Urine oxalic acid content detection system and detection method
阅读说明：本技术 尿液草酸含量检测体系及检测方法 (Urine oxalic acid content detection system and detection method ) 是由 黄性义 于 2021-08-13 设计创作，主要内容包括：本发明公开了尿液草酸含量检测体系及检测方法,检测体系包括预处理试剂盒和检测试剂盒；预处理试剂盒包括：活性炭和样本处理液,检测试剂盒包括R1试剂和R2试剂；所述R1试剂包括以下组分：柠檬酸、DMAB和MBTH；R2试剂包括以下组分：pH值为5.6的缓冲液、草酸氧化酶和过氧化物酶；所述缓冲液在R2试剂中的浓度为0.1M,所述草酸氧化酶在R2试剂中的浓度大于50U/L,所述过氧化物酶在R2试剂中的浓度大于5000U/L；所述R2试剂为干粉状态,使用时采用水复溶。本发明具有较高的检测限,能够用于尿液中草酸含量的检测,同时具有较高的检测灵敏度,且能够排除尿液中维C等物质的干扰,使检测结果更加准确。(The invention discloses a system and a method for detecting the oxalic acid content in urine, wherein the detection system comprises a pretreatment kit and a detection kit; the pretreatment kit comprises: activated carbon and a sample processing solution, wherein the detection kit comprises an R1 reagent and an R2 reagent; the R1 reagent comprises the following components: citric acid, DMAB, and MBTH; the R2 reagent includes the following components: buffer solution with pH value of 5.6, oxalate oxidase and peroxidase; the concentration of the buffer solution in the R2 reagent is 0.1M, the concentration of the oxalate oxidase in the R2 reagent is more than 50U/L, and the concentration of the peroxidase in the R2 reagent is more than 5000U/L; the R2 reagent is in a dry powder state and is redissolved by water when in use. The invention has higher detection limit, can be used for detecting the content of oxalic acid in urine, has higher detection sensitivity, and can eliminate the interference of substances such as vitamin C and the like in the urine, so that the detection result is more accurate.)
1. The urine oxalic acid content detection system is characterized by comprising a pretreatment kit and a detection kit;
the pretreatment kit comprises:
a sample processing tube and a sample processing fluid, the sample processing tube and the sample processing fluid comprising the following components:
a sample processing tube: 0.04-1.5g of activated carbon
Sample treatment liquid: buffer, EDTA and proclin300, pH 5.7-8.0;
the detection kit comprises an R1 reagent and an R2 reagent;
the R1 reagent comprises the following components:
citric acid, DMAB and MBTH, wherein the concentration of the citric acid in the R1 reagent is 0.01-1M, and the concentration of the DMAB in the R1 reagent is 0.1-0.5 mM; the concentration of the MBTH in the R1 reagent is 0.01-0.5 mM;
the R2 reagent includes the following components:
buffer solution with pH value of 3.6-6, oxalate oxidase and peroxidase; the concentration of the buffer solution in the R2 reagent is 0.01-0.5M, the concentration of the oxalate oxidase in the R2 reagent is greater than or equal to 50U/L, and the concentration of the peroxidase in the R2 reagent is greater than or equal to 5000U/L;
the R2 reagent is in a dry powder state and is redissolved by water when in use.
2. The system for detecting the oxalic acid content in urine according to claim 1, wherein the concentration of the buffer solution with pH of 5.7-8.0 in the treating solution is 0.1M, the concentration of EDTA is 1-10Mm, and the mass fraction of proclin300 is 0.01-0.2%.
3. The system for detecting oxalic acid content in urine according to claim 1, wherein the weight of the activated carbon is 0.07-0.5 g.
4. The urine oxalic acid content detection system according to claim 1, wherein the buffer solution of the R2 reagent is a citrate buffer solution, and the citrate buffer solution is obtained by mixing 0.1M citric acid and 0.1M sodium citrate according to a volume ratio of 11: 29.
5. The system for detecting the oxalic acid content in urine as claimed in claim 1, wherein the concentration of citric acid in the R1 reagent is 0.05-0.3M, and the concentration of DMAB in the R1 reagent is 0.15-0.3 mM; the concentration of MBTH in the R1 reagent is 0.09-0.3 mM.
6. The system for detecting the content of oxalic acid in urine as claimed in claim 1, wherein the concentration of the buffer solution in the R2 reagent is 0.05-0.2M, the concentration of the oxalate oxidase in the R2 reagent is 50-1000U/L, and the concentration of the peroxidase in the R2 reagent is 5000-20000U/L.
7. The system for detecting oxalic acid content in urine according to claim 1, wherein the detection kit further comprises an oxalic acid standard.
8. The detection method of the urine oxalic acid content detection system based on any one of claims 1 to 7, characterized by comprising the following steps:
s1, preprocessing:
adding the urine sample and the sample treatment solution into a test tube according to a certain proportion, mixing, adding the mixed solution into the sample treatment tube, performing centrifugal separation, and reserving supernatant to be tested;
s2, detection:
the supernatant obtained in step S1, and the R1 reagent and the R2 reagent were placed at the corresponding positions of the instrument, and the instrument was started to measure directly.
9. The method for detecting the oxalic acid content in urine according to claim 8, wherein in step S1, the ratio of the urine sample to the sample treatment solution is 1: 1.
10. The method for detecting the urine oxalic acid content detection system according to claim 8, wherein the concentration of the buffer solution with the pH of 5.7-8.0 in the sample treatment solution is 0.1M, the concentration of EDTA is 1-10Mm, the mass fraction of proclin300 is 0.01-0.2%, and the mass fraction of activated carbon is 0.04-1.5 g.
Technical Field
The invention relates to the technical field of detection of oxalic acid content in urine, in particular to a system and a method for detecting the oxalic acid content in urine.
Background
Oxalic acid has a strong chelating effect on calcium, so that the absorption and metabolism of calcium by a human body can be influenced. Firstly, it can form a stable water-insoluble chelate with dietary calcium, making it difficult for calcium to enter the human environment and be absorbed by the human body. And secondly, oxalic acid generated by a human body through food intake and metabolism can form precipitates with calcium in the body and accumulate in glomerulus, which is the main cause of urinary calculus, the concentration of oxalic acid in urine is closely related to urinary calculus, and the simple and accurate detection of the content of the urinary oxalic acid has important significance for the basic research and clinical treatment of the urinary calculus. The oxalic acid determination method mainly comprises a titration method, a colorimetric method, a High Pressure Liquid Chromatography (HPLC), an ion chromatography method, a capillary electrophoresis method (CE), an enzyme method and the like.
The titration method comprises the steps of selecting a certain titrant to titrate the oxalic acid extracting solution, indicating a titration end point by potential jump or indicator color change, and calculating the content of oxalic acid by the consumption of a titration solution. The titration method is still widely applied to food analysis as a classical method, but the sensitivity is low, and the operation is more complicated and time-consuming; the colorimetric method is to extract oxalic acid by pre-precipitation, dissolve the oxalic acid by acid, oxidize or reduce the oxalic acid, or directly react the oxalic acid with a certain color developing agent to generate a colored substance, and quantify the substance by colorimetry. The colorimetric method is cheap and has high sensitivity, but the pre-precipitation operation is difficult to completely extract the oxalic acid, the operation is complicated and the repeatability is poor. Ion Chromatography (IC) is an ion qualitative and quantitative analysis technique for ion separation by means of differences in mobility of ions on ion exchange columns or on membranes impregnated with an ion exchanger; the Capillary Electrophoresis (CE) analysis method has high voltage and high flow rate, reduces the retention time of solute in the capillary, reduces zone expansion caused by molecular diffusion, and breakthroughs the bottleneck of analyzing small molecular ions, thus being suitable for micro-detection; the ion chromatography and the capillary electrophoresis have high sensitivity and simple and convenient operation, but the instrument is not popularized yet, is only suitable for micro detection and is not suitable for constant detection. High Performance Liquid Chromatography (HPLC) uses o-phenylenediamine as a derivatization agent, can generate a compound 2, 3-dihydroxyquinoxaline with strong ultraviolet absorption after reacting with oxalic acid in blood and urine, and can detect the concentration of oxalic acid by separating through a reversed phase C18 column. However, the high pressure liquid chromatography has serious interference of inorganic acid in the separation process, expensive instruments, high requirement on operators and difficult popularization, and is suitable for being used in professional departments.
Disclosure of Invention
The invention aims to provide a urine oxalic acid content detection system which has higher detection limit, can be used for detecting the oxalic acid content in urine, has higher detection sensitivity, can eliminate the interference of substances such as vitamin C and the like in the urine and enables the detection result to be more accurate.
In addition, the invention also provides a detection method based on the system for detecting the content of oxalic acid in urine.
The invention is realized by the following technical scheme:
the urine oxalic acid content detection system comprises a pretreatment kit and a detection kit;
the pretreatment kit comprises:
a sample processing tube and a sample processing fluid, the sample processing tube and the sample processing fluid comprising the following components:
a sample processing tube: 0.04-1.5g of activated carbon
Sample treatment liquid: buffer, EDTA and proclin300, pH 5.7-8.0;
the detection kit comprises an R1 reagent and an R2 reagent;
the R1 reagent comprises the following components:
citric acid, DMAB and MBTH, wherein the concentration of the citric acid in the R1 reagent is 0.01-1M, and the concentration of the DMAB in the R1 reagent is 0.1-0.5 mM; the concentration of the MBTH in the R1 reagent is 0.01-0.5 mM;
the R2 reagent includes the following components:
buffer solution with pH value of 3.6-6, oxalate oxidase and peroxidase; the concentration of the buffer solution in the R2 reagent is 0.01-0.5M, the concentration of the oxalate oxidase in the R2 reagent is greater than or equal to 50U/L, and the concentration of the peroxidase in the R2 reagent is greater than or equal to 5000U/L;
the R2 reagent is in a dry powder state and is redissolved by water when in use.
The R1 reagent is colorless transparent liquid, the R2 reagent is white dry powder after being freeze-dried, and the R3578 reagent is colorless or light yellow transparent liquid after being redissolved.
The detection principle of the invention is as follows:
the production of colored complexes by the coupling reaction described below of oxalate in urine can be measured with automated instrumentation;
the core components of the kit are oxalate oxidase and peroxidase, but the most suitable pH values of the oxalate oxidase and the peroxidase are different, the invention ensures higher activity of the two enzymes by reasonably designing the formula of the R2 reagent, thereby achieving the characteristic of high sensitivity, and simultaneously, the whole kit has high sensitivity and high detection limit and can be suitable for detecting oxalic acid in urine by reasonably designing the formula of the R1 reagent.
This application is because having higher linearity, for 180mg/L, when detecting oxalic acid content in the urine, need not to dilute the sample just can directly measure.
In the existing oxalic acid measurement, the detection limit of most detection kits is low, basically 9-45mg/L, and only suitable for detecting the oxalic acid content in blood, when the detection kit is used for detecting the oxalic acid content in urine, a sample needs to be diluted, so that the accuracy of a detection result is reduced.
Meanwhile, the detection system also comprises a pretreatment kit for pretreating the urine sample, wherein the pretreatment kit comprises a sample treatment tube and a sample treatment liquid, the sample treatment liquid is used for maintaining the pH value during pretreatment, and the sample treatment tube adsorbs most of interfering substances in the urine.
According to the invention, the urine sample is pretreated, so that the interference of other substances (such as vitamin C) in the urine on the detection of oxalic acid can be reduced to the greatest extent, the detection is further more stable, and the sensitivity is higher under the condition of ensuring a high linear range.
Furthermore, the concentration of the buffer solution with the pH value of 5.7-8.0 in the treatment solution is 0.1M, the concentration of EDTA is 1-10Mm, and the mass fraction of proclin300 is 0.01-0.2%.
Further, the weight of the activated carbon is 0.07-0.5 g.
Further, the buffer solution of the R2 reagent is a citrate buffer solution, and the citrate buffer solution is obtained by mixing 0.1M citric acid and 0.1M sodium citrate according to the volume ratio of 11: 29.
Further, the concentration of citric acid in the R1 reagent is 0.05-0.3M, and the concentration of DMAB in the R1 reagent is 0.15-0.3 mM; the concentration of MBTH in the R1 reagent is 0.09-0.3 mM.
Further, a buffer solution having a pH of 4 to 6, wherein the concentration of the buffer solution in the R2 reagent is 0.05 to 0.2M, the concentration of the oxalate oxidase in the R2 reagent is 50 to 1000U/L, and the concentration of the peroxidase in the R2 reagent is 5000-
Further, the detection kit also comprises an oxalic acid standard substance.
The detection method based on the urine oxalic acid content detection system comprises the following steps:
s1, preprocessing:
adding the urine sample and the sample treatment solution into a test tube according to a certain proportion, mixing, adding the mixed solution into the sample treatment tube, performing centrifugal separation, and reserving supernatant to be tested;
s2, detection:
the supernatant obtained in step S1, and the R1 reagent and the R2 reagent were placed at the corresponding positions of the instrument, and the instrument was started to measure directly.
The instrument is a BioSystems A400 full-automatic specific protein analyzer.
Further, in step S1, the ratio of the urine sample to the sample processing liquid is 1: 1.
Furthermore, the concentration of the buffer solution with the pH value of 5.7-8.0 in the processing solution is 0.1M, the concentration of EDTA is 1-10Mm, the mass fraction of proclin300 is 0.01-0.2%, and the weight of the activated carbon in the sample processing tube is 0.04-1.5 g.
Compared with the prior art, the invention has the following advantages and beneficial effects:
1. the oxalic acid detection kit of the invention ensures that the oxalate oxidase and the peroxidase both achieve higher activity under the condition of proper PH, thus improving the detection sensitivity.
2. The linear range of the oxalic acid detection kit measured by the invention is 1.8-180 mmol/L.
3. According to the invention, the urine sample is pretreated, so that the interference of other substances (such as vitamin C) in the urine on the detection of oxalic acid can be reduced to the greatest extent, the detection is further more stable, and the sensitivity is higher under the condition of ensuring a high linear range.
4. The detection system provided by the invention has the advantages that the automatic instrument operation is realized, the detection result is accurate, and the operation is more convenient and rapid.
Description of the drawings:
FIG. 1 is a line graph of example 1;
FIG. 2 is a line graph of example 2;
FIG. 3 is a line graph of example 3.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples and accompanying drawings, and the exemplary embodiments and descriptions thereof are only used for explaining the present invention and are not meant to limit the present invention.
Example 1:
the urine oxalic acid content detection system comprises a pretreatment kit and a detection kit;
the pretreatment kit comprises:
the pretreatment kit comprises:
a sample processing tube and a sample processing fluid, the sample processing fluid comprising the following components:
buffer, EDTA and proclin300, pH 5.7; the concentration of a buffer solution with the pH value of 5.7 in the sample treatment solution is 0.1M, the concentration of EDTA is 2Mm, and the mass fraction of proclin300 is 0.2%;
the weight of the activated carbon in the sample processing tube was 0.1 g;
the detection kit comprises an R1 reagent and an R2 reagent;
the R1 reagent comprises the following components:
citric acid at a concentration of 0.07M in R1 reagent, DMAB at a concentration of 0.18mM in R1 reagent, and MBTH; the concentration of the MBTH in the R1 reagent is 0.09 mM;
the R2 reagent includes the following components:
buffer solution with pH value of 5, oxalate oxidase and peroxidase; the concentration of the buffer solution in the R2 reagent is 0.06M, the concentration of the oxalate oxidase in the R2 reagent is 100U/L, and the concentration of the peroxidase in the R2 reagent is 5000U/L; the buffer solution is a citrate buffer solution, and the citrate buffer solution comprises 0.06M of citric acid and 0.06M of sodium citrate according to a volume ratio of 41: 59 mixing to obtain;
the R2 reagent is in a dry powder state and is redissolved by water when in use.
The detection method of the embodiment comprises the following steps:
s1, preprocessing:
adding the urine sample and the sample treatment solution into a test tube according to a certain proportion, mixing, adding the mixed solution into the sample treatment tube, performing centrifugal separation, and reserving supernatant to be tested;
s2, detection:
placing the supernatant obtained in the step S1, and the R1 reagent and the R2 reagent at corresponding positions of an instrument, and starting the instrument to directly measure, wherein the instrument adopts a BioSystems A400 full-automatic specific protein analyzer.
Example 2:
this example is based on example 1, differing from example 1 in the concentration of the individual components, in particular:
the concentration of a buffer solution with the pH of 7.8 in the sample treatment solution is 0.1M, the concentration of EDTA is 9Mm, and the mass fraction of proclin300 is 0.02%;
the weight of the activated carbon in the sample processing tube is 0.07 g;
the detection kit comprises an R1 reagent and an R2 reagent;
the R1 reagent comprises the following components:
citric acid at a concentration of 0.2M in R1 reagent, DMAB at a concentration of 0.29mM in R1 reagent, and MBTH; the concentration of the MBTH in the R1 reagent is 0.2 mM;
the R2 reagent includes the following components:
buffer solution with pH value of 5.8, oxalate oxidase and peroxidase; the concentration of the buffer solution in the R2 reagent is 0.2M, the concentration of the oxalate oxidase in the R2 reagent is 300U/L, and the concentration of the peroxidase in the R2 reagent is 10000U/L; the buffer solution is citrate buffer solution, and the citrate buffer solution is obtained by mixing 0.2M citric acid and 0.2M sodium citrate according to a volume ratio of 47:153
Example 3:
this example is based on example 1, differing from example 1 in the concentration of the individual components, in particular:
the concentration of a buffer solution with the pH value of 6.5 in the sample treatment solution is 0.1M, the concentration of EDTA is 3Mm, and the mass fraction of proclin300 is 0.1%;
the weight of the activated carbon is 0.5 g;
the detection kit comprises an R1 reagent and an R2 reagent;
the R1 reagent comprises the following components:
citric acid at a concentration of 0.15M in R1 reagent, DMAB at a concentration of 0.2mM in R1 reagent, and MBTH; the concentration of the MBTH in the R1 reagent is 0.15 mM;
the R2 reagent includes the following components:
buffer solution with pH value of 5, oxalate oxidase and peroxidase; the concentration of the buffer solution in the R2 reagent is 0.13M, the concentration of the oxalate oxidase in the R2 reagent is 200U/L, and the concentration of the peroxidase in the R2 reagent is 8000U/L; the buffer solution is a citrate buffer solution, and the citrate buffer solution comprises 0.13M of citric acid and 0.13M of sodium citrate according to a volume ratio of 41: 59 mixing to obtain;
comparative example 1:
this comparative example is based on example 1 and differs from example 1 in that the sample treatment liquid has a different pH, specifically a pH value of 2.
Comparative example 2:
this comparative example is based on example 1 and differs from example 1 in that the weight of activated carbon inside the sample treatment tube is different, no activated carbon being added.
Comparative example 3:
this comparative example is based on example 1 and differs from example 1 in that the weight of the activated carbon inside the sample treatment tube is different, in particular 2 g.
Comparative example 4:
this comparative example is based on example 1 and differs from example 1 in that the sample treatment solution has a different pH, specifically a pH of 10.
Accuracy, repeatability, interference tests were performed for examples 1-3, comparative examples 1-4:
1. accuracy data, 90mg/L oxalate solution was prepared and tested for accuracy, with the results shown in Table 1:
TABLE 1
Accuracy of
Example 1
Example 2
Example 3
Comparative example 1
Comparative example 2
Comparative example 3
Comparative example 4
1
90.34
89.44
92.15
13.55
81.31
78.60
17.16
2
89.76
88.86
90.32
8.90
78.09
78.09
24.24
3
87.67
86.79
89.70
11.22
78.03
76.27
22.79
4
91.23
91.73
87.77
4.56
82.11
79.37
12.77
5
90.61
89.29
92.15
9.90
81.55
78.83
9.97
6
88.66
88.52
91.56
15.65
79.79
77.13
16.85
7
89.93
89.03
89.42
15.45
80.94
78.24
9.89
8
87.54
86.66
87.63
12.34
78.79
76.16
13.13
9
86.78
85.91
89.87
13.02
78.10
75.50
16.49
10
89.97
88.52
89.07
14.54
80.07
78.27
10.80
Mean value of
89.25
88.48
89.96
11.91
79.88
77.65
15.41
Relative deviation of
-0.83％
-1.69％
-0.04％
-86.76％
-11.25％
-13.73％
-82.88％
SD
1.49
1.68
1.62
3.42
1.56
1.30
5.08
CV
1.67％
1.90％
1.80％
28.69％
1.95％
1.67％
32.97％
2. Linear data:
urine samples were applied, diluted, and the reagent linearity was verified, and the linearity data of examples 1 to 3 are shown in fig. 1 to 3, respectively.
As can be seen from figures 1-3:
the linear range of the oxalic acid detection kit is 1.8-180 mmol/L.
3. Repeatability data:
the data for the concentration (80mg/L) are shown in Table 2:
TABLE 2
Number of repetitions
Example 1
Example 2
Example 3
Comparative example 1
Comparative example 2
Comparative example 3
Comparative example 4
1
82.32
81.50
83.14
16.46
72.36
74.91
24.70
2
81.55
82.37
80.73
34.67
71.68
75.03
23.12
3
79.66
78.86
80.46
45.32
70.02
74.88
12.34
4
82.23
83.05
81.41
23.43
72.28
74.83
9.13
5
80.19
79.39
80.99
12.32
70.49
72.97
11.23
6
79.33
80.12
78.54
7.99
69.73
72.19
14.56
7
78.98
78.19
79.77
32.23
69.42
71.87
22.45
8
76.89
77.66
76.12
24.32
67.59
69.97
19.02
9
79.08
78.29
79.87
9.19
69.51
71.96
25.44
10
80.23
81.03
79.43
11.98
70.52
73.01
27.99
11
81.90
81.08
82.72
13.67
71.99
74.53
13.23
12
82.22
83.04
81.40
18.32
72.27
74.82
15.34
13
81.45
80.64
82.26
23.31
71.59
74.12
16.35
14
82.11
82.93
81.29
32.11
72.17
74.72
34.55
15
84.44
83.60
85.28
23.12
74.22
76.84
21.67
AVG
80.84
80.78
80.89
21.90
71.06
73.78
19.41
SD
1.88
1.98
2.13
10.66
1.65
1.74
7.10
CV
2.32％
2.45％
2.63％
48.66％
2.32％
2.36％
36.58％
The data for the concentration (10mg/L) are shown in Table 3:
TABLE 3
4. The interference data is shown in table 4:
TABLE 4
The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are merely exemplary embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalent substitutions, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.
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