrate that can be attached to a backing membrane. The substrate can be a polymeric material that is water-swellable and permeable to the ionic species of interest, preferably insoluble in the medium being monitored. Particularly useful substrate polymers include, but are not limited to, ion-permeable cellulosic materials, high molecular weight or cross-linked polyvinyl alcohol (PVA), dextran, cross-linked dextran, polyurethane, quaternized polystyrenes, sulfonated polystyrene, polyacrylamide, polyhydroxyalkyl acrylate, polyvinylpyrrolidone, hydrophilic polyamide, polyester, and mixtures thereof. In some embodiments, the substrate is cellulosic, particularly ion-permeable cross-linked cellulose. In certain embodiments, the substrate comprises a regenerated cellulose membrane (Futamura P5-1 Membrane, Futamura Chemical-Manufacturer) that has been cross-linked with an epoxide such as butanediol diglycidyl ether and further reacted with a diamine to provide pendant amine functional groups from the cellulosic polymer.

[0029] The optical probe 240 includes at least one light source that directs light to the one or more sensors 224. Each of the one or more sensors 224 has a corresponding individual light source. The optical probe 240 also includes at least one photodetector for detecting light emitted from the one or more sensors 224. Each of the one or more sensors 224 has a corresponding individual photodetector. The system 200 includes a signal converter connected to the at least one photodetector. The signal converter provides a digital output signal that varies in response to the amount of light detected by each of the photodetectors.

[0030] 3 , in some embodiments, the sensor 300 can include an enzyme layer 302 and a probe layer 304. The enzyme layer 302 is closer to the interior space than the probe layer 304. Thus, when the blood parameter measuring device 220 is filled with blood or another bodily fluid, the enzyme layer 302 is closer to the bodily fluid (e.g., blood). In some cases, the enzyme layer 302 is directly adjacent to the probe layer 304, such that the enzyme layer 302 is closer to the interior space than the probe layer 304.

[0031] The enzyme layer 302 can be a hydrogel in which an enzyme is entrapped or immobilized. The hydrogel can include an ether-based hydrophilic urethane such as Hydromed™ D4, or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and an ethanol-water mixture. In some cases, the hydrogel can have a pH of about pH 7 to about pH 8 (e.g., about pH 7.4).

[0032] The biological analyte can diffuse from the bodily fluid to at least the enzyme layer 302. The enzyme can generate hydrogen peroxide upon reaction with at least one biological analyte in the bodily fluid (e.g., blood). The enzyme can be any enzyme that generates hydrogen peroxide. Such enzymes can include glucose oxidase (GOx), lactate oxidase (LOx), cholesterol oxidase (ChOx), galactose oxidase (GAOx), pyruvate oxidase (POx), xanthine oxidase (XAOx), monoamine oxidase A (MAO-Ax), monoamine oxidase B (MAO-Bx), D-amino acid oxidase (D-AAOx), L-amino acid oxidase (L-AAOx), lactose oxidase (LOx), and superoxide dismutase (SOD). In some cases, the enzyme includes glucose oxidase (GOx). In some cases, the enzyme includes lactate oxidase (LOx).

[0033] Non-limiting examples of biological analytes that can be measured by blood parameter measuring device 220 are described above. In some cases, the biological analyte is glucose. In some cases, the biological analyte is lactate.

[0034] The hydrogen peroxide generated by the enzyme can diffuse at least as far as the probe layer 304. The probe layer 304 includes a substance that chemically reacts with hydrogen peroxide, such as hydrogen peroxide generated by the enzyme in the enzyme layer 302. One example of a substance includes a europium(III)-tetracycline complex (also known as an "EuTu" complex). The complex europium(III)-tetracycline complex can coordinate with hydrogen peroxide at physiologically relevant pH. This coordination produces EuTu complexes that are detectable at 616 nm. 3+ Aeon 5 D0→ 7 This can result in an increase in the emission intensity of the F2 transition. The light produced by the substance that chemically reacts with hydrogen peroxide can be detected by, for example, optical probe 240.

[0035] Additionally, in some embodiments, the sensor can include one or more additional layers. One such layer is substrate 306. Any suitable substrate can be used. Suitable substrate 306 allows transmission of light between 350 nm and 800 nm. Non-limiting examples of substrate 306 include polyethylene terephthalate (PET) films, such as biaxially oriented PET films / foils (e.g., Mylar®), and polycarbonates. Substrate 306 can be coated with an adhesive (e.g., a pressure-sensitive adhesive). In some embodiments, substrate 306 can have a thickness of about 100 μm to about 150 μm (e.g., 110 μm to about 140 μm, or about 120 μm to about 130 μm). In some embodiments, adhesive-coated substrate 306 can have a thickness of about 150 μm to about 200 μm (e.g., about 175 μm to about 180 μm). Alternatively, a cycloolefin copolymer (COC) based substrate (e.g., cycloolefin copolymer TOPAS type 8007S-04®) can be used. Additionally, the substrate 306 can have a similar polarity to the polymer or polymer cocktails that can coat the substrate 306.

[0036] 4, in some embodiments, exemplary sensor 400 can include enzyme layer 302, probe layer 304, and substrate 306, as described above. Additionally, in some embodiments, sensor 400 can also include protective layer 410. Protective layer 410 can 1) prevent or limit, e.g., sieve, direct contact with bodily fluids (e.g., blood, etc.) as a barrier to limit interference with enzyme or probe function, 2) provide anti-fouling properties, and / or 3) be a non-toxic interface between any of the sensors described herein (sensor 300, sensor 400, sensor 500, sensor 600, sensor 700) and bodily fluids.

[0037] In some embodiments, the sensor 400 can also include at least one intermediate layer 420. The at least one separation layer 420 can be used to separate the enzyme layer 302 and the probe layer 304. This can improve bonding between the enzyme layer 302 and the probe layer 304 and / or limit interference between various solvents used in the different layers. In some embodiments, the intermediate layer 420 can be a polyvinyl acetate (PVAc) layer, a cellulose acetate (CA) layer, or a combination thereof. In some cases, the PVAc, CA, or a combination thereof can be dissolved in dimethylformamide (DMF) / HO.

[0038] Referring to FIG. 5, in some embodiments, any of the sensors described herein may use a probe construction 500 comprising an intermediate layer 420, a probe layer 304, and a substrate 306.

[0039] 6 and 12 , in some embodiments, an exemplary sensor 600 can include an enzyme layer 302 and a substrate 306 as an enzyme construct 610, and a probe construct 500 including an intermediate layer 420, a probe layer 304, and a substrate 306. The enzyme construct 610 can be an annular layer substantially centered on the probe construct 500. Such a structure results in a well-shaped sensor defining an open interior space adjacent to the intermediate layer 420. The open interior space is surrounded by the substrate 306 and the enzyme layer 302. The enzyme construct 610 can be closer to the interior region than the probe construct 500. The enzyme layer 302 can be closer to the interior region than the substrate 306 of the enzyme construct 610. The probe layer 304 can be closer to the interior region than the substrate 306 of the probe construct 500.

[0040] 7, in some embodiments, another exemplary sensor 700 can include the enzyme layer 302 and substrate 306 as enzyme construct 610, as described above, and the probe construct 500 including the intermediate layer 420, the probe layer 304, and the substrate 306. Additionally, in some embodiments, the sensor 700 can include a reference layer 710. The reference layer 710 can be positioned farther from the open interior space than the probe layer 304.

[0041] In some embodiments, the reference layer 710 can include a reference dye. The reference layer 710 can be ratiometric. The reference dye can have an excitation maximum at 400±10 nm and an emission maximum at 450±10 nm. The reference dye can be selected from the group consisting of 9,10 di(phenylethynyl)anthracene (DPEA), CD405M, 1-anilinonaphthalene-8-sulfonic acid, 8-benzyloxy-5,7-diphenylquinoline, 4-methylumbelliferyl acetate, octadecyl 7-hydroxycoumarin-3-carboxylate, perylene, tetracene, H9-40, pyranine, ethyl eosin, coumarin 30, coumarin 153, and CF™ 405M. The inclusion of a reference dye in any of the sensors described herein can prevent the need to calibrate the sensor with each use to produce accurate measurements. Any of the sensors described herein, including but not limited to sensor 400, can include a reference layer 710.

[0042] 8, in some embodiments, another exemplary sensor 800 can include an enzyme construct 610 as described above and an alternative probe construct 810 having an intermediate layer 420 and a combined reference and substrate layer 820. The combined reference and substrate layer 820 can be ratiometric. In some cases, the combined reference and substrate layer 820 includes 9,10 di(phenylethynyl)anthracene (DPEA) and cycloolefin copolymer (COC).

[0043] 9 , in some embodiments, sensor 224 and / or any of the ratiometric sensors described herein can be fabricated using an exemplary method 900. Method 900 can include step 910, which comprises coating a substrate with a ratiometric layer, step 920, which comprises coating the substrate with a probe layer, and step 930, which comprises coating the substrate with an enzyme layer. The substrates can be the same substrate or different substrates. Optionally, if the layers are on different substrates, they can be bonded or physically held together with an intermediate layer (e.g., intermediate layer 420).

[0044] 10 , in some embodiments, sensor 224 and / or any of the sensors described herein having multiple substrates (e.g., sensor 600, sensor 700, or sensor 800) can be manufactured using exemplary method 1000. Method 1000 can include step 1010, comprising coating substrate 306 with enzyme layer 302 to create enzyme construct 610; step 1020, comprising forming enzyme layer 302 into a disk shape defining a hole, such that the enzyme layer 302 becomes an annular layer defining an open interior space; step 1030, comprising coating a second substrate with probe layer 304 to create probe construct 500; and step 1040, comprising positioning annular enzyme construct 610 on probe construct 500.

[0045] Any of the coatings of the substrates described herein can be applied using knife coating and / or other suitable coating techniques. When appropriate, multiple layers of coating can be applied.

[0046] Any of the sensors described herein can also include an intermediate layer (e.g., intermediate layer 420) between any of the enzyme layers described herein (e.g., enzyme layer 302) and any of the probe layers described herein (e.g., probe layer 304).

[0047] Any of the sensors described herein may be included in the blood parameter measuring device 220 and / or used in the HLM 100.

[0048] [Example] [Example 1: Preparation of sensor 800 and measurement with a flow cell] Polymer cocktail: 5-20 wt% D4 in EtOH / HO - Hydromed D4 is dissolved in a mixture of ethanol and water (80-95% ethanol) at a temperature between approximately 20°C and approximately 50°C.

[0049] Polymer cocktail: 5-20 wt% PVAc / CA in cyanic acid (CHON) / HO - CHON is mixed with water (95-99.9% CHON). CA and PVAc (approximately 95-99.9 wt% CA) are dissolved in CHON / HO. The temperature should be between 20°C and 50°C.

[0050] Polymer cocktail: 5-20 wt% PVAc / CA in DMF / HO - Mix DMF with water (80-95% DMF). Dissolve CA and PVAc (approximately 95-99.9 wt% CA) in DMF / HO. Temperature should be between 20°C and 50°C.

[0051] EU 3+ Stock solution - EuCl3·6H2O dissolved in water, can be stored at 4°C under light protection for at least 6 months.

[0052] DPEA stock solution - Dissolve DPEA in toluene. Store the stock in a glass vial with a tightly sealed lid at 4°C and protect from light.

[0053] Tetracycline stock solution - Dissolve tetracycline HCl in water. The solution must be used within 30 minutes and stored light-protected.

[0054] GOx and LOx stock solution in 0.1 M HEPES pH 7.4 - Dissolve GOx or LOx in HEPES buffer with gentle shaking for 10-20 min at 37 °C. The solution must be used immediately and cannot be stored.

[0055] Eu-tetracycline (EuTc) stock solution for sensor cocktail - tetracycline HCl to Eu 3+ Dissolve in stock solution. The solution must be used immediately and cannot be stored.

[0056] Sensor cocktail - D4 in ethanol / HO is mixed with EuTc stock solution at ambient conditions. It is recommended to use the cocktail as soon as possible.

[0057] Mix PVAc / CA in DMF / HO with EuTc stock solution at ambient conditions. It is recommended to use the cocktail as soon as possible.

[0058] Enzyme cocktail - Mix D4 and enzyme stock solutions in ethanol / HO at 37 °C until a homogenous suspension is obtained. It is recommended to use the cocktail immediately within 1-2 h.

[0059] Reference phase cocktail - Topas® 8007S-04 polymer and 0.1-200 µL of DPEA stock in toluene are dissolved in toluene at room temperature under light protection.

[0060] [Preparing the sensor foil] Sensor layer: D4-EuTc / / PVAc / CA A homogeneous layer of the sensor cocktail with a wet thickness of 10-100 μm is obtained on a Mylar® substrate by knife coating. After 30 seconds to 2 minutes, the sensor foil is transferred from the coating device to an oven. The foil is dried at approximately 40°C for 3.0 ± 0.5 hours. A second layer is applied immediately after the drying period.

[0061] A homogeneous protective layer of PVAc / CA cocktail with a wet thickness of 10-100 μm is obtained on the previously coated D4-EuTc sensor layer by knife coating. After 30 s to 2 min, the sensor foil is transferred from the coating device to an oven. The foil is dried at approximately 40 °C for 3.0 ± 0.5 h.

[0062] The foil is washed in excess 0.1 M HEPES pH 7.4 for 15±5 minutes to remove unbound complexes and to rehydrate the dried hydrogel.

[0063] The sheets can then be carefully stacked and stored light-protected at 4 °C in a closed zipper bag until sensor disc preparation, or they can be used immediately for the next step. Sensor foils can be stored for at least 4 weeks under the conditions described above.

[0064] Sensor layer: PVAc / CA-EuTc A homogeneous layer of the sensor cocktail with a wet thickness of 10-100 μm is obtained on the Mylar® substrate by knife coating. After 30 seconds to 2 minutes, the sensor foil is transferred from the coating device to an oven. The foil is dried at approximately 40°C for 3.0 ± 0.5 hours.

[0065] The foil is washed in excess 0.1 M HEPES pH 7.4 for 15±5 minutes to remove unbound complexes and to rehydrate the dried hydrogel.

[0066] The sheets can then be carefully stacked and stored light-protected at 4 °C in a closed zipper bag until sensor disc preparation, or they can be used immediately for the next step. Sensor foils can be stored for at least 4 weeks under the conditions described above.

[0067] Enzyme layer: D4-GOx / LOx A homogeneous layer of the enzyme cocktail with a wet thickness of 10-100 μm is obtained on the Mylar® substrate by knife coating. After 30 seconds to 2 minutes, the sensor foil is transferred from the coating device to an oven. The foil is dried at approximately 40°C for 3.0 ± 0.5 hours.

[0068] The foil is washed in excess 0.1 M HEPES pH 7.4 for 15±5 minutes to remove unbound enzyme and to rehydrate the dried hydrogel.

[0069] The sheets can then be carefully stacked and stored light-protected at 4 °C in a closed zipper bag until sensor disk preparation, or they can be used immediately for the next step. Enzyme foils can be stored for at least 6 months under the conditions described above.

[0070] The same procedure was used for all GOx concentrations and LOx sensor foils.

[0071] Reference layer: DPEA-COC (optional) A homogeneous layer of the reference cocktail with a wet thickness of 10-100 μm is obtained by knife coating on a Mylar® substrate. After 30-60 seconds, the sensor foil is transferred from the coating device to an oven. The foil is dried at approximately 60°C for 4.0 ± 0.5 hours. Subsequent layers can be applied later. The foil is stored at room temperature under light protection.

[0072] [Preparing the sensor disk] MTP assay To fix the disc to the MTP, double-sided adhesive tape is applied to the uncoated side of the Mylar® support. Disks with a diameter of 6 mm are punched out of the foil using a toggle press. A distance of at least 2 mm to the edge is recommended. After removing the protective cover of the adhesive tape, the sensor disc is fixed to the bottom of the MTP well. Typically, n = 4 or n = 8 discs are used for the assay. The diameter of the disc is selected depending on the geometry of the measurement device and can be adjusted accordingly.

[0073] Flow cell assay Punch out 24 mm diameter discs from the foil using a toggle press. A distance of at least 2 mm to the edge is recommended. The prepared discs can be stored at 4°C, protected from light, for 4 weeks or used immediately. The diameter of the disc can be selected depending on the geometry of the measuring device and adjusted accordingly.

[0074] Measurement in MTP format Between all measurements, the samples are orbitally shaken by the plate reader. The samples are excited at 405 / 10 nm, and the emission intensity is measured at 615 / 10 nm at regular intervals at 25.0 ± 0.5 °C. Before measurements, the sensor disk is washed and rehydrated with HEPES buffer for 5 to 15 minutes. Each glucose / HP sample (e.g., 0.5, 1, 2.5, 5, 10, 25, and 50 mM glucose / HP solutions in buffer) is added to the sensor disk in the well of the MTP. The kinetic measurement is initiated. The solution is removed before testing the next concentration.

[0075] Experimental setup for the detection of luminescent sensor disks in a flow cell. Figure 11 shows a schematic diagram with two disks in one disk holder. For sample injection, the pump is stopped, the inlet tube is placed in the sample, and the pump is started again for a certain period of time ("stop-and-go" method). Figure 12 shows a schematic diagram of the combination of a sensor disk (inner part: EuTc-D4 / / PVAc / CA) and an enzyme disk (outer part: D4-GOx / LOx) for measurement.

[0076] Place the inlet tubing in the buffer reservoir and the outlet tubing in the waste, and start the pump at a constant speed. Set the PMT voltage to a suitable level. Set the excitation wavelength to 405 / 8 nm. Detect the emission intensity at 615 / 8 nm. Start a time trace measurement over a period of time with a suitable data acquisition interval. Figure 11 shows the probe construct (D4-EuTc / / PVAc / CA) fixed in the center of the flow cell, surrounded by / overlapping the circular enzyme construct (D4-GOx). The constructs are held together by pressure applied by the screws holding the flow cell together. The constructs can also be glued together by applying a solvent or adhesive to the overlapping areas. After equilibration with HEPES buffer, the sample is injected.

[0077] While this specification contains details of many specific implementations, these should not be construed as limitations on the scope of the invention or what may be claimed, but rather as descriptions of features specific to particular embodiments of a particular invention. Certain features that are described in this specification in the context of separate embodiments may also be implemented in combination in a single embodiment. Conversely, various features that are described in the context of a single embodiment may also be implemented in multiple embodiments separately or in any suitable subcombination. Furthermore, while features may be described above as working in particular combinations, and even initially claimed as such, one or more features from a claimed combination may in some cases be deleted from that combination, and the claimed combinations may be directed to subcombinations or variations of the subcombinations.

[0078] Specific embodiments of the subject matter have been described. Other embodiments are within the scope of the following claims. For example, the actions recited in the claims may be performed in a different order and still achieve desirable results. As one example, the processes illustrated in the accompanying figures do not necessarily require the particular order shown or sequential order to achieve desirable results. In certain implementations, multitasking and parallel processing may be advantageous.

Claims

1. A blood parameter measuring device, comprising: a tubular housing defining an interior space configured to receive blood; a sensor connected to the tubular housing; The sensor comprises:

1. A blood parameter measuring device comprising: (i) a first layer including an enzyme that produces hydrogen peroxide when reacting with at least one biological analyte in the blood; and (ii) a second layer including a substance that chemically reacts with hydrogen peroxide, the first layer being closer to the interior space than the second layer.

2. 2. The device of claim 1, wherein the enzyme is selected from the group consisting of glucose oxidase (GOx), lactate oxidase (LOx), cholesterol oxidase (ChOx), galactose oxidase (GAOx), pyruvate oxidase (POx), xanthine oxidase (XAOx), monoamine oxidase A (MAO-Ax), monoamine oxidase B (MAO-Bx), D-amino acid oxidase (D-AAOx), L-amino acid oxidase (L-AAOx), lactose oxidase (LOx), and superoxide dismutase (SOD).

3. The device of claim 1 or 2, wherein the enzyme comprises glucose oxidase (GOx).

4. The device of any one of claims 1 to 3, wherein the enzyme comprises lactate oxidase (LOx).

5. The device of any one of claims 1 to 4, wherein the substance comprises a europium(III)-tetracycline (EuTu) complex.

6. The device of any one of claims 1 to 5, wherein the first layer and the second layer are immediately adjacent to each other.

7. The device of any one of claims 1 to 6, wherein the sensor further comprises an intermediate layer between the first layer and the second layer.

8. The device of any one of claims 1 to 6, wherein the first layer is an annular layer and is substantially centered on the second layer.

9. The device of any one of claims 1 to 8, wherein the sensor further comprises a protective layer positioned between the first layer and the interior space.

10. The device of any one of claims 1 to 9, wherein the sensor further comprises a reference dye.

11. 11. The device of claim 10, wherein the reference dye is selected from the group consisting of 9,10 di(phenylethynyl)anthracene (DPEA), CD405M, 1-anilinonaphthalene-8-sulfonic acid, 8-benzyloxy-5,7-diphenylquinoline, 4-methylumbelliferyl acetate, octadecyl 7-hydroxycoumarin-3-carboxylate, perylene, tetracene, H9-40, pyranine, ethyl eosin, coumarin 30, coumarin 153, and CF™ 405M.

12. 11. The device of claim 10, wherein the reference dye has an excitation maximum at 400±10 nm and an emission maximum at 450±10 nm.

13. 13. The device of claim 10, wherein the sensor further comprises a reference layer containing the reference dye, the reference layer being positioned further from the interior space than the second layer.

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