阅读说明：本技术 Atp荧光检测试剂 (ATP fluorescence detection reagent ) 是由 李书红 陈杨青 夏放 蒋晓东 于 2019-10-30 设计创作，主要内容包括：本发明提供了一种ATP荧光检测试剂,所述ATP荧光检测试剂,在酶反应基本溶液体系和缓冲体系中加入用于提高所述酶反应基本溶液体系中的荧光素酶热稳定性的保护剂,所述保护剂包含特定浓度的海藻糖和甜菜碱。与相关技术相比,本发明提供的ATP荧光检测试剂的热稳定性更佳且保存时间明显延长。(The invention provides an ATP fluorescence detection reagent, wherein a protective agent for improving the thermal stability of luciferase in an enzyme reaction basic solution system is added into the enzyme reaction basic solution system and a buffer system, and the protective agent comprises trehalose and betaine with specific concentrations. Compared with the related technology, the ATP fluorescence detection reagent provided by the invention has better thermal stability and obviously prolonged storage time.)

1. An ATP fluorescence detection reagent is characterized in that a protective agent for improving the thermal stability of luciferase in an enzyme reaction basic solution system is added into the enzyme reaction basic solution system and a buffer system, the protective agent comprises trehalose and betaine, the concentration of the betaine is 0.18-0.36M, and the concentration of the trehalose is 0.62-1.24M of the betaine.

2. The ATP fluorescence detection reagent of claim 1, wherein the ATP fluorescence detection reagent consists of an enzyme reaction basic solution system, a buffer system and a protective agent.

3. The ATP fluorescence detection reagent of claim 1, wherein the buffer system is a trimethylglycine buffer solution, and the concentration of the trimethylglycine buffer solution is 0.1M.

4. The ATP fluorescence detection reagent according to claim 1, wherein the enzyme reaction basic solution system comprises luciferase, luciferin and metal ions, the luciferase is preferably luciferase, the luciferin is preferably D-luciferin potassium salt, the concentrations of the luciferase and the D-luciferin potassium salt are preferably 10Mg/L and 500Mg/L respectively, and the metal ions are Mg²⁺The metal ion Mg²⁺The concentration of (B) is preferably 15 mM.

5. The ATP fluorescence detection reagent of claim 1, further comprising bovine serum albumin and dithiothreitol, wherein the concentration of bovine serum albumin is preferably 0.05% -0.1%, and the concentration of dithiothreitol is preferably 0.01% -0.05%.

[ technical field ] A method for producing a semiconductor device

The invention relates to the technical field of ATP fluorescence detection, in particular to an ATP fluorescence detection reagent with stable room temperature.

[ background of the invention ]

Luciferase oxidises luciferin and emits fluorescence in the presence of ATP (adenosine triphosphate), and the emitted photons can be detected by a light sensitive element, such as a fluorescence detector or a modified light microscope.

Luciferase is very thermostable and loses activity quickly at room temperature. The improvement of the thermal stability of luciferase can be achieved by changing the gene structure (gene mutation) of luciferase. However, this procedure is cumbersome and many mutations that alter its thermostability have been patented. Another approach is to add specific protective agents to improve the stability of luciferase.

Although various commercially available ATP fluorescence detection reagents improve the thermal stability of a solution reagent to some extent, the storage life and thermal stability of the reagents are not satisfactory.

Therefore, there is a need to provide a new ATP fluorescence detection reagent to solve the above problems.

[ summary of the invention ]

The invention aims to overcome the technical problems and provide an ATP fluorescence detection reagent with good thermal stability, which solves the problems of poor thermal stability and short storage time of the existing commercially available ATP fluorescence detection reagent.

The invention provides an ATP fluorescence detection reagent, wherein a protective agent for improving the thermal stability of luciferase in an enzyme reaction basic solution system is added into the enzyme reaction basic solution system and a buffer system, the protective agent comprises trehalose and betaine, the concentration of the betaine is 0.18-0.36M, and the concentration of the trehalose is 0.62-1.24M of the betaine.

Preferably, the ATP fluorescence detection reagent consists of an enzyme reaction basic solution system, a buffer system and a protective agent.

Preferably, the buffer system is a trimethylglycine buffer solution, and the concentration of the trimethylglycine buffer solution is 0.1M.

Preferably, the enzyme reaction basic solution system comprises luciferase, luciferin and metal ions, the luciferase is preferably luciferase, the luciferin is preferably D-luciferin potassium salt, the concentration of the luciferase and the D-luciferin potassium salt is preferably 10Mg/L and 500Mg/L respectively, and the metal ions are Mg²⁺The metal ion Mg²⁺The concentration of (B) is preferably 15 mM.

Preferably, the ATP fluorescence detection reagent further comprises bovine serum albumin and dithiothreitol, wherein the concentration of the bovine serum albumin is preferably 0.05% -0.1%, and the concentration of the dithiothreitol is preferably 0.01% -0.05%.

Compared with the prior art, the ATP fluorescence detection reagent provided by the invention is added with the protective agent with specific proportion of trehalose and betaine, wherein the betaine effectively prevents protein thermodynamic interference induced by dehydration, and the trehalose and the betaine greatly improve the thermal stability of the ATP fluorescence detection reagent and also obviously prolong the storage time of the ATP detection reagent.

[ description of the drawings ]

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without inventive efforts, wherein:

FIG. 1 is a graph showing fluorescence values of ATP fluorescence detection reagents with trehalose only and without betaine and trehalose at 25 ℃;

FIG. 2 is a graph showing fluorescence values of ATP fluorescence detection reagents with betaine only and without betaine and trehalose at 25 ℃;

FIG. 3 is a graph showing fluorescence values of ATP fluorescence detection reagents with and without trehalose and trehalose added simultaneously with trehalose and betaine at 25 ℃;

FIG. 4 is a graph showing fluorescence values of different amounts of trehalose and betaine added to an ATP fluorescence detection reagent at 35 ℃.

[ detailed description ] embodiments

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.