JP2005245342A - Method for determining atp in cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for determining ATP presenting in cells by a firefly bioluminescence method without getting an influence of a cell-lysis agent. <P>SOLUTION: This method for determining the ATP is characterized by incorporating the cell lysis agent into liposome consisting of a lipid bilayer membrane as a constituting component of the membrane and utilizing the liposome as a sensitizing agent in the firefly bioluminescence. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a method for measuring ATP present in cells by a firefly bioluminescence method without being affected by a cytolytic agent.

  In the firefly bioluminescence reaction, it is known that light is emitted in the process of decarbonylation of luciferin in the presence of luciferin, ATP, and magnesium ions due to the catalytic action of luciferase. Since the amount of luminescence depends on the ATP concentration, the firefly bioluminescence method is widely used as a quantitative method for ATP.

  On the other hand, since a certain amount of ATP exists in living cells, the number of bacteria can be measured by measuring the amount of ATP. Therefore, quantification of ATP dissolved from inside the cell after lysing the cell membrane using a bactericide is performed by the firefly bioluminescence method.

  Surfactants, organic solvents, acids and the like are used as lysing agents, and these lysing agents all show an inhibitory action on luciferase. Therefore, in actual measurement, ATP is measured after a sample containing a lysing agent is diluted until the inhibitory effect on luciferase is lost. As a result, there was a problem that the superior sensitivity of the firefly bioluminescence method was lost by diluting ATP in the lysed sample.

  Thus, in recent years, various improvements have been attempted for surfactants in order to suppress the inhibitory action on luciferase. As one of them, a method has been developed in which mutant luciferase resistant to the surfactant Benzalkonium chloride (BKC) is prepared, and ATP can be quantified without diluting the sample using BKC as a lysing agent. (Patent Document 1, Non-Patent Document 1).

  In addition, after lysis with the cationic surfactant dodecyl trimethylammonium bromide (DTAB), dextran is added to the sample solution, and DTAB is included in dextran, thereby suppressing the inhibitory effect of DTAB on luciferase. It was. As a result, in order to suppress the inhibitory action of DTAB on luciferase, it was necessary to dilute the sample 1000 times, but by adding dextran, 20-fold dilution was required (Patent Document 2, Non-Patent Document 2). ).

Furthermore, after lysis with the cationic surfactant Benzethonium chloride (BZC), a large amount of polyhydric alcohol nonionic surfactant is added to the sample solution, and mixed micelles of BZC and nonionic surfactant are added. By forming it, the inhibitory action of BZC on luciferase can be suppressed (Patent Document 3).
JP2002-191396 US Pat. US Patent 5004684 Analytical Biochemistry, 319, 287-295 (2003) Luminescence, 14, 255-261 (1999)

Therefore, the problem to be solved by the present invention is the following problems that exist in the conventional method for preventing the inhibitory effect on luciferase of surfactants;
(1) It is necessary to take different measures depending on the type of cationic surfactant.
(2) Although the inhibitory action of the cationic surfactant on luciferase can be prevented, there is no effect on the luminescence reaction, and there is no effect of increasing the amount of luminescence.

The present invention is a method for quantifying ATP, the main feature of which is to utilize the following characteristics of liposomes in order to solve the above problems:
(1) Liposomes composed of lipid bilayer membranes can incorporate surfactant molecules as components of the membrane, produce liposomes with positive surface charge, and suppress the inhibitory action of luciferase by surfactants,
(2) The coexistence of liposomes having a positive surface charge produces a sensitizing effect in firefly bioluminescence, enabling highly sensitive measurement of ATP.

That is, the present invention has the following configuration.

1. A method for quantifying ATP, comprising lysing cells with a cytolytic agent, adding liposomes, and measuring adenosine 5 ′ triphosphate (ATP) eluted from the cells by a firefly bioluminescence method.

2. 2. The method according to 1 above, wherein the cytolytic agent is absorbed into the liposome.

3. 2. The method according to 1 above, wherein the luminescence reaction is enhanced by the addition of liposomes.

4. The method according to any one of 1 to 3, wherein a liposome solution containing 20 to 160 mM lipid is added to the reaction solution at an addition rate of 10 to 50% (v / v).

5). 5. The method according to any one of 1 to 4 above, wherein the cell lysing agent is a surfactant.

6). 6. The method according to 5 above, wherein the surfactant is a nonionic surfactant.

7). The method according to 6 above, wherein the nonionic surfactant is a cationic surfactant.

8). 8. The method according to 7 above, wherein the cationic surfactant is selected from BKC, DTAB, or BZC.

9. The constituent component of the liposome is any one of the above 1 to 4 selected from phosphatidylcholine (PC), phosphatidylglycerol (PG), diethylaminoethyl carobamoyl cholesterol (DEAE-Chol) or cholesterol (Chol) The method described.

10. 5. The method according to any one of 1 to 4 above, wherein the liposome is selected from amphoteric liposomes, cationic liposomes or anionic liposomes.

11. A method for measuring the number of cells using the quantitative method according to any one of 1 to 10 above.

12 12. The method according to any one of 1 to 11 above, wherein the cell is derived from bacteria and microorganisms.

13. The reagent kit used for the method of any one of said 1-12.

  The ATP quantification method of the present invention has the advantage of enabling highly sensitive measurement of ATP by suppressing the inhibitory action of luciferase by a surfactant with a liposome and increasing the amount of luminescence in firefly bioluminescence.

Hereinafter, the present invention will be described in detail.

  Examples of the cells in the present invention include cells derived from prokaryotes such as bacteria and microorganisms, and cells derived from eukaryotes such as plants and animals.

  The cell lysis referred to in the present invention means lysis of the cell membrane and cell wall of a living cell. As a means for cell lysis, addition of a cell lysing agent is preferably mentioned.

  The cell lysis agent as used in the field of this invention means the solvent which melt | dissolves the cell membrane and cell wall of a cell, and a surfactant is mentioned as an example.

[Surfactant]
As the surfactant used as the cell lysis agent in the present invention, a nonionic surfactant is preferably exemplified. As the nonionic surfactant in the present invention, a cationic surfactant is desirable. Cationic surfactants include benzalkoronium chloride (BKC), dodecyltrimethylammonium bromide (DTAB) and benzonium chloride (BZC) that are used as cell lysing agents.

The structural formula of benzalkorium chloride (BKC) is shown below.

The structural formula of dodecyltrimethylammonium bromide (DTAB) is shown below.

The structural formula of benzonium chloride (BZC) is shown below.

In the present invention, the addition ratio of the surfactant can be appropriately adjusted at the time of cell lysis, preferably according to the cell type. Generally, BKC is added at a final concentration in the range of 0.01% to 0.06%, preferably 0.06%. Further, DTAB is generally a final concentration ranging from 5x10 ^-3 M of 1x10 ^-3 M, preferably added at 3.5 × 10 ^-3 M. BZC generally a final concentration in the range of 1x10 ^-3 M from 3x10 ^-4 M, preferably added at 6x10 ^-3 M.

[Liposome]
The liposome referred to in the present invention is a particle composed of an artificial lipid membrane, and is formed as a lipid bilayer from phospholipid, glyceroglycolipid, cholesterol and the like. For the preparation, widely known methods such as a surfactant removal method, a hydration method, a super heavy oil method, a reverse phase evaporation method, a freeze-thaw method, an ethanol injection method, an extrusion method, and a high-pressure emulsification method are applied.

  The addition of liposome refers to mixing and contacting liposomes during cell lysis.

  The absorption in the liposome as used in the present invention means that the cell lytic agent is irreversibly incorporated into the liposome or at the liposome interface utilizing the membrane properties of the liposome.

  The constituent components of the liposome used in the present invention include phosphatidylcholine (PC), phosphatidylglycerol (PG), diethylaminoethylcarbamoylcholesterol (DEAE-Chol) and cholesterol (Chol).

The structural formula of phosphatidylcholine (PC) is shown below.

The structural formula of phosphatidylglycerol (PG) is shown below.

The structural formula of diethylaminoethyl-carbamoyl cholesterol (DEAE-Chol) is shown below.

The structural formula of cholesterol (Chol) is shown below.

  The surface charge of the single membrane liposome used in the present invention is preferably amphoteric, cationic and anionic liposome, more preferably amphoteric liposome. Zwitterionic liposomes are prepared with PC and Chol. Cationic liposomes are prepared with PC and DEAE-Chol. In addition, anionic liposomes are prepared with PC and PG.

As a specific example, a single membrane zwitterionic liposome is prepared as follows:
With pear shaped flask to prepare a Chol PC and 1.0 x10 ^-3 M of 1.0 x10 ^-3 M in chloroform. Next, using a rotary evaporator, chloroform is evaporated to dryness to form a thin film of phospholipid on the surface of the flask. Next, 2 ml of 25 mM HEPES buffer solution (pH 7.75) is added to the flask and vigorously stirred with a vortex mixer to produce multilamellar liposomes. Finally, a single membrane liposome (VET) containing 20 mM lipid is prepared by passing 20 times through a polycarbonate filter having an average pore size of 100 nm.

[Firefly bioluminescence method]
The firefly bioluminescence method of the present invention includes a luminescence measurement method involving luciferin and luciferase. Examples of the luminescence measurement method include a method for measuring the amount of ATP in a sample by measuring luminescence generated by adding luciferin, luciferase, and magnesium ions to the sample.

  In the present invention, the enzyme solution used for measuring ATP by the firefly bioluminescence method is derived from 1 mg luciferase, 0.6 mM luciferin, 24 mM magnesium acetate, 3 mg calf in 50 ml of 25 mM HEPES buffer solution (pH 7.75). Prepare by dissolving albumin, 2 mM dithiothreitol and 2 mM EDTA. The mixed solution of the enzyme solution and the liposome is prepared by mixing 100 μl of the enzyme solution and 50 μl of the liposome solution. The ratio of the liposome solution added to the reaction solution can be appropriately adjusted depending on the concentration of the surfactant. Specifically, a liposome solution containing 20 to 160 mM lipid is added to the reaction solution at an addition rate of 10 to 50% (v / v), preferably 33% (v / v).

  The firefly bioluminescence method of the present invention may be used in any industrial field. Examples of the industrial field include a food processing field, a medical field, and an academic research field. For example, according to the present invention, ATP present in bacteria and microorganisms is measured by a firefly bioluminescence method without being affected by a surfactant that is a cell lysing agent in a cleanness test in the food processing field, etc. The number of microorganisms can be measured.

[Reagent kit]
The ATP quantification method of the present invention can be applied to a reagent kit. That is, one of the aspects of the present invention is a reagent kit containing at least the following components: (1) a cytolytic agent, (2) a liposome, and (3) luciferase. The reagent kit of the present invention can be combined with other components involved in the firefly bioluminescence reaction. The other components involved in the firefly bioluminescence reaction are preferably luciferin, ATP, and divalent metal ions.

  Next, embodiments of the present invention will be specifically described by way of examples. However, the present invention is not limited to the examples. The measurement of the luminescence response curve was performed using a chemiluminescence counter BLD-100HU (manufactured by Tohoku Electronics Industry). The amount of luminescence during the measurement time was measured by the number of photons per second (countspersecond: cps). The maximum light emission amount in the light emission response curve was defined as the light emission intensity.

[ATP measurement]
ATP was measured as follows, and a comparative study of relative luminescence intensity was conducted. 250 μl of a solution containing 1.0 × ^{10 −10} M ATP and various concentrations of cationic surfactant was placed in a glass cuvette and the cuvette was placed in a luminometer. With an autoinjector, 150 μl of an enzyme solution containing zwitterionic liposomes was injected from the outside of the luminometer, and the luminescence response curve was measured. Zwitterionic liposomes were prepared by dissolving 10 mM each of PC and Chol in chloroform. The final concentration of BKC after mixing all solutions was in the concentration range of 1.0 × 10 ⁻³ to 0.1%. The final DTAB concentration ranged from 1.0 × 10 ⁻⁵ M to 1.0 × 10 ⁻² M. The final concentration of BZC was in the concentration range of 1.0 × 10 ⁻⁶ M to 1.0 × 10 ⁻² M.

(Comparative Example 1)
250 μl of a solution containing 1.0 × ^{10 −10} M ATP and various concentrations of cationic surfactant was placed in a glass cuvette and the cuvette was placed in a luminometer. Using an autoinjector, 150 μl of a mixed solution of an enzyme solution (100 μl) and a buffer solution (50 μl) was injected from the outside of the luminometer, and a luminescence response curve was measured.

(result)
1 to 4 show the relative luminescence intensity under each measurement condition. The relative luminescence intensity in FIGS. 1 and 2 is a value obtained by dividing the luminescence intensity obtained in this example by the luminescence intensity obtained from the mixing of only 1.0 × ^{10 −10} M ATP and the enzyme solution. The relative luminescence intensity in FIG. 2 is a value obtained by dividing the luminescence intensity obtained in this comparative example by the luminescence intensity obtained from the mixture of only 1.0 × ^{10 −10} M ATP and the enzyme solution.

As is clear from FIGS. 1 and 2, in any cationic surfactant, when zwitterionic liposomes were present, a surfactant concentration region in which the luminescence intensity increased rapidly was developed. As is clear from FIG. 3 and FIG. 4, when the surfactant concentration increases, the amount of luminescence decreases due to the inhibitory action of luciferase by the surfactant, and almost no luminescence occurs at the surfactant concentration used as the cytolytic agent. Not observed. The surfactant concentration used as a cell lysing agent is the concentration after mixing with the sample solution containing cells, BKC is about 0.1%, DTAB is about 1x10 ^{-1 to} 1x10 ^-2 M, BZC is about 1x10 ^-3 M . Comparing FIGS. 1 to 4, luminescence is observed due to the presence of zwitterionic liposomes even when the luciferase is deactivated by the surfactant and no luminescence is observed, and the relative luminescence intensity is 1 or more. I found out.

  From the above results, it was found that the liposome can prevent the inhibitory action of the surfactant on luciferase by incorporating the surfactant as a membrane constituent of the liposome. Moreover, since the relative luminescence intensity was 1 or more, it was found that the liposome produced by incorporating a cationic surfactant exhibits a sensitizing effect on firefly bioluminescence. In addition, it turned out that since the quantity of surfactant which is not taken in into a liposome increases when surfactant concentration increases, emitted light intensity reduces.

[Examination of optimum measurement conditions for surface charge of liposomes]
ATP was measured as follows, and the surface charge of the liposomes used was examined. 250 μl of a solution containing 1.0 × ^{10 −10} M ATP and various concentrations of BKC was placed in a glass cuvette and the cuvette was placed in a luminometer. Using an autoinjector, 150 μl of an enzyme solution containing cationic or anionic liposomes was injected from the outside of the luminometer, and the luminescence response curve was measured. Cationic liposomes were prepared by dissolving 10 mM each of PC and DEAE-Chol in chloroform. Anionic liposomes were prepared by dissolving PC, PG and Chol in chloroform at 10, 2 and 8 mM, respectively. The final concentration of BKC after mixing all solutions was in the concentration range of 1.0 × 10 ⁻³ to 0.1%.

(result)
FIG. 5 shows the emission intensity obtained in this example. When any type of liposome was used, the luminescence intensity reached its maximum when the BKC concentration was around 0.01%. However, when the surface charges of the liposomes used differed, the luminescence intensity changed greatly, and the luminescence intensity increased in the order of zwitterionic liposome> cationic liposome> anionic liposome. Therefore, it was found that amphoteric liposomes are desirable for the liposomes used.

[Examination of optimal measurement conditions for optimal concentration of amphoteric liposomes]
ATP was measured as follows, and the optimum concentration of the zwitterionic liposome to be used was examined. 250 μl of a solution containing 1.0 × ^{10 −10} M ATP and various concentrations of cationic surfactant was placed in a glass cuvette and the cuvette was placed in a luminometer. Using an autoinjector, 150 μl of an enzyme solution containing zwitterionic liposomes of various concentrations was injected from the outside of the luminometer, and the luminescence response curve was measured. Zwitterionic liposomes containing 20 mM, 40 mM, 80 mM and 160 mM lipids were prepared. The PC / Chol concentration ratio was 1: 1. The final concentration of BKC after mixing all solutions was in the concentration range of 1.0 × 10 ⁻³ to 0.1%.

(result)
FIG. 6 shows the emission intensity obtained in this example. As the lipid concentration used increased, the BKC concentration at which a sensitizing effect was expressed shifted to a higher concentration side. Since BKC is in the concentration range of 0.01 to 0.06% in the mixed solution of the sample solution containing cells and the enzyme solution, it is necessary to change the amount of liposomes used depending on the concentration of BKC used for the cell lysing agent. I understood.

[Creation of ATP calibration curve]
ATP was measured as follows, and a calibration curve for ATP was prepared. 250 μl of a solution containing various concentrations of ATP and 0.06% BKC at a final concentration was placed in a glass cuvette and the cuvette was placed in a luminometer. With an autoinjector, 150 μl of an enzyme solution containing zwitterionic liposomes was injected from the outside of the luminometer, and the luminescence response curve was measured. Zwitterionic liposomes were prepared by dissolving 80 mM each of PC and Chol in chloroform. The concentration range of ATP was 4.0 × 10 ⁻¹² M to 1.0 × 10 ⁻⁹ M.

(Comparative Example 2)
Place 250 μl of solution containing various concentrations of ATP into a glass cuvette. Place the cuvette in the luminometer. Using an autoinjector, 150 μl of a mixed solution of an enzyme solution (100 μl) and a buffer solution (50 μl) was injected from the outside of the luminometer, and a luminescence response curve was measured. The concentration range of ATP was 4.0 × 10 ⁻¹² M to 1.0 × 10 ⁻⁹ M.

(result)
A straight line 1 in FIG. 7 is a calibration curve of ATP when an enzyme solution containing liposomes is mixed with an ATP solution containing BKC based on this example. A good linear relationship was obtained in the range of 4.0 × 10 ⁻¹² M to 1.0 × 10 ⁻⁹ M, the lower limit of quantification. A straight line 2 in FIG. 7 is a calibration curve of ATP when an enzyme solution containing no liposome is mixed with a solution containing only ATP based on this comparative example. A good linear relationship was obtained in the range of 4.0x10 ^-11 M to 4.0x10 ^-9 M, the lower limit of quantification. From the above results, it was found that even when BKC was contained in the ATP solution, the detection sensitivity of ATP was improved 10 times due to the sensitization effect when liposomes coexisted.

[Measurement of E. coli number]
The number of E. coli was measured as follows, and the effect of liposomes on the measurement of the number of E. coli was examined. BKC is added to a solution containing E. coli in the range of 10 ⁵ to 10 ⁷ to lyse, and ATP is extracted. Place 250 μl of the solution in a glass cuvette. Place the cuvette in the luminometer. With an autoinjector, 150 μl of an enzyme solution containing zwitterionic liposomes was injected from the outside of the luminometer, and the luminescence response curve was measured. Zwitterionic liposomes were prepared by dissolving 80 mM each of PC and Chol in chloroform. The final concentration of BKC after mixing all the solutions was 0.06% (w / v).

(Comparative Example 3)
BKC was added to a solution containing E. coli in the range of 10 ⁵ to 10 ⁷ to lyse, and ATP was extracted. The solution was diluted 1/6, 250 μl of the diluted solution was placed in a glass cuvette, and the cuvette was placed in a luminometer. Using an autoinjector, 150 μl of a mixed solution of an enzyme solution (100 μl) and a buffer solution (50 μl) was injected from the outside of the luminometer, and a luminescence response curve was measured. The concentration of BKC after mixing all the solutions was 0.01% (w / v).

(result)
Line 1 in FIG. 8 shows the relationship between the number of E. coli and the luminescence intensity when an enzyme solution containing liposomes is mixed with a solution containing BKC and extracted ATP, based on this example. In the range of the number of E. coli cells examined, a good linear relationship was obtained between the number of cells and the luminescence intensity. On the other hand, line 2 in FIG. 8 shows the relationship between the number of E. coli and luminescence intensity when an enzyme solution containing no liposome is mixed with a solution containing BKC and extracted ATP, based on this comparative example. When liposomes were not included, a luminescent reaction was performed after diluting a solution containing BKC and extracted ATP 1/10 in order to prevent the inhibitory action of BKC on luciferase. Therefore, it was found that the number of E. coli can be measured 10 times more sensitively by using liposomes.

It is a graph which shows the relationship between surfactant concentration and relative luminescence intensity. It is a graph which shows the relationship between surfactant concentration and relative luminescence intensity. It is a graph which shows the relationship between surfactant concentration and relative luminescence intensity. It is a graph which shows the relationship between surfactant concentration and relative luminescence intensity. It is a graph which shows the relationship between surfactant density | concentration and emitted light intensity. It is a graph which shows the relationship between BKC density | concentration and emitted light intensity. It is a graph which shows the relationship between ATP density | concentration and luminescence intensity. It is a graph which shows the relationship between E. coli body number and luminescence intensity.

Claims (13)

  A method for quantifying ATP, comprising lysing cells with a cytolytic agent, adding liposomes, and measuring adenosine 5 ′ triphosphate (ATP) eluted from the cells by a firefly bioluminescence method.   The method according to claim 1, wherein the cytolytic agent is absorbed into the liposome.   2. The method according to claim 1, wherein the luminescence reaction is enhanced by the addition of liposomes.   The method according to any one of claims 1 to 3, wherein a liposome solution containing 20 to 160 mM lipid is added to the reaction solution at an addition rate of 10 to 50% (v / v).   The method according to any one of claims 1 to 4, wherein the cytolytic agent is a surfactant.   The method of claim 5, wherein the surfactant is a nonionic surfactant.   The method according to claim 6, wherein the nonionic surfactant is a cationic surfactant.   The method according to claim 7, wherein the cationic surfactant is selected from BKC, DTAB, or BZC.   The constituent component of the liposome is selected from phosphatidylcholine (PC), phosphatidylglycerol (PG), diethylaminoethyl carobamoyl cholesterol (DEAE-Chol) or cholesterol (Chol). The method described in 1.   The method according to any one of claims 1 to 4, wherein the liposome is selected from amphoteric liposomes, cationic liposomes or anionic liposomes.   The cell number measuring method using the quantification method of any one of Claims 1-10.   The method according to any one of claims 1 to 11, wherein the cells are derived from bacteria and microorganisms.   The reagent kit used for the method of any one of Claims 1-12.