CN114350626A - Luciferase freeze-dried powder dilution reaction liquid - Google Patents
Luciferase freeze-dried powder dilution reaction liquid Download PDFInfo
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- CN114350626A CN114350626A CN202111575835.6A CN202111575835A CN114350626A CN 114350626 A CN114350626 A CN 114350626A CN 202111575835 A CN202111575835 A CN 202111575835A CN 114350626 A CN114350626 A CN 114350626A
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- Prior art keywords
- luciferase
- freeze
- reaction solution
- dried powder
- dilution reaction
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- 108060001084 Luciferase Proteins 0.000 title claims abstract description 35
- 239000005089 Luciferase Substances 0.000 title claims abstract description 34
- 239000000843 powder Substances 0.000 title claims abstract description 24
- 239000012895 dilution Substances 0.000 title claims abstract description 23
- 238000010790 dilution Methods 0.000 title claims abstract description 23
- 239000012295 chemical reaction liquid Substances 0.000 title abstract description 6
- 238000006243 chemical reaction Methods 0.000 claims abstract description 24
- 239000000243 solution Substances 0.000 claims abstract description 24
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims abstract description 16
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims abstract description 16
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 16
- 229920001223 polyethylene glycol Polymers 0.000 claims abstract description 16
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims abstract description 14
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 claims abstract description 12
- 239000002202 Polyethylene glycol Substances 0.000 claims abstract description 10
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims abstract description 10
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 claims abstract description 8
- 229910001629 magnesium chloride Inorganic materials 0.000 claims abstract description 8
- 239000001103 potassium chloride Substances 0.000 claims abstract description 8
- 235000011164 potassium chloride Nutrition 0.000 claims abstract description 8
- 239000011780 sodium chloride Substances 0.000 claims abstract description 8
- 239000004471 Glycine Substances 0.000 claims abstract description 7
- 108010024636 Glutathione Proteins 0.000 claims abstract description 6
- 229960003180 glutathione Drugs 0.000 claims abstract description 6
- ZYVIIEFODRWQGD-UHFFFAOYSA-N 2-piperazin-1-ylethanol;propane-1-sulfonic acid Chemical compound CCCS(O)(=O)=O.OCCN1CCNCC1 ZYVIIEFODRWQGD-UHFFFAOYSA-N 0.000 claims abstract description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 239000008176 lyophilized powder Substances 0.000 claims description 5
- 239000008223 sterile water Substances 0.000 claims description 3
- 229940057838 polyethylene glycol 4000 Drugs 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 6
- OWXMKDGYPWMGEB-UHFFFAOYSA-N HEPPS Chemical compound OCCN1CCN(CCCS(O)(=O)=O)CC1 OWXMKDGYPWMGEB-UHFFFAOYSA-N 0.000 description 6
- 239000007996 HEPPS buffer Substances 0.000 description 6
- 239000007983 Tris buffer Substances 0.000 description 6
- 238000001514 detection method Methods 0.000 description 4
- IGXWBGJHJZYPQS-SSDOTTSWSA-N D-Luciferin Chemical compound OC(=O)[C@H]1CSC(C=2SC3=CC=C(O)C=C3N=2)=N1 IGXWBGJHJZYPQS-SSDOTTSWSA-N 0.000 description 3
- 238000005415 bioluminescence Methods 0.000 description 2
- 230000029918 bioluminescence Effects 0.000 description 2
- 238000004020 luminiscence type Methods 0.000 description 2
- CYCGRDQQIOGCKX-UHFFFAOYSA-N Dehydro-luciferin Natural products OC(=O)C1=CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 CYCGRDQQIOGCKX-UHFFFAOYSA-N 0.000 description 1
- 108090000331 Firefly luciferases Proteins 0.000 description 1
- BJGNCJDXODQBOB-UHFFFAOYSA-N Fivefly Luciferin Natural products OC(=O)C1CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 BJGNCJDXODQBOB-UHFFFAOYSA-N 0.000 description 1
- DDWFXDSYGUXRAY-UHFFFAOYSA-N Luciferin Natural products CCc1c(C)c(CC2NC(=O)C(=C2C=C)C)[nH]c1Cc3[nH]c4C(=C5/NC(CC(=O)O)C(C)C5CC(=O)O)CC(=O)c4c3C DDWFXDSYGUXRAY-UHFFFAOYSA-N 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000013024 dilution buffer Substances 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000003223 protective agent Substances 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
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Abstract
The invention provides luciferase freeze-dried powder dilution reaction liquid which comprises the following components: 0.1-3mM fluorescein, 0.1-1mM polyethylene glycol, 0.1-1mM tris (hydroxymethyl) aminomethane, 0.5-2mM 4-hydroxyethyl piperazine propanesulfonic acid, 0.1-0.5mM magnesium chloride, 0.01-0.3mM potassium chloride, 0.05-0.15mM sodium chloride, 0-3mM glycine, 0-20mM glutathione. The luciferase freeze-dried powder dilution reaction solution provided by the invention can effectively embody the activity of luciferase while maintaining the stability of luciferase dry powder.
Description
Technical Field
The invention relates to the technical field of luciferase dry powder, and in particular relates to luciferase freeze-dried powder dilution reaction liquid.
Background
The firefly luciferase catalyzes luminescence to ATP, luciferin and Mg2+In the presence of a yellow-green fluorescence. Most scientists generally consider that the luciferase is catalyzed by D-luciferin as a catalyst, the D-luciferin emits light by oxidation, and the emission intensity of the fluorescence is directly and positively correlated with the concentration of ATP under certain conditions.
Although the principle of luciferase-catalyzed luminescence is basically known to researchers, the instability of luciferase limits its application prospects, and therefore most of the researchers' major research efforts have been focused on stability studies, such as the study of protective agents or the expression of more stable luciferases by site-directed mutagenesis. The luciferase dry powder has good stability, and can be preserved for a long time without losing activity. However, the buffer solution for diluting the dry powder needs a good proportion to better embody the activity of the luciferase. In order to solve the above situation, the application provides a luciferase freeze-dried powder dilution reaction solution.
Disclosure of Invention
The invention aims to provide a luciferase freeze-dried powder dilution reaction solution, which maintains the stability of luciferase dry powder and can effectively embody the activity of luciferase.
The invention adopts the following technical scheme to solve the technical problems:
a luciferase freeze-dried powder dilution reaction solution comprises the following components: comprises the following components: 0.1-3mM fluorescein, 0.1-1mM polyethylene glycol, 0.1-1mM tris (hydroxymethyl) aminomethane, 0.5-2mM 4-hydroxyethyl piperazine propanesulfonic acid, 0.1-0.5mM magnesium chloride, 0.01-0.3mM potassium chloride, 0.05-0.15mM sodium chloride, 0-3mM glycine, 0-20mM glutathione.
Further, the water content in the diluted reaction liquid is not more than 90%.
Further, the water in the diluted reaction solution is sterile water.
Further, the polyethylene glycol is polyethylene glycol 2000 or polyethylene glycol 4000.
The invention has the advantages that:
(1) the luciferase freeze-dried powder dilution reaction solution can well embody the activity of luciferase in a dry powder dilution state.
(2) The luciferase freeze-dried powder dilution reaction solution provided by the invention is simple in formula and can be suitable for large-scale production.
Drawings
FIG. 1 is a summary diagram of the results of diluted reaction solutions in various embodiments of the present invention.
Detailed Description
The invention is further illustrated by the following examples, which are intended to illustrate, but not to limit the invention further.
In the invention, the water content in the diluted reaction solution is not more than 90 percent, and the water is sterile water;
in the following examples, PEG was polyethylene glycol, Tris was Tris (hydroxymethyl) aminomethane, and HEPPS was 4-hydroxyethylpiperazine propanesulfonic acid. In the following embodiments, the polyethylene glycol is polyethylene glycol 2000, the control group distribution ratios are different, and the optimal ratio is measured by an ATP bioluminescence detector.
The invention is further illustrated by the following specific examples:
example 1
The detection method comprises the following steps: dissolving luciferase lyophilized powder 100MG in a centrifuge tube with prepared dilution buffer solution to reach volume of 10ML, and collecting 10ul 1 × 10-9Adding 500ul of dissolved reaction solution into the M ATP standard solution in a reaction tube, placing the reaction tube into a handheld ATP bioluminescence detector for detection, and recording detection data. The above detection steps were repeated 10 times to obtain 10 sets of data. The results are summarized in table 1 below:
table 1: test results obtained in example 1
Example 2
table 2: test results obtained in example 2
Example 3
table 3: test results obtained in example 3
Example 4
table 4: test results obtained in example 4
Example 5
table 5: test results obtained in example 5
The results of the above examples show that, after comprehensive comparison, the luciferase lyophilized powder provided in example 3 is diluted to an optimal ratio.
However, the average of the 10 data in example 1 was 4541.6RLU, the average of the 10 data in example 2 was 6439.7RLU, the average of the 10 data in example 3 was 7582.7RLU, the average of the 10 data in example 4 was 7637.2RLU, and the average of the 10 data in example 5 was 6391.7RLU, in terms of the average ATP content; the average value of the detected ATP content is the highest in the embodiment 4, and the ratio of the diluted reaction solution of the luciferase freeze-dried powder is the most excellent; therefore, the luciferase activity can be well embodied by the luciferase freeze-dried powder dilution reaction liquid formula provided by the application.
Finally, it should be noted that: the above embodiments are only used to illustrate the present invention and do not limit the technical solutions described in the present invention; it will be understood by those skilled in the art that the present invention may be modified and equivalents may be substituted; all such modifications and variations are intended to be included herein within the scope of this disclosure and the present invention and protected by the following claims.
Claims (4)
1. A luciferase freeze-dried powder dilution reaction solution is characterized by comprising the following components: 0.1-3mM fluorescein, 0.1-1mM polyethylene glycol, 0.1-1mM tris (hydroxymethyl) aminomethane, 0.5-2mM 4-hydroxyethyl piperazine propanesulfonic acid, 0.1-0.5mM magnesium chloride, 0.01-0.3mM potassium chloride, 0.05-0.15mM sodium chloride, 0-3mM glycine, 0-20mM glutathione.
2. The luciferase lyophilized powder dilution reaction solution as claimed in claim 1, wherein the water content in the dilution reaction solution is not more than 90%.
3. The luciferase freeze-dried powder dilution reaction solution as claimed in claim 2, wherein water in the dilution reaction solution is sterile water.
4. The luciferase lyophilized powder dilution reaction solution as claimed in claim 1, wherein the polyethylene glycol is polyethylene glycol 2000 or polyethylene glycol 4000.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202111575835.6A CN114350626A (en) | 2021-12-21 | 2021-12-21 | Luciferase freeze-dried powder dilution reaction liquid |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202111575835.6A CN114350626A (en) | 2021-12-21 | 2021-12-21 | Luciferase freeze-dried powder dilution reaction liquid |
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| CN114350626A true CN114350626A (en) | 2022-04-15 |
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| CN202111575835.6A Pending CN114350626A (en) | 2021-12-21 | 2021-12-21 | Luciferase freeze-dried powder dilution reaction liquid |
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5283179A (en) * | 1990-09-10 | 1994-02-01 | Promega Corporation | Luciferase assay method |
| WO1994011528A1 (en) * | 1992-11-17 | 1994-05-26 | Celsis Limited | Bioluminescence reagent formulation |
| CN101126718A (en) * | 2006-08-16 | 2008-02-20 | 中国科学院电子学研究所 | Surface Cleanliness Detection Reagents for Hygiene Monitoring |
| CN101218354A (en) * | 2005-05-13 | 2008-07-09 | 珀金埃尔默生命与分析科学有限公司 | Application of Ammonium and Phosphate Ions in Improved Luciferase Assays |
| CN103757089A (en) * | 2014-01-10 | 2014-04-30 | 广东省微生物研究所 | Adenosine triphosphate (ATP) bioluminescent reagent for detecting hygienic quality of drinking water and surface sanitation of GMP factory, method and kit |
| CN105018457A (en) * | 2015-07-20 | 2015-11-04 | 宁波美成生物科技有限公司 | Luciferase stabilizer |
-
2021
- 2021-12-21 CN CN202111575835.6A patent/CN114350626A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5283179A (en) * | 1990-09-10 | 1994-02-01 | Promega Corporation | Luciferase assay method |
| WO1994011528A1 (en) * | 1992-11-17 | 1994-05-26 | Celsis Limited | Bioluminescence reagent formulation |
| CN101218354A (en) * | 2005-05-13 | 2008-07-09 | 珀金埃尔默生命与分析科学有限公司 | Application of Ammonium and Phosphate Ions in Improved Luciferase Assays |
| CN101126718A (en) * | 2006-08-16 | 2008-02-20 | 中国科学院电子学研究所 | Surface Cleanliness Detection Reagents for Hygiene Monitoring |
| CN103757089A (en) * | 2014-01-10 | 2014-04-30 | 广东省微生物研究所 | Adenosine triphosphate (ATP) bioluminescent reagent for detecting hygienic quality of drinking water and surface sanitation of GMP factory, method and kit |
| CN105018457A (en) * | 2015-07-20 | 2015-11-04 | 宁波美成生物科技有限公司 | Luciferase stabilizer |
Non-Patent Citations (1)
| Title |
|---|
| 薛大权 等: "《聚乙二醇在医药学领域的应用与技术》", 华中科技大学出版社, pages: 43 * |
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