Novel creatinine quantification device

Abstract

The present invention relates to a strip for detecting creatinine and a method for measuring the concentration of creatinine in a biological sample using same. According to the present disclosure, creatinine and creatine concentrations can be simultaneously measured only with a single loading of a small amount of bodily fluid using a strip having a simple structure, thereby enabling accurate creatinine quantification without distortion caused by creatine concentrations. Therefore, the present invention can be advantageously applied to a point of care testing (POCT) in which a patient himself / herself simply and directly measures the concentration of creatinine, which is a key marker of renal function.

Description

A novel creatinine quantification device

[0001] The present invention relates to a creatinine detection strip for rapidly and accurately measuring creatinine in a body fluid using an enzyme colorimetric method, and a method for detecting and / or quantifying creatinine using the same.

[0002]

[0003] Creatine is synthesized in the liver and pancreas from three amino acids: arginine, glycine, and methionine. It is used as a means of storing ATP (adenosine triphosphate) in muscles and nerve tissue. Creatinine, which is produced through dehydration of creatine, is a waste product produced in muscles and is excreted through urine. Normally, the total amount excreted is proportional to the body's muscle mass, with an average daily excretion of 20-26 mg / kg for adult men and 14-22 mg / kg for adult women. Because creatinine is mostly excreted through the kidneys in urine, it is an important indicator of kidney function. High creatinine levels indicate that the kidneys are not properly filtering waste products. Measuring creatinine has become a very important issue in assessing and treating kidney function abnormalities. For example, elevated serum creatinine is a marker of the end-stage of chronic kidney disease.

[0004] Meanwhile, the development of point-of-care testing (POCT) methods is actively underway to facilitate rapid, early diagnosis. These methods allow patients to conduct simple tests themselves to predict the presence of disease. It is crucial to overcome the limitations of existing diagnostic methods that rely on expensive equipment and provide rapid, sensitive, and affordable tests. Demand for creatinine point-of-care testing is rapidly increasing, necessitating the development of efficient, portable, and compact measuring devices that are inexpensive, simple to use, and offer high diagnostic reliability.

[0005] Numerous papers and patents are referenced and cited throughout this specification. The disclosures of these cited papers and patents are incorporated herein by reference in their entirety to provide a clearer understanding of the state of the art and the scope of the present invention.

[0006]

[0007] The present inventors have made extensive research efforts to develop a novel point-of-care diagnostic (POCT) method for rapidly and reliably measuring the creatinine concentration in body fluids, which is a key indicator of renal dysfunction. As a result, when a first reaction pad containing a creatine detection agent and a second reaction pad containing both creatine and creatinine detection agents are physically contacted at the ends of a sample pad for loading a sample to be analyzed, the loaded sample simultaneously diffuses from the sample pad to the first and second reaction pads by capillary action without a separate external force, and an accurate creatinine concentration corrected for the creatine concentration can be measured through a simple process of arithmetically subtracting the color development intensity of the first reaction pad from the color development intensity of the second reaction pad.

[0008] Accordingly, the purpose of the present invention is to provide a strip for detecting creatinine and a method for measuring the concentration of creatinine in a biological sample using the strip.

[0009] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below.

[0010]

[0011] Various embodiments of the present invention are described with reference to the drawings. In the following description, various specific details, such as specific configurations, compositions, and processes, are set forth to provide a thorough understanding of the present invention. However, certain embodiments may be practiced without one or more of these specific details, or in conjunction with other known methods and configurations. In other instances, well-known processes and manufacturing techniques are not described in specific detail so as not to unnecessarily obscure the present invention. Reference throughout this specification to an embodiment means that a particular feature, configuration, composition, or characteristic described in connection with the embodiment is included in one or more embodiments of the present invention. Thus, the appearance of an embodiment in various places throughout this specification does not necessarily indicate the same embodiment of the present invention. Additionally, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.

[0012]

[0013] According to one aspect of the present invention, the present invention provides a strip for detecting creatinine comprising:

[0014] (a) a sample pad to which a biological sample to be analyzed is applied;

[0015] (b) a first reaction pad placed in contact with one end of the sample pad and containing a preparation capable of detecting creatine; and

[0016] (c) A second reaction pad, which is placed in contact with one end of the sample pad and includes a preparation capable of detecting creatine and a preparation capable of detecting creatinine.

[0017] The present inventors have made extensive research efforts to develop a novel point-of-care diagnostic method for rapidly and reliably measuring the creatinine concentration in body fluids, a key indicator of kidney function. As a result, when a first reaction pad containing a creatine detection agent and a second reaction pad containing both creatine and creatinine detection agents are brought into contact with the ends of a sample pad for loading a sample to be analyzed, the creatine concentration is measured by the first reaction pad and the creatine and creatinine concentrations are measured simultaneously by the second reaction pad through capillary diffusion of the loaded sample, thereby enabling the measurement of an accurate creatinine concentration corrected for the creatine concentration with only a single sample loading, thereby completing the present invention.

[0018] In this specification, the term “sample pad” refers to a region where a biological sample to be analyzed is loaded, which absorbs and uniformly distributes a liquid sample and allows the received sample to spread to a reaction pad that is in contact with one end of the sample pad.

[0019] The term “biological sample” as used herein means any sample containing creatine or its dehydrate, creatinine, obtained from a mammal, including a human, including, but not limited to, body fluid, tissue, organ, cell or cell culture.

[0020] According to a specific embodiment of the present invention, the biological sample used in the present invention is a body fluid, more specifically urine, whole blood, serum or plasma, and most specifically whole blood, serum or plasma.

[0021] The term “creatine-detectable agent” or “creatinine-detectable agent” as used herein means an agent that can provide information on whether creatine or creatinine is present in a biological sample or information on a quantitative value of the creatine or creatinine concentration, and for example, means an agent that can provide visual information on the presence or concentration of creatine or creatinine through an enzymatic reaction, a chemical colorimetric reaction, or a combination thereof.

[0022] According to a specific embodiment of the present invention, the agent capable of detecting creatine included in the first reaction pad includes creatinase, sarcosine oxidase, and HRP (Horseradish Peroxidase).

[0023] According to a more specific embodiment of the present invention, the first reaction pad or the second reaction pad additionally includes 4-AAP (4-aminoantipyrine) and TOPS (N-ethyl-N-(3-sulfopropyl)-3-methylaniline).

[0024] Creatine is converted into sarcosine and urea by creatine hydrolase (CI), and sarcosine, the product of hydrolysis, produces formaldehyde, glycine, and hydrogen peroxide by sarcosine oxidase (SOx), and hydrogen peroxide, the product of oxidation, is reduced by horseradish peroxidase (HRP) and reacts with a chromogenic compound to produce a color with an intensity proportional to the concentration.

[0025]

[0026]

[0027] The chromogenic compound used in the present invention includes, for example, 4-AAP (4-aminoantipyrine) and TOPS (N-ethyl-N-(3-sulfopropyl)-3-methylaniline), MAOS (N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline, sodium salt monohydrate), ESPT (N-Ethyl-Sulfopropyl-m- Toluidine) and TODB (N,N-Bis(4-sulfobutyl)-3-methylaniline disodium salt) which exhibit color development through oxidative coupling reaction therewith, but is not limited thereto, and any generating compound that emits color with an intensity proportional to the amount of the product of a continuous hydrolysis / oxidation reaction using creatine as a starting material may be used.

[0028] According to a specific embodiment of the present invention, the agent capable of detecting creatinine included in the second reaction pad includes creatininase, creatinase, sarcosine oxidase, and HRP (Horseradish Peroxidase).

[0029] The second reaction pad may additionally include creatinine hydrolase (Ce) for quantifying creatinine compared to the first reaction pad for quantifying creatine. Creatinine hydrolase hydrolyzes creatinine in the sample to be analyzed into creatine, and then reacts with Ce, sarcosine oxidase, HRP, and a chromogenic compound (e.g., 4-AAP and TOPS) in the same manner as in the first reaction pad to produce a purple color.

[0030]

[0031]

[0032] According to a specific embodiment of the present invention, the sample pad comprises ascorbate oxidase.

[0033] More specifically, the ascorbate oxidase is included at a concentration of 800 to 3000 Units / ml. Even more specifically, the ascorbate oxidase is included at a concentration of 800 to 2000 Units / ml, even more specifically, the ascorbate oxidase is included at a concentration of 800 to 1600 Units / ml, even more specifically, the ascorbate oxidase is included at a concentration of 800 to 1500 Units / ml, even more specifically, the ascorbate oxidase is included at a concentration of 900 to 1200 Units / ml, and most specifically, the ascorbate oxidase is included at a concentration of about 1000 Units / ml.

[0034]

[0035] According to a specific embodiment of the present invention, the sample pad; and the first reaction pad and the second reaction pad are arranged to overlap each other at the contact portion.

[0036] As shown in FIG. 1, the creatinine detection strip (100) of the present invention includes a sample pad (120) on which a sample to be analyzed, specifically, a body fluid, is loaded; a first reaction pad (130a) for detecting creatine in the sample; and a second reaction pad (130b) for detecting creatine and creatinine, wherein the sample pad (120), the first reaction pad (130a), and the second reaction pad (130b) are arranged to overlap at one end, so that a liquid body fluid sample (e.g., blood) loaded on the sample pad (120) moves to the first reaction pad (130a) and the second reaction pad (130b) by a capillary phenomenon without a separate external force, thereby enabling simultaneous detection of creatine and creatinine with only one sample loading.

[0037] According to a specific embodiment of the present invention, the length of the section where the sample pad (120) and the first reaction pad (130a) and the second reaction pad (130b) overlap is 1 to 5 mm, more specifically 1 to 4 mm, even more specifically 1 to 3 mm, and most specifically about 2 mm.

[0038] According to a specific embodiment of the present invention, the strip additionally includes a support member that fixes the sample pad, the first reaction pad, and the second reaction pad as a length member.

[0039] As shown in Fig. 1, the creatinine detection strip (100) of the present invention has a support member (110) of a length member arranged at the bottom to fix and support the first reaction pad (130a), the second reaction pad (130b), and the sample pad (120). The support member (110) may be composed of any material as long as it can support and fix the first reaction pad (130a), the second reaction pad (130b), and the sample pad (120), but may be liquid-impermeable to prevent leakage of the liquid sample moving from the sample pad (120) to the first reaction pad (130a) and the second reaction pad (130b), and specifically, may be composed of a glass material or a hydrophobic polymer.

[0040] As used herein, the term “hydrophobic polymer” refers to a synthetic or natural polymer compound in which monomers of the same or different types are continuously bonded and which is insoluble in water or other polar solvents. Accordingly, polymers that can be used in the liquid-impermeable support of the present invention include, but are not limited to, acrylic, epoxy, polyethylene, polystyrene, polyvinyl chloride, polytetrafluoroethylene, polydimethylsiloxane, polyester, or polyurethane.

[0041]

[0042] According to one aspect of the present invention, the present invention provides a method for measuring creatinine concentration in a biological sample, comprising the following steps:

[0043] (a) loading a biological sample into a sample pad within a creatinine detection strip of claim 1;

[0044] (b) a step of measuring the color development intensity of the first reaction pad and the second reaction pad;

[0045] (c) A step of subtracting the color development intensity of the first reaction pad from the color development intensity of the second reaction pad.

[0046] According to the present invention, by arithmetically removing the color development intensity of the first reaction pad, which reflects only the concentration of creatine, from the color development intensity of the second reaction pad, which reflects both the concentration of creatinine and its hydrolysis product, creatine, the accuracy of creatinine measurement through an enzymatic colorimetric method is reduced, thereby allowing creatinine, an important marker for diagnosing kidney disease, to be quantified with high reliability.

[0047] The term “diagnosis” as used herein includes determining an individual’s susceptibility to a specific disease, determining whether an individual currently has a specific disease, and determining a prognosis for an individual suffering from a specific disease. Since the present invention measures the concentration of creatinine, a waste product produced in muscles, in body fluids to determine whether kidney disease or kidney dysfunction has occurred or to predict the possibility of onset, the “method for measuring creatinine concentration” of the present invention has the same meaning as “a method for providing information necessary for diagnosing kidney disease” or “a method for providing information necessary for measuring kidney function.”

[0048]

[0049] According to one aspect of the present invention, the present invention provides a detection device for detecting creatinine, comprising:

[0050] Application portion to which the biological sample to be analyzed is applied;

[0051] A first reaction unit comprising a preparation capable of detecting creatine from the sample application unit; and

[0052] A second reaction unit comprising a preparation capable of detecting creatine from the sample application unit and a preparation capable of detecting creatinine;

[0053]

[0054] The term “detection device” as used herein refers to a device capable of detecting a specific indicator in vitro based on a biological sample such as urine, whole blood, serum, or plasma. For example, the detection device may include an application portion to which a sample is applied, and one or more reaction portions (e.g., a first reaction portion and a second reaction portion), and there is no limitation on the specific structure or configuration thereof as long as it is capable of detecting a desired marker, value, or specific indicator (e.g., the concentration of creatine or creatinine, etc.) from the biological sample through the components.

[0055] In this specification, the term "application portion" means a portion where a biological sample to be analyzed is introduced into the device, and is a region where the sample first comes into contact, and is configured to move or transfer the sample to a strip or other component of the detection device (e.g., a reaction portion). For example, the application portion may be implemented in the form of a sample pad, a sample pad, etc.

[0056] It refers to a region including one or more detection agents that can selectively react with a specific analyte (e.g., creatine, creatinine, etc.) among the biological samples delivered from the above application unit.

[0057] The term "reaction unit" in this specification may include a first reaction unit for detecting creatine and a second reaction unit capable of detecting both creatine and creatinine. The reaction unit generally has a function of reacting with the corresponding detection agents to generate a signal, such as a color change, thereby enabling the presence and concentration of the analyte to be confirmed.

[0058]

[0059] In addition, it should be understood that the effects of the present invention are not limited to the above-described effects, but include all effects that can be inferred from the composition of the invention described in the detailed description or claims of the present invention.

[0060] The features and advantages of the present invention are summarized as follows:

[0061] (a) The present invention provides a strip for detecting creatinine and a method for measuring the concentration of creatinine in a biological sample using the strip.

[0062] (b) The present invention enables accurate measurement of creatinine concentration without distortion due to creatine concentration by simultaneously measuring creatinine and creatine concentrations with only one loading of a small amount of body fluid using a strip of a simple structure.

[0063] (c) Accordingly, the present invention can be usefully applied to point-of-care testing (POCT) in which patients themselves simply measure the creatinine concentration, a key indicator of kidney function.

[0064]

[0065] FIG. 1 is a perspective view (FIG. 1a), a plan view (FIG. 1b), a case (FIG. 1c), and a flowchart (FIG. 1d) of a creatinine sensor strip according to one embodiment of the present invention.

[0066] Figure 2 illustrates the results of color reaction signal analysis according to creatinine concentration of the creatinine sensor strip of the present invention, where R1 represents the first reaction pad and R2 represents the second reaction pad.

[0067] Figure 3 illustrates the results of analysis of the creatinine color reaction signal in blood of the creatinine sensor strip of the present invention.

[0068] Figure 4 shows the results confirming that the creatinine sensor strip of the present invention has high selectivity for creatinine and that there is almost no color reaction signal for substances other than creatinine.

[0069] Figure 5 illustrates the results of color reaction signal analysis according to the concentration of ascorbate oxidase of the creatinine sensor strip of the present invention.

[0070] Figure 6 illustrates the results of color reaction signal analysis according to the presence or absence of ascorbate oxidase in the creatinine sensor strip of the present invention.

[0071] Figure 7 illustrates the results of color reaction signal analysis according to the concentration of PBS (pH 7.4) of the creatinine sensor strip of the present invention.

[0072]

[0073] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples, in accordance with the gist of the present invention.

[0074]

[0075] Example

[0076] Manufacturing Example 1: Creatinine Sensor Strip Manufacturing

[0077] A creatinine sensor strip was manufactured as illustrated in Fig. 1. More specifically, the support was cut to a width of 2 mm, a length of 13 mm, a sample pad to a length of 10 mm, and the first and second reaction pads to a length of 5 mm. At this time, the length of the overlapping section between the sample pad and the first and second reaction pads was set to 2 mm.

[0078] The sample pad used was PALL's Vivid GX (Vivid Plasma Separation GX membrane). Ascorbate oxidase was completely dissolved in 1X PBS (pH 7.4), and 10 Units / 10 ㎕ was dropped onto the sample pad (Vivid GX) and dried at 37°C for 10 minutes.

[0079] The first reaction pad used ADVANTEC's CA 0.45 (Cellulose Acetate 0.45 μm), and 0.5 μl of a solution containing 1000 Unit / ml of Creatinase, 200 Unit / ml of Sarcosine oxidase, 200 Unit / ml of HRP (Horseradish Peroxidase), 20 mM of 4-AAP (4-aminoantipyrine), 20 mM of TOPS (N-ethyl-N-(3-sulfopropyl)-3-methylaniline), and 4 wt% of PVA (Polyvinyl alcohol) was dropped onto the first reaction pad (CA 0.45) and dried at 37°C for 10 minutes.

[0080] The second reaction pad used ADVANTEC's CA 0.45 (Cellulose Acetate 0.45 μm), and 0.5 μl of a solution containing 1500 Unit / ml of creatininase, 1000 Unit / ml of creatinase, 200 Unit / ml of sarcosine oxidase, 200 Unit / ml of HRP (Horseradish Peroxidase), 20 mM of 4-AAP (4-aminoantipyrine), 20 mM of TOPS (N-ethyl-N-(3-sulfopropyl)-3-methylaniline), and 4 wt% of PVA (Polyvinyl alcohol) was dropped onto the second reaction pad (CA 0.45) and dried at 37°C for 10 minutes.

[0081]

[0082] Experimental Example 1: Analysis of color reaction signals according to creatinine concentration

[0083] After preparing the creatinine sensor strip of the above Manufacturing Example 1, Sigma's creatinine was completely dissolved in distilled water (DW), diluted with Bio-Rad's Human serum based control level 1 (creatinine 0.8 mg / dl), and 15 μl of creatinine at concentrations of 0, 0.5, 1, 2, 5, 10, and 20 mg / dl was injected onto the sample pad. As a result of analyzing the intensity of the color reaction 10 minutes after sample injection, it was confirmed that the color reaction signal increased depending on the creatinine concentration (Fig. 2).

[0084]

[0085] Experimental Example 2: Analysis of the colorimetric reaction signal of creatinine in blood

[0086] 15 μl of human serum with a creatinine level of 1.5 mg / dl or less (normal) and human serum with a creatinine level of more than 1.5 mg / dl were injected onto the sample pad of the creatinine sensor strip of Manufacturing Example 1. As a result of analyzing the intensity of the color reaction 10 minutes after sample injection, it was confirmed that the color reaction signal of human serum with a creatinine level of more than 1.5 mg / dl (Abnormal) increased compared to human serum with a creatinine level of 1.5 mg / dl or less (Normal) (see Table 1, Table 2, and Fig. 3).

[0087] Normal (≤ 1.5 mg / dl)No.R1R2R2-R1No.R1R2R2-R1N151,26351,33875N2050,45550,820365N250,41550,903488N 2151,01751,173157N351,08151,13049N2250,78551,208423N451,23251,29058N2350,80950,9851 76N550,27150,755484N2451,60852,201593N650,50351,070567N2550,72951,158429N750,63851 ,093455N2650,40850,43223N850,52350,997474N2753,48953,956467N951,27651,33457N2852,28 952,872583N1051,22551,352127N2952,92953,543614N1150,82950,92191N3052,49752,882385N 1250,24450,28239N3152,28852,797508N1351,45951,51859N3252,63952,968329N1451,46151,88 6425N3352,50652,715209N1550,45150,992540N3452,58452,902318N1650,21050,367158N3551, 79252,465673N1750,10850,275167N3652,20252,24543N1850,80550,920115N1951,04351,265222

[0088] Abnormal (> 1.5 mg / dl)No.R1R2R2-R1No.R1R2R2-R1C149,95751,9501,993C2048,61049,458848C250,61352,0721,460C2151,548 52,9861,438C350,66152,1001,439C2250,67052,9032,233C450,38552,0491,663C2350,72752,6671,940C550,9 4253,9923,050C2449,82250,519697C651,39554,4693,074C2549,92550,582657C750,89151,748857C2649,6075 1,3151,708C850,81351,607794C2750,60451,8001,195C949,98751,5241,537C2849,43850,424986C1050,57652 ,3411,766C2952,18754,0401,852C1147,72548,8861,162C3054,96856,5141,545C1247,07647,998922C3152,84 056,4523,612C1349,00149,619618C3251,57353,2951,722C1448,04548,970924C3351,36154,0992,738C1547,8 4950,2482,399C3451,92657,4485,522C1651,10252,2021,101C3550,78853,8293,040C1747,92450,1882,264C3 651,98457,6665,682C1849,28551,7862,501C3751,17056,6675,497C1949,12949,918788C3851,65156,3964,745

[0089]

[0090] Experimental Example 3: Creatinine Selectivity Analysis

[0091] After preparing the creatinine sensor strip of the above Manufacturing Example 1, each interference substance was completely dissolved in distilled water (DW) and prepared at concentrations of 4 mg / dl creatinine, 4 mg / dl creatine, 10 mg / dl ascorbic acid, 100 mg / dl glucose, 10 mg / dl glycine, and 10 mg / dl urea, and 15 μl of each substance was injected onto the sample pad. As a result of analyzing the intensity of the color reaction 10 minutes after sample injection, it was confirmed that there was almost no color reaction signal for substances other than creatinine (Fig. 4).

[0092]

[0093] Manufacturing Example 2: Manufacturing of a creatinine sensor strip according to ascorbate oxidase concentration

[0094] Manufacturing Example 2-1

[0095] The same procedure as in Manufacturing Example 1 was performed, but ascorbate oxidase was not added to the sample pad.

[0096] Manufacturing Example 2-2

[0097] The same procedure as in Manufacturing Example 1 was performed, but 5 Units / 10 μl of ascorbate oxidase was added to the sample pad.

[0098] Manufacturing Example 2-3

[0099] The same procedure as in Manufacturing Example 1 was performed, but 20 Units / 10 μl of ascorbate oxidase was added to the sample pad.

[0100] Manufacturing Example 2-4

[0101] The same procedure as in Manufacturing Example 1 was performed, but 50 Units / 10 μl of ascorbate oxidase was added to the sample pad.

[0102]

[0103] Experimental Example 4: Analysis of color reaction signals according to ascorbate oxidase concentration

[0104] After preparing the creatinine sensor strips of the above Manufacturing Example 1 and Manufacturing Examples 2-1 to 2-4, Sigma's creatinine was completely dissolved in distilled water (DW), diluted in Bio-Rad's Human serum based control level 1 (creatinine 0.8 mg / dl), and 15 μl of 4 mg / dl creatinine was injected onto the sample pad. As a result of analyzing the color reaction signal 10 minutes after sample injection, it was confirmed that the color reaction signal intensity of the creatinine sensor strip of Manufacturing Example 1, in which the ascorbate oxidase concentration was 10 Unit / 10 μl, was the best (Fig. 5).

[0105]

[0106] Experimental Example 5: Analysis of color reaction signals according to the presence or absence of ascorbate oxidase.

[0107] After preparing the creatinine sensor strips of the above Manufacturing Example 1 and Manufacturing Example 2-1, Sigma's creatinine and ascorbic acid were completely dissolved in distilled water (DW) and then diluted in Bio-Rad's Human serum based control level 1 (creatinine 0.8 mg / dl) to prepare concentrations of 4 mg / dl creatinine and 0, 0.5, 1, 5, 10, 50, and 100 mg / dl ascorbic acid. 15 μl of a solution containing various concentrations of ascorbic acid and 4 mg / dl creatinine was injected onto the sample pad. As a result of analyzing the color reaction signal 10 minutes after sample injection, it was confirmed that the sample pad with ascorbate oxidase added maintained a stable color reaction signal intensity regardless of the ascorbic acid concentration, but the sample pad without ascorbate oxidase added showed a rapid decrease in the color reaction signal intensity as the ascorbic acid concentration increased (Fig. 6).

[0108]

[0109] Manufacturing Example 3: Creatinine sensor strip production according to PBS (pH 7.4) concentration

[0110] Manufacturing Example 3-1

[0111] The same procedure as in Manufacturing Example 1 was followed, but ascorbate oxidase was completely dissolved in 2X PBS (pH 7.4) and used.

[0112] Manufacturing Example 3-2

[0113] The same procedure as in Manufacturing Example 1 was followed, but ascorbate oxidase was completely dissolved in 5X PBS (pH 7.4) and used.

[0114]

[0115] Experimental Example 6: Analysis of color reaction signals according to PBS (pH 7.4) concentration

[0116] After preparing the creatinine sensor strips of the above Manufacturing Example 1 and Manufacturing Examples 3-1 to 3-2, Sigma's creatinine was completely dissolved in distilled water (DW), diluted in Bio-Rad's Human serum based control level 1 (creatinine 0.8 mg / dl), and 15 μl of 4 mg / dl creatinine was injected onto the sample pad. As a result of analyzing the color reaction signal 10 minutes after sample injection, it was confirmed that the color reaction signal intensity of the creatinine sensor strip of Manufacturing Example 1 with a 1X concentration of PBS (pH 7.4) was the best (Fig. 7).

[0117] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. Creatinine detection strip containing: (a) a sample pad to which a biological sample to be analyzed is applied; (b) a first reaction pad placed in contact with one end of the sample pad and containing a preparation capable of detecting creatine; and (c) A second reaction pad, which is placed in contact with one end of the sample pad and includes a preparation capable of detecting creatine and a preparation capable of detecting creatinine.

2. In paragraph 1, A creatinine detection strip characterized in that the agent capable of detecting creatine included in the first reaction pad comprises creatinase, sarcosine oxidase, and HRP (Horseradish Peroxidase).

3. In paragraph 1, A creatinine detection strip characterized in that the agent capable of detecting creatinine contained in the second reaction pad comprises creatininase, creatinase, sarcosine oxidase, and HRP (Horseradish Peroxidase).

4. In paragraph 2 or paragraph 3, A strip for detecting creatinine, characterized in that the first reaction pad or the second reaction pad additionally contains 4-AAP (4-aminoantipyrine) and TOPS (N-ethyl-N-(3-sulfopropyl)-3-methylaniline).

5. In paragraph 1, A strip for detecting creatinine, characterized in that the sample pad contains ascorbate oxidase.

6. In paragraph 5, A strip for detecting creatinine, characterized in that the above ascorbate oxidase is contained in a concentration of 800 to 3000 Unit / ml.

7. In paragraph 5, A strip for detecting creatinine, characterized in that the above ascorbate oxidase is contained at a concentration of 800 to 2000 Unit / ml.

8. A strip for detecting creatinine, characterized in that the sample pad in the first paragraph; and the first reaction pad and the second reaction pad are arranged to overlap each other at the contact portion.

9. In paragraph 1, A strip for detecting creatinine, characterized in that the strip additionally includes a support for fixing the sample pad, the first reaction pad, and the second reaction pad as a length member.

10. In paragraph 1, A strip for detecting creatinine, characterized in that the biological sample is urine, whole blood, serum or plasma. 11.(a) A step of loading a biological sample into a sample pad in a creatinine detection strip of claim 1; (b) a step of measuring the color development intensity of the first reaction pad and the second reaction pad; (c) A method for measuring creatinine concentration in a biological sample, comprising the step of subtracting the color development intensity of the first reaction pad from the color development intensity of the second reaction pad.

12. In paragraph 10, A method characterized in that the biological sample is urine, whole blood, serum or plasma.

13. To detect creatinine, Application portion to which the biological sample to be analyzed is applied; A first reaction unit comprising a preparation capable of detecting creatine from the sample application unit; and A detection device comprising a second reaction unit comprising a preparation capable of detecting creatine from the sample application unit and a preparation capable of detecting creatinine.

14. In paragraph 13, A detection device comprising a preparation capable of detecting creatine in the first reaction section, comprising creatine hydrolase, sarcosine oxidase, and HRP (Horseradish Peroxidase).

15. In paragraph 13, A detection device comprising a preparation capable of detecting creatinine in the second reaction section, comprising creatinine hydrolase, creatinase, sarcosine oxidase, and HRP (Horseradish Peroxidase).

16. In paragraph 13, A detection device, wherein the above application portion includes ascorbate oxidase.

17. In paragraph 16, A detection device wherein the above ascorbate oxidase is included at a concentration of 800 to 2000 Unit / ml.

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