阅读说明：本技术 一种二氧化碳酶法测定试剂盒 (Carbon dioxide enzyme method determination kit ) 是由 吴年芬 赵畅 梁艳 龚婷 凡速朋 舒芹 张雪娇 于 2020-06-16 设计创作，主要内容包括：本发明公开了一种二氧化碳酶法检测试剂盒,它含有磷酸烯醇丙酮酸、磷酸烯醇丙酮酸羧化酶、苹果酸脱氢酶、缓冲液、反应加速剂、防腐剂、表面活性剂、保护剂以及由葡萄糖、葡萄糖脱氢酶和氧化型3-乙酰吡啶腺嘌呤二核苷酸组成的循环再生系统,所述循环再生系统中各成分在试剂盒中的含量分别为：葡萄糖：1-10g/L；葡萄糖脱氢酶：50-1000U/L；氧化型3-乙酰吡啶腺嘌呤二核苷酸：0.3-0.6g/L。本发明通过对循环再生系统中各成分的比例和用量进行优化,进一步选择合适的表面活性剂、保护剂以及检测体系的pH条件,从而更好地控制了循环系统的反应速率,使其在提高试剂盒稳定性的基础上,同时提高了低浓度二氧化碳检测结果的准确性。(The invention discloses a carbon dioxide enzyme method detection kit, which contains phosphoenolpyruvate, phosphoenolpyruvate carboxylase, malate dehydrogenase, buffer solution, a reaction accelerator, a preservative, a surfactant, a protective agent and a circulating regeneration system consisting of glucose, glucose dehydrogenase and oxidized 3-acetylpyridine adenine dinucleotide, wherein the contents of all components in the circulating regeneration system in the kit are respectively as follows: glucose: 1-10 g/L; glucose dehydrogenase: 50-1000U/L; oxidized 3-acetylpyridine adenine dinucleotide: 0.3-0.6 g/L. According to the invention, the proportion and the dosage of each component in the circulating regeneration system are optimized, and the proper surfactant, protective agent and pH condition of the detection system are further selected, so that the reaction rate of the circulating system is better controlled, and the accuracy of the detection result of the low-concentration carbon dioxide is improved on the basis of improving the stability of the kit.)

1. a carbon dioxide enzyme method detection kit comprises phosphoenolpyruvate, phosphoenolpyruvate carboxylase, malate dehydrogenase, buffer solution, a reaction accelerator, a preservative, a surfactant, a protective agent and a circulating regeneration system consisting of glucose, glucose dehydrogenase and oxidized 3-acetylpyridine adenine dinucleotide, and is characterized in that: the contents of all components in the circulating regeneration system in the kit are respectively as follows:

glucose: 1-10g/L

Glucose dehydrogenase: 50-1000U/L

Oxidized 3-acetylpyridine adenine dinucleotide: 0.3-0.6 g/L.

2. The enzymatic capnometry assay kit of claim 1, wherein: the contents of all components in the circulating regeneration system in the kit are respectively as follows:

glucose: 5g/L

Glucose dehydrogenase: 100U/L

Oxidized 3-acetylpyridine adenine dinucleotide: 0.4 g/L.

3. The enzymatic capnometry assay kit of claim 1, wherein: the surfactant is one or more selected from castor oil, queen flower 709, queen flower A90, and queen flower 430.

4. The enzymatic capnometry assay kit of claim 3, wherein: the surfactant is castor oil and queen bee A90, and the weight ratio of the castor oil to the queen bee A90 is 1: 1.

5. the enzymatic capnometry assay kit of claim 4, wherein: the content of the surfactant in the kit is 0.5-5 g/L.

6. The enzymatic capnometry assay kit of claim 1, wherein: the pH value of the kit is 7.2-7.6.

7. The enzymatic capnometry assay kit of claim 1, wherein: the buffer solution is HEPES buffer solution.

8. The enzymatic capnometry assay kit of claim 1, wherein: the reaction accelerator is magnesium sulfate.

9. The enzymatic capnometry assay kit of claim 1, wherein: the preservative is sodium azide.

10. The enzymatic capnometry assay kit of claim 1, wherein: the protective agent is one or more of serine, L-serine, glycine, EDTA-2 Na, bovine serum albumin, mannitol, dextran, and trehalose.

Technical Field

The invention belongs to the field of biochemical detection, and relates to a carbon dioxide determination kit.

Background

CO in blood₂Mainly HCO₃ ^-The form of the composition is about 95 percent, is the main buffering alkali for maintaining acid-base balance of the organism, is one of the common indexes for judging the acid-base metabolism condition, provides important basis for the process and curative effect judgment of the acid-base balance disorder disease, is very common in clinical application, and is one of the indispensable emergency treatment projects.

Determination of CO₂The method (2) mainly uses an enzyme method and an electrode method. Electrode methods are generally combined with electrolyte analyzers, CO₂The electrode film needs to be replaced periodically, which is inconvenient to use. The enzyme method determination is suitable for an automatic biochemical analyzer and is more convenient and faster to use. The basic principle of the enzymatic method for detecting the carbon dioxide is as follows: phosphoenolpyruvate with HCO under the action of phosphoenolpyruvate carboxylase₃ ^-Reacting to generate oxaloacetate, reacting oxaloacetate with reduced coenzyme to generate malic acid under the action of malate dehydrogenase, oxidizing the reduced coenzyme into oxidized coenzyme to cause absorbance decrease, and detecting the absorbance decrease degree at a certain wavelength to calculate the concentration of carbon dioxide in blood. For example, CN1763536A discloses a method and a diagnostic kit for detecting the content of carbon dioxide according to the above principle.

However, the reagent kit is easy to be stored with CO in the air when being stored in the opening₂The reaction occurs, the substrate concentration is reduced, the reagent is poor in open bottle stability, for this reason, CN103558370A and CN107966556A both report the carbon dioxide enzyme method determination reagent kit using the recycling regeneration system, and the principle is that: the oxidized coenzyme reacts with glucose under the enzymatic action of glucose dehydrogenase, the oxidized coenzyme is reduced, and the reduced coenzyme is recycled, so that the kit can not be used for the enzymatic reaction of glucose dehydrogenaseThe absorption of external carbon dioxide results in a decrease in stability. At present, the method is widely applied to a kit, but the method also has the great defect that the accuracy of detection of low-concentration carbon dioxide is reduced because the reaction rate of cyclic regeneration is not easy to control and the main reaction of carbon dioxide detection is interfered.

Disclosure of Invention

The invention aims to provide a kit for detecting low-concentration carbon dioxide aiming at the defects of the existing determination kit for the circular regeneration carbon dioxide enzyme method, and the accuracy of a detection result is greatly improved by optimizing the component types and the proportion of the kit.

The above purpose is realized by the following technical scheme:

a carbon dioxide enzyme method detection kit comprises phosphoenolpyruvate, phosphoenolpyruvate carboxylase, malate dehydrogenase, a buffer solution, a reaction accelerator, a preservative, a surfactant, a protective agent and a circulating regeneration system consisting of glucose, glucose dehydrogenase and oxidized 3-acetylpyridine adenine dinucleotide, wherein the contents of all components in the circulating regeneration system in the kit are respectively as follows:

glucose: 1-10g/L

Glucose dehydrogenase: 50-1000U/L

Oxidized 3-acetylpyridine adenine dinucleotide: 0.3-0.6 g/L.

Preferably, the contents of the components in the cyclic regeneration system in the kit are respectively as follows:

glucose: 5g/L

Glucose dehydrogenase: 100U/L

Oxidized 3-acetylpyridine adenine dinucleotide: 0.4 g/L.

Preferably, the surfactant is one or more selected from castor oil, queen flower 709, queen flower A90 and queen flower 430.

Further preferably, the surfactant is castor oil and queen bee a90, and the weight ratio of the castor oil to the queen bee a90 is 1: 1.

preferably, the content of the surfactant in the kit is 0.5-5 g/L.

Preferably, the pH of the kit is 7.2-7.6.

Preferably, the buffer is HEPES buffer.

Preferably, the reaction accelerator is magnesium sulfate.

Preferably, the preservative is sodium azide.

Preferably, the protective agent is one or more of serine, L-serine, glycine, EDTA-2 Na, bovine serum albumin, mannitol, dextran and trehalose.

According to the specific embodiment of the invention, the carbonic acid gas enzyme method detection kit comprises the following more specific components:

the detection principle of the invention is as follows:

1) glucose and oxidized 3-acetylpyridine adenine dinucleotide (APAD)⁺) Under the action of glucose dehydrogenase, producing glucal and reduced 3-acetylpyridine adenine dinucleotide (APADH);

2) phosphoenolpyruvate and HCO₃ ^-Oxaloacetate and phosphate are generated under the action of phosphoenolpyruvate carboxylase;

3) oxaloacetate and reduced 3-acetylpyridine adenine dinucleotide (APADH) are generated into malic acid and oxidized 3-acetylpyridine adenine dinucleotide (APAD) under the action of malate dehydrogenase⁺)；

4) The degree of decrease in absorbance was measured at a wavelength of 405nm to calculate the concentration of carbon dioxide in blood.

The invention has the beneficial effects that:

according to the invention, the proportion and the dosage of each component in the circulating regeneration system are optimized, and the proper surfactant, protective agent and pH condition of the detection system are further selected, so that the reaction rate of the circulating system is better controlled, and the accuracy of the detection result of the low-concentration carbon dioxide is improved on the basis of improving the stability of the kit.

The invention also has the advantages of good thermal stability, high detection sensitivity and precision and the like.

Detailed Description

The present invention will be described in detail below with reference to specific examples. The queen flower surfactants used in the following examples were all available from queen flower company, EMULGEN 709 was a polyoxyethylene alkyl ether as the major component, EMULGEN a-90 was a polyoxyethylene distyrenated phenyl ether as the major component, and EMULGEN 430 was a polyoxyethylene oleyl ether as the major component.