阅读说明：本技术 基于血红蛋白与四碳或五碳二元酸复合物试剂盒制备方法 (Preparation method of kit based on hemoglobin and four-carbon or five-carbon dicarboxylic acid compound ) 是由 黄昊文 花欣怡 汪志芳 谢潇雪 阳秀梅 于 2019-11-05 设计创作，主要内容包括：本发明公开了一种基于血红蛋白与四碳或五碳二元酸复合物试剂盒制备方法。本发明的技术要点是,将四碳二元酸或五碳二元酸与血液样品混合,四碳二元酸或五碳二元酸与血红蛋白作用生成具有过氧化物酶性质的复合物；向前述体系中加入双氧水和四甲基联苯胺,溶液变蓝,根据血液中血红蛋白浓度不同,TMB颜色深浅也不一样,再根据TMB在波长652nm处吸光度值与血红蛋白浓度的关系,能计算得到待测血液样品中血红蛋白的含量；向体系中加入葡萄糖氧化酶和TMB溶液,根据血液中葡萄糖浓度不同,TMB显色程度不同,根据氧化态TMB在波长652nm处吸光度值与葡萄糖浓度的关系,能计算得到待测血液样品中葡萄糖的含量。(The invention discloses a preparation method of a kit based on a hemoglobin and four-carbon or five-carbon dicarboxylic acid compound. The technical key points of the invention are that four-carbon dibasic acid or five-carbon dibasic acid is mixed with a blood sample, and the four-carbon dibasic acid or the five-carbon dibasic acid and hemoglobin react to generate a compound with peroxidase property; adding hydrogen peroxide and tetramethyl benzidine into the system, changing the solution into blue, changing the color depth of TMB according to different hemoglobin concentrations in blood, and calculating the content of hemoglobin in the blood sample to be detected according to the relation between the absorbance value of TMB at the wavelength of 652nm and the hemoglobin concentration; adding glucose oxidase and TMB solution into the system, calculating to obtain the content of glucose in the blood sample to be detected according to the relation between the absorbance value of the oxidation state TMB at the position of 652nm of wavelength and the glucose concentration according to the difference of the glucose concentration in the blood and the different color development degree of the TMB.)

1. A preparation method of a kit based on hemoglobin and four-carbon or five-carbon dibasic acid compound is characterized by comprising the following steps:

(1) mixing four-carbon dibasic acid or five-carbon dibasic acid with different blood samples, and allowing the four-carbon dibasic acid or the five-carbon dibasic acid to react with hemoglobin to generate a compound with peroxidase property;

(2) adding hydrogen peroxide and tetramethylbenzidine (TMB solution) into the system in the step (1), changing the solution into blue, wherein the color of the TMB is different according to the difference of the hemoglobin concentration in blood, and then calculating to obtain the content of the hemoglobin in the blood sample to be detected according to the relation between the absorbance value of the TMB at the wavelength of 652nm and the hemoglobin concentration; the limitation of time limitation and temperature on the blood sample to be detected is avoided when the hemoglobin is detected;

(3) and (2) adding glucose oxidase and TMB solution into the system in the step (1), wherein the content of hydrogen peroxide catalytically decomposed by hemoglobin and four-carbon or five-carbon dicarboxylic acid complex is different according to the difference of the glucose concentration in blood, the color development degree of TMB is different, and the content of glucose in the blood sample to be detected can be calculated according to the relation between the absorbance value of the oxidation state TMB at the position of 652nm of wavelength and the glucose concentration.

2. The method for preparing a kit based on hemoglobin and four-or five-carbon dicarboxylic acid complex according to claim 1, wherein: in the step (1), the four-carbon dibasic acid or five-carbon dibasic acid includes tartaric acid, succinic acid, glutaric acid and glutamic acid.

3. The method for preparing a kit based on hemoglobin and four-or five-carbon dicarboxylic acid complex according to claim 1, wherein: in the step (2), the concentration of hydrogen peroxide is 2.5mmol/L, the concentration of TMB is 5mmol/L, and the volume ratio of the two solutions is 1: 1.

4. the method for preparing a kit based on hemoglobin and four-or five-carbon dicarboxylic acid complex according to claim 1, wherein: in the step (2), the limitation on the blood sample to be detected in the hemoglobin detection process and the temperature are not limited, and the blood sample to be detected is a fresh liquid blood sample, or a dry blood stain stored indoors and outdoors at normal temperature for less than one year, or a blood stain heated at high temperature for two hours in the hemoglobin content determination process.

Technical Field

The invention belongs to the technical field of chemical and biological sensing and biological detection, and particularly relates to a preparation method of a hemoglobin-four-carbon and five-carbon dicarboxylic acid compound-based kit.

Background

Hemoglobin is a major component of red blood cells and is capable of binding oxygen, transporting oxygen and carbon dioxide. The hemoglobin content reflects the degree of anemia well. The hemoglobin increase and decrease has great reference value for clinical detection of certain diseases, such as pathological increase of hemoglobin, which is commonly seen in severe congenital and acquired cardiopulmonary diseases and vascular malformation, such as tetrad of French, cyanotic congenital heart disease, obstructive pulmonary emphysema, pulmonary heart disease, pulmonary arterial fistula or pulmonary venous fistula, and abnormal hemoglobin diseases with low oxygen carrying capacity; also in certain tumors or kidney diseases, such as renal cancer, hepatocellular carcinoma, nephroblastoma and hydronephrosis, polycystic kidney, etc.; pathological reduction, commonly seen in hematopoietic failure of bone marrow, such as aplastic anemia, anemia associated with myelofibrosis; anemia due to hematopoietic deficiency or impaired utilization, such as iron deficiency anemia, and megaloblastic anemia due to folic acid and vitamin B12 deficiency; anemia caused by excessive destruction of erythrocytes due to genetic defects of erythrocyte membranes, enzymes or external factors, such as hereditary spherocytosis, marine anemia, paroxysmal nocturnal hemoglobinuria, abnormal hemoglobinopathy, immune hemolytic anemia, hemolytic anemia caused by major surgery of cardiac extracorporeal circulation or certain biological and chemical factors, and anemia caused by certain acute or chronic blood loss. Therefore, the detection of the content of hemoglobin in blood has important scientific significance.

Currently, clinical hemoglobin detection is generally determined by a colorimetric method, which includes: methemoglobin cyanide (HiCN) assay, sodium dodecyl sulfate hemoglobin (SDS) assay, methemoglobin azide (HiN) ₃) The method, the alkali-hemoglobin method, the bromohexadecyltrimethylamine (CTAB) hemoglobin measurement method, and the like. These methods all require a freshly collected blood sample for detection, and some have the problems of toxicity, public nuisance, complex operation and low accuracy, so that a reagent which is simple and easy to obtain, non-toxic and non-public nuisance, simple and convenient in determination method and low in accuracy is requiredHigh accuracy hemoglobin measurement method.

Disclosure of Invention

The invention aims to provide a preparation method of a kit based on hemoglobin and four-carbon or five-carbon dicarboxylic acid compound, which is simple, convenient, green, safe and low in cost, and can be used for detecting hemoglobin and blood sugar.

The above object of the present invention is achieved by the following technical solutions: the preparation method of the kit based on the hemoglobin and the four-carbon or five-carbon dicarboxylic acid compound comprises the following steps:

(1) mixing four-carbon dibasic acid or five-carbon dibasic acid with different blood samples, and allowing the four-carbon dibasic acid or the five-carbon dibasic acid to react with hemoglobin to generate a compound with peroxidase property;

(2) adding hydrogen peroxide and tetramethylbenzidine (TMB solution) into the system in the step (1), changing the solution into blue, wherein the color of the TMB is different according to the difference of the hemoglobin concentration in blood, and then calculating to obtain the content of the hemoglobin in the blood sample to be detected according to the relation between the absorbance value of the TMB at the wavelength of 652nm and the hemoglobin concentration; the limitation of time limitation and temperature on the blood sample to be detected is avoided when the hemoglobin is detected;

(3) and (2) adding glucose oxidase and TMB solution into the system in the step (1), wherein the content of hydrogen peroxide catalytically decomposed by hemoglobin and four-carbon or five-carbon dicarboxylic acid complex is different according to the difference of the glucose concentration in blood, the color development degree of TMB is different, and the content of glucose in the blood sample to be detected can be calculated according to the relation between the absorbance value of the oxidation state TMB at the position of 652nm of wavelength and the glucose concentration.

Specifically, in the step (1), the four-carbon dicarboxylic acid or five-carbon dicarboxylic acid includes tartaric acid, succinic acid, glutaric acid and glutamic acid.

Specifically, in the step (2), the concentration of hydrogen peroxide is 2.5mmol/L, the concentration of TMB is 5mmol/L, and the volume ratio of the two solutions is 1: 1.

specifically, in the step (2), the blood sample to be measured is not limited in time and temperature during hemoglobin measurement, and the blood sample to be measured is either a fresh liquid blood sample, or a dried blood stain stored indoors and outdoors at normal temperature for less than one year, or a blood stain heated at high temperature for two hours during hemoglobin measurement.

The invention utilizes the characteristic that hemoglobin-four-carbon and five-carbon dicarboxylic acid compound catalytically decomposes hydrogen peroxide, and then achieves the aim of detecting hemoglobin or glucose according to the relation between the TMB color development degree and the concentration of the hemoglobin or the hydrogen peroxide; the high-sensitivity technology for detecting the hemoglobin and the glucose is successfully prepared, has obvious phenomenon, simple and convenient operation, high sensitivity, greenness, safety and low cost, and has no requirement on the storage condition and time of the blood.

The invention is a high sensitivity technology for detecting hemoglobin, which can replace biological enzyme in enzyme-linked immunosorbent reaction in a kit. The four-carbon and five-carbon dibasic acid comprises tartaric acid, succinic acid, glutaric acid, glutamic acid and the like, is low in price, has incomparable advantages of biological enzyme macromolecules in the aspects of price, stability and the like, and more importantly, the hemoglobin-four-carbon and five-carbon dibasic acid compound does not have the problem that biological protease easily loses biological activity, can be placed for a long time and still keeps the activity, and obviously improves the stability and the long time property of detection. On the other hand, different colors are shown in different states of the introduced TMB, and visual detection is established.

TMB solutions with different colors can be obtained by the treatment of the method, the color change can be regulated according to the concentration of hydrogen peroxide, and the ultra-trace detection of hemoglobin or glucose can be realized through the change of the color or the absorbance value. Compared with the prior art, the method is simple, low in cost, high in sensitivity and strong in stability, and can realize rapid and accurate detection; and the blood can be detected after being placed for a long time, and the sample can quickly react to generate a compound with strong peroxidase property no matter the sample is fresh blood or blood placed for a long time or dry blood stain. Based on the advantages, the hemoglobin in fresh blood can be quickly detected, trace detection can be carried out on long-time blood records, and the positions and the trends of blood traces on the blood records can be quickly positioned. The method can quantitatively detect the content of hemoglobin or glucose, can also qualitatively detect blood traces, and has wide application prospect.

Drawings

FIG. 1 is a circular dichroism chart of hemoglobin in different states.

FIG. 2 is a color picture demonstrating the peroxidase property of hemoglobin-tartaric acid; wherein, the test tube a is developed by tartaric acid-hemoglobin, and the test tube b is developed by hemoglobin.

FIG. 3 is the ultraviolet absorption spectrum at 652nm of ox-TMB generated by oxidation of TMB with hydrogen peroxide catalyzed by hemoglobin-tartaric acid complex.

FIG. 4 is a diagram illustrating the effect of different external conditions on hemoglobin; wherein, the test tubes from left to right respectively show the color development of the hemoglobin and the tartaric acid compound after water bath for 1h at 25 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃, 90 ℃ and 100 ℃.

FIG. 5 is an ultraviolet absorption spectrum at 652nm of ox-TMB produced by oxidation of TMB with hydrogen peroxide catalyzed by a compound produced by reacting hemoglobin with tartaric acid in water bath at 25 deg.C, 40 deg.C, 50 deg.C, 60 deg.C, 70 deg.C, 80 deg.C, 90 deg.C and 100 deg.C for 1 h.

Fig. 6 is at i and an enlarged view in fig. 5.

FIG. 7 is a graph of the stability of peroxidase-like properties of hemoglobin-tartaric acid complexes.

FIG. 8 is a graph of the linear equation for hemoglobin concentration versus the absorbance of ox-TMB at 652 nm. I

FIG. 9 is a graph of the linear equation of blood glucose concentration versus the absorbance of ox-TMB at 652 nm.

Detailed Description

The following are specific examples of the present invention. Unless otherwise specified, all the medicines and instruments used are conventional chemical laboratory medicines and instruments.