A urea detection reagent

Abstract

The application discloses a urea detection reagent, which comprises the following components: a buffer, reduced coenzyme I, alpha-ketoglutaric acid, ADP, urease, glutamate dehydrogenase, an excipient, a stabilizer, a surfactant and a preservative. The urea detection reagent has good thermal stability, is not prone to deterioration during storage, has a relatively wide effective testing range of urea, and can test a maximum concentration of urea of about 35 mmol / L.

Description

Technical Field

[0001] This invention relates to the field of in vitro diagnostic reagents, and more specifically to a urea detection reagent. Background Technology

[0002] In the human body, amino acids are deaminated into α-keto acids and NH3. NH3 enters the urea cycle in liver cells and reacts with CO2 to form urea. Urea produced in the liver is mainly excreted by the kidneys via the bloodstream. Under normal circumstances, non-protein nitrogen in the blood is primarily filtered by the glomeruli and excreted in the urine. Non-protein nitrogen in the blood refers to nitrogenous compounds other than plasma proteins, such as urea, uric acid, creatine, creatinine, and amino acids, of which urea nitrogen accounts for approximately 50%. When the renal parenchyma is damaged, the glomerular filtration rate decreases, leading to an increase in the concentration of non-protein nitrogen in the blood. Therefore, measuring the level of non-protein nitrogen in the blood can provide a rough assessment of glomerular filtration function. In renal insufficiency, because the increase in urea concentration is faster and more pronounced than that of non-protein nitrogen, urea concentration is more sensitive to glomerular filtration function than non-protein nitrogen. Blood urea measurement is one of the main diagnostic tests for kidney disease.

[0003] Currently, the common method for determining urea is to first decompose urea into ammonia using urease in a urea assay reagent, then measure the amount of ammonia using different methods and convert it into the amount of urea. However, in implementing this method, the urea assay reagent has poor thermal stability, making it prone to deterioration during storage, and it also has a relatively narrow range of detectable urea levels. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a urea detection reagent to solve the problems of poor thermal stability and narrow measurement range in existing technologies.

[0005] To solve the above problems, the present invention adopts the following solution:

[0006] A urea detection reagent comprises the following components: buffer solution, reduced coenzyme I (NADH), α-ketoglutarate, ADP, urease, glutamate dehydrogenase, excipient, stabilizer, surfactant, and preservative.

[0007] Furthermore, the concentration of the buffer solution is 50-200 mmol / L;

[0008] The concentration of the reduced coenzyme I (NADH) is 1-20 mmol / L;

[0009] The concentration of the α-ketoglutaric acid is 10-100 mmol / L;

[0010] The concentration of ADP is 1-50 mmol / L;

[0011] The concentration of the urease is 10-100 KU / L;

[0012] The concentration of the glutamate dehydrogenase is 0.5-50 KU / L;

[0013] The concentration of the excipient is 10-100 g / L;

[0014] The concentration of the stabilizer is 1-10 g / L;

[0015] The concentration of the surfactant is 0.01-0.2 g / L;

[0016] The concentration of the preservative is 0.01-0.1 g / L.

[0017] Furthermore, the buffer solution includes at least one of Tris buffer, PBS buffer, imidazole buffer, and diethanol buffer.

[0018] Furthermore, the excipients include at least one of sorbitol, mannitol, PEG 6000 (polyethylene glycol 6000), PEG 8000 (polyethylene glycol 8000), dextran 10,000, dextran 40,000, and dextran 50,000.

[0019] Furthermore, the stabilizer includes at least one of bovine serum albumin, trehalose, and sucrose.

[0020] Furthermore, the surfactant includes at least one of Tween 20, Tween 80, Brij L23 (polyoxyethylene lauryl ether), Tritom X-100, SDS (sodium dodecyl sulfonate), and SDBS (sodium dodecylbenzene sulfonate);

[0021] Furthermore, the preservative includes at least one of Kathon, Proclin 300 (PC-300 biological preservative and antibacterial agent), triclocarban, and sodium azide.

[0022] The present invention adopts the above technical solution and has the following advantages:

[0023] The urea detection reagent of this invention has good thermal stability, is not easily deteriorated during storage, has a wide effective testing range for urea, and can test a maximum urea concentration of about 35 mmol / L. Attached Figure Description

[0024] Figure 1 The linear range graph of the detection reagents for experimental group 1 is shown.

[0025] Figure 2 The linear range graph of the detection reagents in experimental group 2 is shown.

[0026] Figure 3 The linear range graph of the detection reagents in experimental group 3 is shown.

[0027] Figure 4 Correlation diagram of reagents in experimental group 1;

[0028] Figure 5 Correlation diagram of the detection reagents in experimental group 2;

[0029] Figure 6 The correlation diagram of the detection reagents in experimental group 3 is shown. Detailed Implementation

[0030] The following examples further illustrate the present invention, but are not intended to limit the invention. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments. However, any modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

[0031] The detection reagent of this invention is prepared in the form of microspheres and placed inside a chip to form a detection chip. Urea is detected by the detection chip. The preparation method of the microsphere-form detection reagent is as follows:

[0032] The buffer solution, reduced coenzyme I (NADH), α-ketoglutarate, ADP, urease, glutamate dehydrogenase, excipients, stabilizers, surfactants and preservatives were mixed in proportion with water as solvent and adjusted to the preset pH value. The mixture was then dropped into liquid nitrogen to form ice balls, which were then freeze-dried to obtain the detection microspheres.

[0033] In an environment with an air humidity of 15-20%, the microspheres prepared by the detection reagent of this invention are loaded into the chip body of the microfluidic chip, the detection sample is injected into the microfluidic chip, and then the automatic biochemical analyzer is used to detect the change value of NADH absorbance at 37°C. The change value is positively correlated with the concentration of urea.

[0034] Specifically, test reagents for experimental groups 1-6 were prepared according to the components and contents shown in Table 1 below, and the components were prepared into microspheres for testing urea and performance evaluation.

[0035] Table 1. Detection reagents for different components

[0036]

[0037] The performance of the detection reagents prepared by this invention is described in conjunction with specific test experiments, including linear range, correlation, accuracy, precision and stability.

[0038] 1. Linear range test

[0039] The test method is as follows: a low-concentration sample close to the lower limit of the linear range (0.9-3.5 mmol / L) and a high-concentration sample close to the upper limit of the linear range (30-35 mmol / L) are mixed in a certain proportion to form 5 samples. The mixing ratio of each sample is shown in Table 2.

[0040] Table 2 shows the mixing ratio of high-concentration and low-concentration samples in the sample.

[0041] Sample number 1 2 3 4 5 High concentration samples 0 20％ 40％ 60％ 100％ low concentration sample 100％ 80％ 60％ 40％ 0

[0042] The urea concentration in five diluted plasma samples was tested using the detection reagents in experimental groups 1-3 of this invention. Each diluted plasma sample was tested three times, and the average urea concentration (yi) of the five diluted plasma samples was calculated. A linear regression equation was derived using the concentration (xi) of each diluted sample as the independent variable and the average concentration (yi) of each sample as the dependent variable, as follows: Figure 1-3 As shown, the upper limit of the linear range of the detection reagents in experimental groups 1-3 is close to 35mM, which is a significant improvement over the existing technology in terms of the effective detection range of urea.

[0043] 2. Correlation and accuracy tests

[0044] Fifteen plasma samples with different urea concentrations (low: 0.9-6 mmol / L, medium: 6-15 mmol / L, high: 15-35 mmol / L) were prepared. The urea concentration of each plasma sample in the 15 samples was detected using microfluidic chips prepared with reagents from experimental groups 1-6. The urea concentration of each plasma sample in the samples was detected using wet reagents from a Beckman AU480 biochemical analyzer as the target value. The deviation between the concentration measured by this method and the target value was calculated to analyze the accuracy of this method (absolute deviation was calculated for the low value range, and relative deviation was calculated for the medium and high value ranges). The detection results are shown in Table 3.

[0045] Table 3: Accuracy test results of experimental groups 1-6 of this invention

[0046]

[0047]

[0048] Compared with the wet reagent test results, the absolute deviation of low-value samples in experimental groups 1-6 of this invention was less than 1 mmol / L, and the relative deviation of medium- and high-value samples was less than 10%, indicating good accuracy of this method. Using the concentrations measured in experimental groups 1-3 as the independent variable and the urea concentration in each plasma sample detected by the Beckman AU480 biochemical analyzer wet reagent test as the dependent variable, a linear regression equation was derived, as follows: Figure 4-6 As shown, the correlation coefficients (r) are all greater than 0.99, indicating that this method has good correlation.

[0049] 3. Repeatability

[0050] Using the same batch of test reagents (experimental groups 1-6 of this invention), 10 repeated measurements were performed on low, medium, and high concentration plasma (low value 0.9-6 mmol / L, medium value 6-15 mmol / L, high value 15-35 mmol / L), and the average value of each measurement was calculated. The standard deviation (S) and the coefficient of variation (CV) within the batch were calculated, and the results are shown in Table 4.

[0051] Table 4: Repeatability test results for samples of different concentrations

[0052]

[0053]

[0054] As shown in Table 4, the detection reagents provided in experimental groups 1-6 of this invention showed good repeatability, with CV < 3% for high-value samples and CV < 8% for low-value samples.

[0055] 4. Stability

[0056] High-temperature stability: The microfluidic chips prepared with the test reagents from experimental groups 1-6 of this invention were sealed in bags and stored at 37°C in a light-protected environment for 0, 1, 3, 5, and 7 days in an environment with 15-20% humidity. The accuracy of the test reagents from experimental groups 1-6 of this invention was tested using low, medium, and high concentration plasma (low value 0.9-6 mmol / L, medium value 6-15 mmol / L, high value 15-35 mmol / L) as test samples. The relative deviation should be within ±10.0%. The results are shown in Table 5.

[0057] Table 5: Results of high-temperature stability testing of microspheres in experimental groups 1-6 of this invention

[0058]

[0059]

[0060] The detection reagents provided in experimental groups 1-6 of this invention, after being stored in a high-temperature (37°C) environment for 1, 3, 5 and 7 days, still showed an absolute decrease of less than 10%. Therefore, they have good thermal stability and can ensure the accuracy of their detection results even after being stored in a high-temperature environment for many days.

[0061] Low-temperature stability was tested by sealing the microfluidic chips prepared with the test reagents in experimental groups 1-6 of this invention in an environment with a humidity of 15-20% and storing them in a light-protected environment at 2-8℃ for 0, 3, 6, 12, and 18 months. The accuracy of the microfluidic chips in the experimental groups of this invention was tested using plasma of different concentrations as test samples. The relative deviation should be within ±10%, and the results are shown in Table 6.

[0062] Table 6: Results of Low-Temperature Stability Testing of Microspheres in Experimental Groups 1-6 of this Invention

[0063]

[0064] The detection reagents provided in experimental groups 1-6 of this invention, after being stored at 2-8°C for 0, 3, 6, 12 and 18 months, still showed an absolute decrease of less than 10%. Therefore, they have good real-time stability and can ensure the accuracy of their detection results even after long-term storage in the environment.

[0065] Existing urea detection reagents often suffer from excessively high initial absorbance due to the high concentration of reducing coenzyme, exceeding the instrument's detection range. These reagents typically exhibit narrow linear ranges, with a maximum detectable concentration of only 20 mmol / L. However, the urea detection reagent beads prepared using the method described in this invention have a wider effective detection range, demonstrating good detection performance for serum and plasma samples with urea concentrations ranging from 0.9 mmol / L to 35 mmol / L. Furthermore, the urea detection reagent beads provided by this invention exhibit good thermal stability and are not easily degraded during storage. When prepared as microspheres, the detection reagents of this invention possess excellent morphology and remelting solubility, along with good accuracy, a wide linear range, and high stability and precision.

[0066] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A urea detection reagent, characterized in that, It includes the following components: buffer solution, reduced coenzyme I, α-ketoglutarate, ADP, urease, glutamate dehydrogenase, excipients, stabilizers, surfactants, and preservatives.

2. The urea detection reagent according to claim 1, characterized in that, The concentration of the buffer solution is 50-200 mmol / L; The concentration of the reduced coenzyme I is 1-20 mmol / L; The concentration of the α-ketoglutaric acid is 10-100 mmol / L; The concentration of ADP is 1-50 mmol / L; The concentration of the urease is 10-100 KU / L; The concentration of the glutamate dehydrogenase is 0.5-50 KU / L; The concentration of the excipient is 10-100 g / L; The concentration of the stabilizer is 1-10 g / L; The concentration of the surfactant is 0.01-0.2 g / L; The concentration of the preservative is 0.01-0.1 g / L.

3. The urea detection reagent according to claim 1, characterized in that, The buffer solution includes at least one of Tris buffer, PBS buffer, imidazole buffer, and diethanol buffer.

4. The urea detection reagent according to claim 1, characterized in that, The excipients include at least one of sorbitol, mannitol, PEG 6000, PEG 8000, dextran 10,000, dextran 40,000, and dextran 50,000.

5. The urea detection reagent according to claim 1, characterized in that, The stabilizer includes at least one of bovine serum albumin, trehalose, and sucrose.

6. The urea detection reagent according to claim 1, characterized in that, The surfactant includes at least one of Tween20, Tween 80, Brij L23, Tritom X-100, SDS, and SDBS.

7. The urea detection reagent according to claim 1, characterized in that, The preservative includes at least one of Kathon, Proclin 300, triclocarban, and sodium azide.

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