DESC:FIELD OF THE INVENTION
[0001] The present disclosure relates to diagnostic reagent formulation and devices for measuring analytes in biological fluids. In particular, the present disclosure relates to reagent formulation for measuring creatinine and urea concentrations in bodily fluids and diagnostic devices comprising the same.

BACKGROUND OF THE INVENTION
[0002] Background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
[0003] Concentrations of creatinine and urea in blood and/or urine are widely used for characterizing kidney function. Creatinine is a protein produced naturally by muscles with a concentration that is relatively stable in healthy people. Each day, 1-2% of muscle creatine is converted to creatinine. Men tend to have higher levels of creatinine than women because, in general, they have a greater mass of skeletal muscle. Increased dietary intake of creatine or eating a lot of meat can increase daily creatinine excretion. Creatinine is filtered from the bloodstream by kidneys in relatively constant amounts every day. During kidney dysfunction or muscle disorder, creatinine concentration in serum/plasma may raise to levels many times the normal levels. Measurement of creatinine levels in serum and determination of renal clearance are used for laboratory diagnosis of renal and muscular function.
[0004] Urea is the main and final product of protein metabolism. Besides its role as carrier of waste nitrogen, urea also plays a role in the countercurrent exchange system of nephrons, that allows for re-absorption of water and critical ions from the excreted urine. Urea concentration in blood responses the assimilation and dissimilation catabolism of protein and secretion of adrenalin. Hence, the urea concentration in blood or urine is an important health index of human body and is also an important data of the kidney function in clinical diagnosis.
[0005] There have been substantial efforts in the prior art to provide methods and systems for determining creatinine and/or urea concentration in bodily fluids such as, whole blood, plasma, serum, urine or saliva. One method employs an electrochemical biosensor that relates creatinine and/or urea content to a measured electrical current. The magnitude of the electrical signal is correlated with creatinine and/or urea content. Electrochemical test sensors are based on enzyme-catalyzed chemical reactions involving the analyte of interest. The transfer of redox potential and/or electrochemical response from the site of chemical reaction catalyzed by an enzyme to an electrode surface is accomplished using electron transfer mediators.
[0006] The reagent formulations used in biosensors have proven to be highly susceptible to the environmental conditions including temperature and moisture, which result in test sensor reagents of low stability. For example, during storage, reduced mediator may be produced by interactions between oxidized mediator and enzyme system. The larger the amount of mediator or enzyme, the larger the amount of reduced mediator that is produced. The background current will generally increase towards the end of shelf-life of the sensor because of high concentration of the reduced mediator. The increased background current may decrease precision and accuracy of the measurements of test sensor and thus provide a limited shelf-life for the test sensors.
[0007] Further, electrochemical biosensors utilizing known reagent formulations require calibration of the sensor both before and after measurement and require large volume of test sample. Interference of other electrochemically active species present in the test sample may lead to false signals. For example, potential interferents such as, ascorbic acid, uric acid, glucose, potassium (K+) and sodium (Na+) ions present in blood sample may interfere with electrochemical response of urea biosensor and affect the selectivity of the sensor.
[0008] There is thus a need in the art for a new and improved reagent formulation for use in electrochemical kidney profile biosensors that is very precise, selective, accurate and highly stable during shelf-life.
[0009] The present invention satisfies the existing needs, as well as others, and generally overcomes the deficiencies found in the prior art.

OBJECTS OF THE INVENTION
[0010] It is an object of the present disclosure to provide a reagent formulation for high-precision quantitative estimation of creatinine and urea in biological fluids.
[0011] It is a further object of the present disclosure to provide a reagent formulation for selective detection and quantification of creatinine and urea in biological fluids.
[0012] It is another of the present disclosure to provide a reagent formulation that is highly stable and provides a longer shelf-life for an electrochemical biosensor.
[0013] It is another object of the present disclosure to provide a reagent formulation that can facilitate high accuracy quantification of creatinine and urea in biological fluids.
[0014] It is another object of the present disclosure to provide an electrochemical biosensor comprising a reagent formulation for quantitative estimation of creatinine and urea in biological fluids.
[0015] It is another object of the present disclosure to provide an electrochemical biosensor comprising a reagent formulation that allows for multiplexed detection and quantification of creatinine and urea in biological fluids.
[0016] It is another object of the present disclosure to provide a method for quantitative estimation of creatinine and urea in biological fluids.

SUMMARY OF THE INVENTION
[0017] The present disclosure provides a reagent formulation capable of being used for quantitative estimation of creatinine and urea in biological fluids, wherein the reagent formulation can include (a) at least one enzyme responsive to at least one analyte of interest, (b) an electron transfer mediator, (c) at least one crystalline polymer, and (d) a buffer.
[0018] In an embodiment, the reagent formulation of the present disclosure can include at least one enzyme responsive to creatinine which can be selected from the group consisting of creatininase, creatinase, sarcosine oxidase and mixtures thereof.
[0019] In another embodiment, the reagent formulation of the present disclosure can include at least one enzyme responsive to urea which can be selected from urease.
[0020] In another embodiment, the reagent formulation of the present disclosure can include hexammineruthenium chloride as electron transfer mediator.
[0021] In another embodiment, the reagent formulation of the present disclosure can include microcrystalline cellulose as crystalline polymer and citrate as buffer.
[0022] In another aspect, the present disclosure provides a method for measuring a concentration of creatinine or urea in a biological fluid, the method can include the steps of:
contacting a sample of the biological fluid with a biosensor, the biosensor comprising at least one electrode deposited on its surface with a single film formed of a reagent formulation comprising (a) at least one enzyme responsive to creatinine or urea, (b) an electron transfer mediator, (c) at least one crystalline polymer, and (d) a buffer;
detecting an electrical signal from the biosensor; and
measuring the electrical signal to determine the concentration of the creatinine or urea in the biological fluid.
[0023] In another aspect, the present disclosure provides an electrochemical biosensor for measuring concentration of creatinine or urea in a biological fluid, wherein the biosensor can include
at least one electrode; and
a reagent formulation coating at least a portion of the at least one electrode, wherein the reagent formulation can include: (a) at least one enzyme responsive to creatinine or urea, (b) an electron transfer mediator, (c) at least one crystalline polymer, and (d) a buffer.
[0024] Various objects, features, aspects and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiments, along with the accompanying drawing figures in which like numerals represent like components.

BRIEF DESCRIPTION OF THE DRAWINGS
[0025] The accompanying drawings are included to provide a further understanding of the present disclosure, and are incorporated in and constitute a part of this specification. The drawings illustrate exemplary embodiments of the present disclosure and, together with the description, serve to explain the principles of the present disclosure.
[0026] FIG. 1 illustrates a graph plotting current measurements against creatinine concentrations in blood serum, in accordance with an aspect of the present disclosure.
[0027] FIG. 2 illustrates a graph plotting current measurements against urea concentrations in blood serum, in accordance with an aspect of the present disclosure.

DETAILED DESCRIPTION OF THE INVENTION
[0028] The following is a detailed description of embodiments of the disclosure. The embodiments are in such detail as to clearly communicate the disclosure. However, the amount of detail offered is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims.
[0029] Each of the appended claims defines a separate invention, which for infringement purposes is recognized as including equivalents to the various elements or limitations specified in the claims. Depending on the context, all references below to the "invention" may in some cases refer to certain specific embodiments only. In other cases it will be recognized that references to the "invention" will refer to subject matter recited in one or more, but not necessarily all, of the claims.
[0030] Unless the context requires otherwise, throughout the specification which follow, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense that is as “including, but not limited to.”
[0031] Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.
[0032] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. It should also be noted that the term “or” is generally employed in its sense including “and/or” unless the content clearly dictates otherwise.
[0033] In some embodiments, the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term “about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range.
[0034] Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.
[0035] The headings and abstract of the invention provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.
[0036] The following discussion provides many example embodiments of the inventive subject matter. Although each embodiment represents a single combination of inventive elements, the inventive subject matter is considered to include all possible combinations of the disclosed elements. Thus if one embodiment comprises elements A, B, and C, and a second embodiment comprises elements B and D, then the inventive subject matter is also considered to include other remaining combinations of A, B, C, or D, even if not explicitly disclosed.
[0037] Various terms as used herein are shown below. To the extent a term used in a claim is not defined below, it should be given the broadest definition persons in the pertinent art have given that term as reflected in printed publications and issued patents at the time of filing.
[0038] In one aspect, the present disclosure provides a reagent formulation capable of being used for quantitative estimation of creatinine or urea in biological fluids, wherein the reagent formulation can include (a) at least one enzyme responsive to at least one analyte of interest, (b) an electron transfer mediator, (c) at least one crystalline polymer, and (d) a buffer.
[0039] As used herein, the term "biological fluid" can include, but not limited to, a whole blood sample, a blood serum sample, a blood plasma sample, other body fluids such as ISF (interstitial fluid), urine, saliva, and non-body fluids.
[0040] The reagent formulation in accordance with aspects of the present disclosure can include at least one enzyme for converting creatinine or urea in a biological fluid sample into a chemical species that is electrochemically measurable in terms of an electrical current it produces and/or in terms of a change in electronic properties of the sensor electrodes it induces. The at least one enzyme that can be used in the present disclosure can vary depending on an analyte to be detected in a biological fluid and can be selected from, but not limited to, for example hydrolase enzyme, redox enzyme and mixtures thereof. For example, if creatinine is to be detected and quantified in a biological fluid, the at least one enzyme can be selected from, but not limiting to, for example creatininase, creatinase, sarcosine oxidase and mixtures thereof. Alternatively, if urea is to be detected, the at least one enzyme can be selected from urea sensitive enzymes such as, but not limited to, urease. The reagent formulation may further comprise one or more coenzymes.
[0041] In an embodiment of the present disclosure, the at least one enzyme that can be used in the reagent formulation for measuring creatinine in a blood sample can preferably be a mixture comprising hydrolase enzymes (e.g. creatininase and creatinase) and redox enzyme (e.g. sarcosine oxidase).
[0042] In another embodiment of the present disclosure, the at least one enzyme that can be used in the reagent formulation for measuring urea concentration in a blood sample can preferably be a hydrolase enzyme such as, urease.
[0043] In an embodiment of the present disclosure, hydrolase enzyme(s) can convert creatinine to an oxidizable substrate that can be a source of electrons that are transferred to an electron transfer mediator by a redox enzyme, and from there, the electrons can be directly transferred to electrode, wherein the electrode is held in a narrow range of potentials. This scheme of enzymes and mediator can make the biosensor selective to only a particular analyte of interest, creatinine. Concentration of electron transfer mediator in the reagent formulation can remain constant and can permit direct measurement of current generated by enzymatic reaction. Changes in dissolved oxygen, temperature, pH, or other stimuli, that can have adverse effect on selectivity, responsiveness, and accuracy of the biosensor, can be avoided in biosensors utilizing the reagent formulation of the present disclosure. The mechanism by which current can be generated at an electrode in an electrochemical biosensor is by oxidation of a creatinine derived substrate (e.g., sarcosine) generated by a cascade of enzymatic reactions. The resulting current can be correlated directly and is proportional to creatinine concentration.
[0044] Ionic products, such as ammonium and bicarbonate ions that can result from metabolism of urea with enzymatic reaction of urea sensitive enzymes, can interact with the electron transfer mediator to induce a change in conductivity of the mediator and can alter electronic properties of an electrode, which can be observed and correlated directly to urea concentration.
[0045] The electron transfer mediator in accordance with aspects of the present disclosure can preferably be a chemical moiety and can be selected from transition metal complexes that can enable accurate, reproducible, quick and continuous detection and quantification of multiple analytes in biological fluids. Further, the transition metal complex electron mediators can be stable under ambient light, humidity, oxygen levels and at the temperatures encountered in use, and thereby enhances shelf-life of the sensor even when stored at ambient temperature.
[0046] In an exemplary embodiment of the present disclosure, organometallic complexes of ruthenium such as, but not limited to, hexammineruthenium chloride can be used as electron transfer mediator to formulate the reagent formulation.
[0047] The reagent formulation in accordance with the aspects of the present disclosure can include crystalline polymer particles. The crystalline polymer can include a cellulose-based crystalline polymer such as microcrystalline cellulose. The crystalline polymer particles assist in thermally stabilizing the enzyme and the electron transfer mediator present in the reagent formulation, and providing adequate viscosity so that the reagent formulation, when dried, stays in its original position on a sensor electrode.
[0048] A pH buffer can be added to the reagent formulation in accordance with the aspects of the present disclosure for the purpose of effectively enhancing the enzyme catalyzed reaction of creatinine and urea. The inclusion of pH buffer can also stabilize the reagent formulation and helps in uniformly dispersing the reagent formulation on the sensor electrode without any cracks upon drying.
[0049] In an exemplary embodiment, the pH buffer that can be used to formulate the reagent formulation of the present disclosure can be a citrate buffer.
[0050] The reagent formulation of the present disclosure can further include one or more stabilizers, one or more polymeric binders and optionally a surfactant. The stabilizer can be any suitable material that is known to stabilize enzymes in the relevant art, and can be selected from bovine serum albumin (BSA), trehalose and mixtures thereof. Suitable binders can include, but not limited to, hydroxy ethyl cellulose (HEC).
[0051] In one exemplary embodiment of the present disclosure, the reagent formulation for measuring creatinine concentration in blood serum can include a mixture of creatininase, creatinase and sarcosine, hexammineruthenium chloride mediator, microcrystalline cellulose particles, a citrate buffer having a pH of about 7.2 to 7.4, a stabilizer or combination of stabilizers, a polymeric binder and optionally a surfactant.
[0052] According to embodiment of the present disclosure, the reagent formulation can be custom formulated using the same components with different weight ratios depending upon desired performance characteristics of biosensor. The inventors have found that when the reagent formulation contains the components in a proper weight ratio, the formulation can enable high precision quantitative estimation of target analytes in biological fluids.
[0053] In a more preferred exemplary embodiment, the reagent formulation for measuring creatinine concentration in blood sample can include (a) creatininase, creatinase and sarcosine oxidase in a weight ratio of 1:1:2 respectively, (b) from 100 to 300 millimolar (mM) of hexammineruthenium chloride, (c) microcrystalline cellulose, and (d) a citrate buffer.
[0054] In one exemplary embodiment of the present disclosure, the reagent formulation for measuring urea concentration in blood serum can include urease enzyme, hexammineruthenium chloride mediator, microcrystalline cellulose particles, a citrate buffer having a pH of about 7.2 to 7.4, a stabilizer or combination of stabilizers, a polymeric binder and optionally a surfactant.
[0055] In a more preferred exemplary embodiment, the reagent formulation for measuring urea concentration in blood sample can include (a) from 5 to 12 % by weight of urease, preferably 10 % by weight, (b) from 300 to 600 millimolar (mM) of hexammineruthenium chloride, (c) microcrystalline cellulose, and (d) a citrate buffer.
[0056] In another aspect, the present disclosure provides a method for measuring a concentration of creatinine or urea in a biological fluid, the method can include the steps of: (i) contacting a sample of the biological fluid with a biosensor, the biosensor comprising at least one electrode deposited on its surface with a single film formed of a reagent formulation comprising (a) at least one enzyme responsive to creatinine or urea, (b) an electron transfer mediator, (c) at least one crystalline polymer, and (d) a buffer; (ii) detecting an electrical signal from the biosensor; and (iii) measuring the electrical signal to determine the concentration of the creatinine or urea in the biological fluid.
[0057] In an exemplary embodiment, the biological fluid used in the method of the present disclosure can be selected from whole blood, blood serum or blood plasma.
[0058] In an embodiment, the single film formed on the electrode surface for measuring creatinine in whole blood can include an enzyme system comprising a mixture of creatininase, creatinase and sarcosine, hexammineruthenium chloride mediator, microcrystalline cellulose particles, a citrate buffer.
[0059] Alternatively, the single film formed on the electrode surface for measuring urea in whole blood can include an enzyme system comprising urease, hexammineruthenium chloride mediator, microcrystalline cellulose particles, a citrate buffer.
[0060] In another aspect, the present disclosure provides an electrochemical biosensor for measuring concentration of creatinine or urea in a biological fluid, wherein the biosensor can include at least one electrode; and a reagent formulation coating at least a portion of the at least one electrode, wherein the reagent formulation can include: (a) at least one enzyme responsive to creatinine or urea, (b) an electron transfer mediator, (c) at least one crystalline polymer, and (d) a buffer.
[0061] The electrochemical biosensor of the embodiments of the present disclosure can comprise a plurality of electrodes. The plurality of electrodes can include at least one working electrode and at least one reference electrode that can be deposited on the substrate. The electrochemical biosensor of the present disclosure may further comprise a counter electrode and it may support the electrochemical activity at the working electrode of the biosensor. The working electrode and the counter electrode can be formed of any suitable electrode material known in the relevant art.
[0062] The plurality of electrodes can be disposed on a substrate that can act as a bottom support for the electrodes. The substrate can be formed of any suitable insulating material.
[0063] In an embodiment, the substrate can be a polymeric material in the form of a base plate that acts as a bottom support for sensor electrodes.
[0064] In accordance with embodiments of the present disclosure, a plurality of nanostructures can be deposited over the working electrode that can increase the surface area of the working electrode. The increase in surface area of the working electrode can increase the sensitivity and accuracy of the assay to be performed even with very low quantities of analyte present in a biological sample.
[0065] As used herein, the term "nanostructures" refer to solid particles or hollow-core particles, which can have particle size less than 500 nm, preferably less than 100 nm, more preferably less than 50 nm.
[0066] The non-limiting exemplary nanostructures according to the present disclosure can be selected from carbon nanotubes (CNTs) or gold nanoparticles.
[0067] In an exemplary embodiment, the nanostructures can be gold nanoparticles deposited over the working electrode using the electro deposition technique.
[0068] In another exemplary embodiment, the nanostructures can be carboxylated carbon nanotubes and the percentage of carboxylation of carbon nanotubes can range from 3% to 5%.
[0069] In accordance with embodiments of the present disclosure, the working electrode of the biosensor can be ablated in the form of concentric arcs, circle, spiral, helix or any polygonal shape to increase the deposition of nanostructures. The "polygonal" shape can be a multi-sided, closed planar shape. The polygons may include trigons (or triangles), tetragons (or quadrilaterals), pentagons, hexagons, heptagons, octagons, and the like. Tetragons may include squares and rectangles, which can have four sides connected at four right angles. Tetragons also may include rhombi (e.g. diamond- shaped polygons or parallelograms), which do not include four right angles.
[0070] In a more preferred exemplary embodiment, the working electrode can be ablated in the form of concentric arcs. The concentric arcs of the working electrode can be in a hill-valley type arrangement providing better deposition of the nanostructure. The working electrode can have a diameter in the range of 2mm to 8mm.
[0071] In an exemplary embodiment, the present disclosure provides an electrochemical biosensor for measuring concentration of creatinine or urea in a biological fluid, wherein the biosensor can include a substrate; at least one working electrode and at least one reference electrode disposed on the substrate; a plurality of nanostructures deposited over the working electrode for increasing the surface area of the working electrode; and a reagent formulation coating at least a portion of the nanostructures, wherein the reagent formulation comprises: (a) at least one enzyme responsive to creatinine or urea, (b) an electron transfer mediator, (c) at least one crystalline polymer, and (d) a buffer.
[0072] The reagent formulation of the present disclosure can be deposited on the electrodes by simply applying the reagent formulation to surfaces of the electrodes. The reagent formulation may be deposited only on the working electrode surface or it may be deposited on both working electrode and reference electrode surfaces. The deposition of reagent formulation on the electrode surface may produce a dry reagent layer on the electrode at micrometer or sub-micrometer thickness. After deposition, the reagent formulation may preferably be dried and the electrodes deposited with reagent formulation may be stored in a desiccated container before final lamination to form the sensor. The amount of reagent formulation deposited on the electrode surface can preferably be in the range from 1 to 100 µl.
[0073] The reagent formulation of the present disclosure can reduce the change in signal intensity caused by interfering substances present in biological fluid samples. The electrochemical biosensor utilizing the reagent formulation of the present disclosure can allow for multiplexing and enable simultaneous detection and quantification of multiple analytes with very low sample volume.

EXAMPLES
[0074] The present invention is further explained in the form of following examples. However it is to be understood that the foregoing examples are merely illustrative and are not to be taken as limitations upon the scope of the invention. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications may be made without departing from the scope of the invention.

Example 1: Preparation of reagent formulation of the present disclosure and sensor comprising the same, and measurement of creatinine concentration in blood sample.

[0075] The reagent formulation for measuring creatinine concentration in a blood sample was prepared by making a stock solution, hereinafter referred to as "solution A", comprising a mixture of 0.1 M citrate buffer, 3 wt. % bovine serum albumin (BSA), 150mM trehalose, 2 wt. % microcrystalline cellulose (AVICEL®), 1 wt. % hydroxy ethyl cellulose and 0.05 wt. % TRITONTMX-100. Solution A was homogenized with a homogenizer at 10,000 rpm for 10 min and the resulting Solution A was observed to contain a pH of 7.4.
[0076] An enzyme solution was prepared by mixing 350 units of creatininase, 350 units of creatinase and 700 units of sarcosine oxidase in 1 ml of solution A (1:1:2 ratio). 300mM of hexammineruthenium chloride mediator was combined with the enzyme solution to prepare the reagent formulation.
[0077] About 7-15µl of the reagent formulation was deposited on a working electrode and dried at 35-50°C for 5-15 min followed by storage in a desiccated container before final lamination to form the sensor.
[0078] About 15-25µl of blood serum sample was contacted with the sensor treated with the reagent formulation. Chronoamperometric response was measured as a function of concentration of creatinine at a fixed bias voltage. As shown in FIG.1, the current for the serum sample was plotted against the creatinine concentration (mg/dL). Data was collected in duplicate with concentration range starts from 0.5 to 2.54 mg/dl as shown in Table I below. The correlation coefficient and linear regression equations are calculated from the obtained results versus urea concentration values. Results shows that system has achieved high degree of linearity (R² = 0.995).
[0079] The linear dose response profile indicates relatively high sensitivity of the present sensors and the y-intercepts indicates low background noise levels. These results indicate that accurate readings can be achieved using the sensors including the reagent formulation described herein.

Table I
Chronoamperometry,0.24 V
Total Creatinine Concentration (mg/dl) Current (uA)
0.51 1.85
1.02 1.98
1.52 2.21
2.03 2.57
2.54 3.03

Example 2: Preparation of reagent formulation of the present disclosure and sensor comprising the same, and measurement of urea concentration in blood sample.

[0080] An enzyme solution was prepared by mixing 7.5 wt % urease in 1 ml of solution A (as prepared in Example 1). 600mM of hexammineruthenium chloride mediator was combined with the enzyme solution to prepare the reagent formulation.
[0081] About 7-10µl of the reagent formulation was deposited on a working electrode and dried at 35-50°C for 5-15 min followed by storage in a desiccated container before final lamination to form the sensor.
[0082] About 15-25µl of blood serum sample was contacted with the sensor treated with the reagent formulation. Chronoamperometric response was measured as a function of concentration of urea at a fixed bias voltage. As shown in FIG.2, the current for the serum sample was plotted against the urea concentration (mg/dL). Data was collected in duplicate with concentration range starts from 8 to 65 mg/dl as shown in Table II below. The correlation coefficient and linear regression equations are calculated from the obtained results versus urea concentration values. Results shows that system has achived high degree of linearity (R² = 0.972).
[0083] The linear dose response profile and the low background noise levels indicate relatively high sensitivity of the present sensors. These results indicate that accurate readings may be achieved using the sensors including the reagent formulation described herein.

Table II
Chronoamperometry,0.24 V
Total urea Concentration (mg/dl) Current (uA)
8.2 15.26
16.4 25.51
32.8 40.51
49.2 53.40
65.6 60.01

ADVANTAGES OF THE PRESENT INVENTION
[0084] The present disclosure provides a reagent formulation for use in electrochemical biosensor for high-precision quantitative analysis of creatinine and urea in biological fluids that can be easily and quickly conducted.
[0085] The present disclosure provides a reagent formulation that is highly stable and provides a longer shelf-life for the biosensor.
[0086] The present disclosure provides an electrochemical biosensor that has excellent storage stability even at ambient temperature.
[0087] The present disclosure provides an electrochemical biosensor that has good reproducibility and multiplexing capabilities.
[0088] The present disclosure provides an electrochemical biosensor that requires relatively lower sample volumes than known sensors.
[0089] The present disclosure provides an electrochemical biosensor that enables simultaneous detection and quantification of multiple analytes in biological fluids.
[0090] The present disclosure provides an electrochemical biosensor that overcomes the drawbacks of the prior art.
[0091] The present disclosure provides a simple and highly accurate method for quantitative estimation of creatinine and urea in biological fluids.
,CLAIMS:1. A reagent formulation for measuring concentration of creatinine or urea in a biological fluid, said formulation comprising:
at least one enzyme responsive to creatinine or urea;
an electron transfer mediator;
at least one crystalline polymer; and
a buffer.

2. The formulation of claim 1, wherein said at least one enzyme responsive to creatinine is selected from the group consisting of creatininase, creatinase, sarcosine oxidase and mixtures thereof.

3. The formulation of claim 1, wherein said at least one enzyme responsive to creatinine is a mixture comprising creatininase, creatinase and sarcosine oxidase.

4. The formulation of claim 1, wherein said at least one enzyme responsive to urea is urease.

5. The formulation of claim 1, wherein said electron transfer mediator is an organometallic complex of ruthenium.

6. The formulation of claim 5, wherein said organometallic complex of ruthenium is hexammineruthenium chloride.

7. The formulation of claim 1, wherein said crystalline polymer is cellulose based crystalline polymer.

8. The formulation of claim 7, wherein said cellulose based crystalline polymer is microcrystalline cellulose.

9. The formulation of claim 1, wherein said buffer is citrate buffer.

10. The formulation of claim 1 further comprises at least one additive selected from stabilizer, binder or surfactant.

11. A reagent formulation for measuring concentration of creatinine in a biological fluid, said formulation comprising:
creatininase, creatinase and sarcosine oxidase, wherein the weight ratio of creatininase to to creatinase to sarcosine oxidase is 1:1:2 ;
from 100 to 300 millimolar (mM) of hexammineruthenium chloride;
microcrystalline cellulose; and
a citrate buffer.

12. A reagent formulation for measuring concentration of urea in a biological fluid, said formulation comprising:
from 5 to 12 % by weight of urease;
from 300 to 600 millimolar (mM) of hexammineruthenium chloride;
microcrystalline cellulose; and
a citrate buffer.

13. A method for measuring concentration of creatinine or urea in a biological fluid, said method comprising the steps of:
contacting a sample of said biological fluid with a biosensor, said biosensor comprising at least one electrode deposited on its surface with a single film formed of a reagent formulation comprising (a) at least one enzyme responsive to creatinine or urea, (b) an electron transfer mediator, (c) at least one crystalline polymer, and (d) a buffer;
detecting an electrical signal from said biosensor; and
measuring said electrical signal to determine the concentration of said creatinine or urea in said biological fluid.

14. The method of claim 13, wherein said biological fluid is selected from whole blood, blood serum or blood plasma.

15. A biosensor for measuring concentration of creatinine or urea in a biological fluid, said biosensor comprising:
at least one electrode; and
a reagent formulation coating at least a portion of said at least one electrode, wherein said reagent formulation comprises: (a) at least one enzyme responsive to creatinine or urea, (b) an electron transfer mediator, (c) at least one crystalline polymer, and (d) a buffer.

16. A biosensor for measuring concentration of creatinine or urea in a biological fluid, said biosensor comprising:
a substrate;
at least one working electrode and at least one reference electrode disposed on said substrate;
a plurality of nanostructures deposited over said working electrode for increasing the surface area of said working electrode; and
a reagent formulation coating at least a portion of said nanostructures, wherein said reagent formulation comprises: (a) at least one enzyme responsive to creatinine or urea, (b) an electron transfer mediator, (c) at least one crystalline polymer, and (d) a buffer.