Patent Description:
The ability to accurately measure creatinine and creatine levels in a patient's blood is an important indicator of renal health. In particular, serum creatinine is an important indicator of renal health because it is excreted unaltered by the kidneys, and can readily be measured. For example, elevated levels of blood serum creatinine is a late marker of chronic kidney disease, and is generally only observed when significant kidney damage has already occurred.

Creatinine and creatine concentrations in a sample (e.g., a patient's blood) may be measured via electrochemical sensors. For example, current creatinine sensors may include an enzymatic biosensor containing three enzymes-creatininase, creatinase, and sarcosine oxidase-that catalyze the production of glycine, formaldehyde, and hydrogen peroxide from creatinine and water. These three enzymes may be immobilized on the surface of a platinum electrode, and the final reaction product of hydrogen peroxide (H<NUM>O<NUM>) may then be electrochemically oxidized on the platinum electrode under a constant polarization potential and used to measure creatinine/creatine in a patient's blood. Due to the presence of creatine in clinical samples, an additional sensor for creatine measurement is required for correcting the creatine response of the creatinine sensor. Such creatine sensors include an enzymatic biosensor containing two enzymes-creatinase and sarcosine oxidase.

On both creatinine and creatine sensors, there is a diffusion control membrane (also referred to as an outer membrane) on top of the enzyme layer. The diffusion control membrane limits the flux of creatinine and creatine substrates entering the enzyme layer to ensure that the signals generated by the hydrogen peroxide are proportional to the substrate concentrations of the sample.

Theoretically, the creatinine measuring system described above can quantitatively measure creatinine in biological samples, however several factors can create challenges to obtaining accurate creatinine measurements. One factor, creatine interference, is typically dealt with by using a separate creatine sensor and subtracting the creatine concentration as a correction measure. However, large errors can be introduced when creatine concentrations are high (e.g., [creatine]>>[creatinine]). Another factor, slow baseline recovery, can create errors if unreacted substrates are not removed immediately after sample measurement because unremoved substrates will continue to generate current signal. A third factor relates to biological sample matrix effect and biocompatibility. For example, when exposed to a biological sample matrix such as whole blood, some outer diffusion membranes can exhibit severe sensitivity and baseline change due to protein fouling or surface hydrophobicity changes and micro clot formation.

Currently available outer diffusion control membranes for creatinine sensors include polymeric materials such as polycarbonate, poly <NUM>-hydroxyethyl methacrylate (polyHEMA) and polyurethane. However, such diffusion membranes do not address all three of the limitations mentioned above. Accordingly, there is an urgent unmet need to identify and develop new compositions to improve outer membrane properties to address problems due to creatine interference, slow baseline recovery and biological sample matrix effects and biocompatibility.

In this context, <CIT> describes a cross-linked enzyme matrix and uses thereof.

In one aspect, the present invention provides a biosensor comprising an electrode, a plurality of enzymes immobilized on the electrode and a diffusion membrane, wherein the plurality of enzymes is selected from the group consisting of creatininase, creatinase and sarcosine oxidase, wherein the diffusion membrane comprises <NUM>% w/w of <NUM>% water uptake polyurethane and <NUM>% w/w of <NUM>% water uptake polyurethane, and wherein the diffusion membrane has a creatinine to creatine diffusion ratio of at least <NUM>. The biosensor containing the diffusion membrane is a creatinine/creatine biosensor. The terms "diffusion membrane" and "outer membrane" are used interchangeably throughout the present disclosure. Also, the terms "biosensor" and "sensor" are used interchangeably throughout the present disclosure.

The diffusion membrane of the biosensor includes two different polyurethanes. The two different polyurethanes have different water uptake percentages. The w/w amounts of the two different polyurethanes are adjusted to result in a diffusion membrane having a creatinine to creatine diffusion ratio of at least <NUM>, preferably <NUM>-<NUM>, more preferably <NUM>-<NUM> and most preferably at least <NUM>. The w/w amounts of the two different polyurethanes are also adjusted to result in a diffusion membrane having a creatinine slope of at least <NUM>, preferably at least <NUM> and more preferably at least <NUM>.

Herein, the diffusion membrane of the biosensor comprises <NUM>% w/w of <NUM>% water uptake polyurethane and <NUM>% w/w of <NUM>% water uptake polyurethane.

The diffusion membrane of the biosensor may be obtained by a method including the steps of: a) dissolving the two different polyurethanes in an organic solvent or mixture of solvents to create a polyurethane mixture; b) casting a layer of the polyurethane mixture onto a support material; c) allowing the solvent or mixture of solvents to evaporate; and d) repeating steps b) and c) <NUM>-<NUM> times.

In the above method, the two different polyurethanes may be dissolved in organic solvent(s) including methylene chloride, dimethylformamide, dimethylacetamide, tetrahydrofuran, cyclohexanone, isopropanol or mixtures thereof.

In the above method, the support material may be the electrode having the plurality of enzymes immobilized thereon.

In the above method, steps b) and c) may be repeated two times.

The biosensor provided herein includes an electrode, a plurality of enzymes immobilized on the electrode and a diffusion membrane, with the plurality of enzymes immobilized on the electrode forming an enzyme layer and the diffusion membrane disposed onto the surface of the enzyme layer.

In certain embodiments of the biosensor, the electrode is made of carbon, graphite or carbon nanotubes. In other embodiments, the electrode is made of metal. Representative examples of metals of the electrodes include platinum, gold, palladium, or alloys of platinum, gold and palladium. In some embodiments of the biosensor, the plurality of enzymes is creatininase, creatinase and sarcosine oxidase. The enzyme layer is positioned between the outer diffusion membrane and the electrode. The biosensor is configured to measure creatine and/or creatinine in a body fluid sample such as blood, plasma or serum.

In a further aspect, the present invention provides a disposable cartridge housing the biosensor described herein. In some embodiments of the disposable cartridge, the biosensor described herein is one sensor in a sensor array. In some embodiments of the disposable cartridge, with the biosensor thereof including an electrode, a plurality of enzymes immobilized on the electrode forming an enzyme layer and a diffusion membrane disposed onto the surface of the enzyme layer, the diffusion membrane is adjacent to a body fluid sample flow chamber.

Other features and advantages of the present disclosure will be apparent from the following detailed description of the preferred embodiments thereof. 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 the present disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. The scope of the present invention is only limited by the appended claims.

Where applicable or not specifically disclaimed, any one of the embodiments described herein are contemplated to be able to combine with any other one or more embodiments, even though the embodiments are described under different aspects of the present disclosure.

These and other embodiments are disclosed and/or encompassed by the following detailed description.

The present disclosure is based, at least in part, on the discovery that diffusion control membranes (i.e., outer membranes) made from blends of two different polyurethanes can limit creatine interference, allow for quick baseline recovery and limit biological sample matrix effects. The diffusion membrane of the biosensor described herein includes two different polyurethanes and has a creatinine to creatine diffusion ratio of at least <NUM>.

Current creatinine sensors in a creatine/creatinine system (e.g., GEM PAK cartridge) include an enzymatic biosensor containing three enzymes. These enzymes are immobilized on the surface of a platinum electrode. The creatinine detection is based on three enzyme cascade reaction:
<CHM>
<CHM>
<CHM>.

The product hydrogen peroxide (H<NUM>O<NUM>) is then electrochemically oxidized on the platinum electrode under the constant polarization potential.

The presence of creatine in clinical samples necessitates an additional sensor for creatine measurement to correct the creatine response of the creatinine sensor. The creatine sensor includes steps (<NUM>) and (<NUM>) of the above enzyme cascade reactions.

In order to determine the respective concentrations of creatinine and creatine in a biological sample(s), the creatinine and creatine sensors need to be calibrated in order to determine their respective sensitivities. This may be achieved by comparing the readings of the creatinine and creatine sensors in calibration solutions containing pre-determined concentrations of creatinine and creatine. Once the sensitivities of the creatinine and creatine sensors are determined, the concentrations of creatine and creatinine in any biological sample can be estimated by adjusting the measured readings with the results determined from the calibration process.

Both the creatine and creatinine sensors also have a diffusion control membrane (also referred to as an outer membrane) on top of the enzyme layers. The diffusion control membrane limits the flux of creatinine and creatine substrates entering the enzyme layer to ensure that the signals generated by the hydrogen peroxide are proportional to the substrate concentrations of the sample.

Theoretically, a creatinine measurement system as described above may quantitatively measure the concentration of creatinine in biological samples. However, there are several practical issues associated with sensor variations that result from typical manufacturing processes, calibration and other analyzer operation specific variations, and biological sample matrix variations that present significant challenges with respect to measuring creatinine accurately including, for example, the following:.

The biosensor described herein comprises a polyurethane-based outer membrane. The membrane has a permeability for creatinine that is <NUM> to <NUM> times higher than the permeability for creatine. This differential in permeability provides an advantageous Crea/Cr signal ratio for the biosensor. The outer membrane of the biosensor described herein also facilitates fast removal of the substrate(s) from the enzyme layer and has a quick baseline recovery after sample exposure and wash. In addition, the outer membrane of the biosensor described herein exhibits superior biocompatibility having little or no micro clot formation, and minimizing protein fouling and permeability changes when in contact with whole blood samples.

Polyurethane (PU) is a polymer having superior biocompatibility in many successful in vivo and in vitro applications in medical devices. Hydrophilic medical grade polyurethane families were used in the compositions of the outer membrane of the biosensor described herein, including Tecophlic™ and Tecoflex™ from Lubrizol (Ohio, USA) and HydroMed™ D series from AdvanSource Biomaterials (Massachusetts, USA). These commercially available polymeric resins or solutions are aliphatic, polyether-based polyurethane, which can be dissolved in organic solvents or mixtures of solvents such as methylene chloride, dimethylformamide (DMF), dimethylacetamide (DMA), tetrahydrofuran (THF), cyclohexanone, isopropanol, etc. Within these polyurethane families, there are different grades of materials available with various combinations of hardness and water uptake levels.

The permeability of a substance going through a mass transfer polymer membrane mainly depends on its molecular structure, hydrophobicity and charge. The polymer membrane of the biosensor described herein is a mixture of more than one polymer, and the permeability of a substance through such membrane also depends on the molecular structure, hydrophobicity and charge of the polymer membrane composition.

As used herein, "creatine (a. , <NUM>-[carbamimidoyl(methyl)amino]acetic acid, N-carbamimidoyl-N-methylglycine, or methylguanidoacetic acid)" refers to an organic compound that produces energy for the cells through the recycling of adenosine triphosphate (ATP) by converting adenosine diphosphate (ADP) back to ATP by donating phosphate groups. Creatine has the following chemical structure:
<CHM>.

As used herein, "creatinine" refers to the enzymatic breakdown by-product of creatine, and is generally found in two major tautomeric forms, which are shown below. <CHM>
<CHM>.

As shown in the diagram above, the significant difference in molecular structure of creatinine (cyclic compound) versus creatine (linear compound) makes it possible to modify or regulate the diffusion rate of creatinine and creatine through a polymer membrane composition based on the polymer material properties (structure, hydrophobicity and charge), the mix ratio of multiple polymers and the casting process (solid concentrations, number of layers and solvent selection).

Outer membrane compositions that not only have the best diffusion rate for creatinine but also have a maximum Crea/Cr ratio to minimize creatine interference were developed by varying critical factors of the membrane composition: polyurethane type (hardness <NUM>-<NUM>, water uptake: <NUM>% to <NUM>%), % w/v solid of casting solution (<NUM>-<NUM>%), solvent type and solvent ratio (for example, THF, cyclohexanone, and the mixture of the two with a ratio between <NUM>-<NUM>%). It should be noted that the solvents play an important role when the membrane is formed through a solvent casting process. That is, since different solvents evaporate at different rates, the evaporation rate will impact the morphology and orientation of internal polymer chains as well as the adhesion of the membrane to a support material.

A group of formulations were obtained through experiments that meet the requirements of having high sensitivity for creatinine and a Crea/Cr ratio ≥ <NUM>. Subsequent screening tests were conducted to evaluate the sensor performance stability upon exposure to whole blood samples. The accuracy of reported creatinine concentration compared to a reference method and the stability of sensor sensitivity over the use life were evaluated. Some of the outer membrane formulations had initial high sensitivity but showed significant changes after exposure to whole blood samples. Without wishing to be bound by theory, some explanations for this phenomenon are that the total weight % solid material was too low so that pin holes may have formed in the membrane during drying or that the composition of the mixed polyurethanes was too hydrophilic and the diffusion rate changed significantly when protein and lipid in the blood coated on the surface which led to a drop in sensor sensitivity. The outer membrane composition formulations are discussed in more detail in the following examples.

Similarly, when values are expressed as approximations, by use of the antecedent "about," it is understood that the particular value forms another aspect. It is further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as "about" that particular value in addition to the value itself. It is also understood that throughout the present specification, data are provided in a number of different formats and that these data represent endpoints and starting points and ranges for any combination of the data points. Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of <NUM> to <NUM> is understood to include any number, combination of numbers, or sub-range from the group consisting <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM> as well as all intervening decimal values between the aforementioned integers such as, for example, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and <NUM>. With respect to sub-ranges, "nested sub-ranges" that extend from either end point of the range are specifically contemplated. For example, a nested sub-range of an exemplary range of <NUM> to <NUM> may comprise <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, and <NUM> to <NUM> in one direction, or <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, and <NUM> to <NUM> in the other direction.

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 the present disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Reference will now be made in detail to exemplary embodiments of the present disclosure. While the present disclosure will be described in conjunction with the exemplary embodiments, it will be understood that it is not intended to limit the scope of the present invention to those embodiments. Standard techniques well known in the art or the techniques specifically described below were utilized.

Four polyurethane materials (PU1-PU4) were selected from Tecophlic™, Tecoflex™ and HydroMed™ D families covering Shore Hardness from <NUM>-<NUM>, and water uptake levels from <NUM>-<NUM>%. Experiments were designed to investigate the relationship of the permeability of each component and various <NUM>-, <NUM>- and <NUM>-component mixtures with creatinine and creatine slopes. Since there are other factors that may play roles in sensor slope, all of the test PU solutions were kept at a concentration of <NUM>% w/v and a solvent ratio of <NUM>/<NUM> of THF/cyclohexanone. The casting conditions were also kept constant at two layers for each testing PU solution. The outer membranes were cured at room temperature for <NUM>-<NUM> minutes for each layer for solvent evaporation.

A simplex-centroid mixture design using four types of polyurethane (PU1-PU4) was applied to <NUM> design points (F1-F18) are listed in Table <NUM> related to the PU raw material characteristics (hardness and water uptake). The concentration ranges tested for each PU component were <NUM>-<NUM>%w/v. Twelve sensors were prepared with each of the <NUM> PU formulations and the mean slope values (in units of picoampere per milligram/deciliter, pA/mg/dL) towards creatinine and creatine for each formulation are summarized in Table <NUM>.

The results in Table <NUM> indicate that the creatinine permeability is directly proportional to the water uptake level of the PU material tested. PU2, with extremely low water uptake of <NUM>%, blocks the diffusion of creatinine and creatine and will not generate useful sensor signals if an outer membrane composition contains <NUM>% or higher of PU2.

On the other hand, in addition to water uptake, creatine diffusion is also PU-type dependent. Creatine has highest permeability with the PU4 membrane (mean slope of <NUM> pA/mg/dL) versus the PU3 membrane (mean slope of <NUM> pA/mg/dL); the PU4 membrane having a <NUM>% water uptake and the PU3 membrane having a <NUM>% water uptake. This difference in permeability between creatinine and creatine provides the possibility of maximizing the Crea/Cr signal ratio while still maintaining a high creatinine signal by adjusting the PU material and composition. For example, F5 provides a similarly high creatinine slope to F3 (<NUM> vs. <NUM> pA/mg/dL), but F5 has a Crea/Cr ratio that is two times more than that of F3 (<NUM> vs. <NUM>), thus providing a better sensor in terms of less creatine interference effect.

Based on the test results of the <NUM> PU formulations shown in Table <NUM>, the creatinine slope is a function of the PU mixtures. Thus, a target creatinine sensor slope for creatinine and creatine can be achieved with a mixture of two to four polyurethanes. For example, with a set target of <NUM> pA/mg/dL for creatinine slope, various formulations can be obtained using regression modeling (Table <NUM>).

Formulation F20 applied onto a creatinine sensor demonstrated stable sensitivities toward both creatinine and creatine, and had a Crea/Cr signal ratio of about <NUM> as shown in Table <NUM>. Both sensor sensitivities and the Crea/Cr ratio were stable over a <NUM>-month shelf life at ambient storage.

Sensors (N=<NUM>) with outer membrane formulation, F20, demonstrated a creatinine sensor sensitivity remaining consistent over multiple batches over a <NUM>-week use life after a <NUM>-month room temperature storage. All sensors showed stable creatinine slopes (<FIG>) and stable creatine slopes (<FIG>), and a high Crea/Cr ratio as listed in Table <NUM>. The data in Table <NUM> also demonstrate that the preferred Crea/Cr signal ratios were stable over a <NUM>-month shelf life.

Whole blood creatinine in clinical samples was measured by creatinine sensors with outer membrane formulation, F20, and compared to plasma creatinine measured by chemistry analyzer, Roche Cobas. The sample biases were well within clinical requirements (dashed lines) (<FIG>).

The results demonstrated the biocompatibility of outer membrane formulation, F20, when continuously exposed to clinical whole blood samples and exhibited stable performance compared to plasma results from a clinical laboratory analyzer. F20 also exhibited fast baseline recovery of a creatinine sensor during blood sample measurement. Creatinine accuracy was maintained throughout the measured sample range of <NUM>-<NUM>/dL, indicating that the impact from sample carryover was minimal.

Claim 1:
A biosensor comprising an electrode, a plurality of enzymes immobilized on the electrode and a diffusion membrane,
wherein the plurality of enzymes is selected from the group consisting of creatininase, creatinase and sarcosine oxidase,
wherein the diffusion membrane comprises <NUM>% w/w of <NUM>% water uptake polyurethane and <NUM>% w/w of <NUM>% water uptake polyurethane, and
wherein the diffusion membrane has a creatinine to creatine diffusion ratio of at least <NUM>.