JPH0262943A - Method of measuring material in cell - Google Patents

Abstract

PURPOSE:To simplify a treating stage and to shorten the time for analysis as well as to allow the analysis of a desired material with high accuracy by irradiating a soln. contg. cells with ultrasonic waves. CONSTITUTION:The cell membranes are broken and the material to be measured in the cells is extracted by irradiating the soln. contg. the cells with the ultrasonic waves. The material to be measured is then measured. For example, a freeze-preserved sample liquid contg. coliform bacilli is thawed and 0.6ml sample liquid is fraction-collected into a test tube by using a manual dispenser such as pipette. After 5.7ml buffer soln. of 0.1ml/l phosphoric acid (7.0 pH) is added to this test tube, the liquid is irradiated with 30W ultrasonic waves for 1 to 2 minutes to break the cell membranes of the coliform bacilli and to extract ATP. In succession, 0.5ml this soln. is fraction-collected into a vial bottle to be exclusively used for a measuring instrument by a pipette and 0.5ml reaction reagent contg. a light emitting reagent is automatically dispensed thereon. The quantity of the light emitted for 30 seconds right after the dispensing is measured.

Description

DETAILED DESCRIPTION OF THE INVENTION A. Field of Industrial Application The present invention relates to a method for measuring substances present in somatic cells and microbial cells.

B2 Overview of the Invention The present invention extracts the substance to be measured inside the cell by destroying the cell membrane, and then irradiates this substance with, for example, optical ultrasonic waves to destroy the cell membrane. The purpose is to make the process short and easy to operate.

C1 Conventional Techniques Effective knowledge can be obtained by qualitatively or conventionally measuring substances existing within cells or microbial cells.

For example, in living cells, adenone triphosphate (AT), which plays a role in phosphate metabolism and energy metabolism, is present in living cells.
P) is always present, and it is known that ATP is not present in dead cells. Also, ATP present in one cell
ffi, ATP exists at the same concentration in the same cell.

Therefore, if ATP can be quantified, it will be possible to determine the number of cells, determine whether living cells are dead cells, and measure the activity of cells.

In addition, various cancer-related substances are contained in raw water water and airborne dust, and the Ames method or its modified reverse mutation test is widely used for monitoring and detecting these substances. . These short-term tests are simple and quantitative. Recently, several effective short-term tests have been proposed to quickly detect chemical DNA damage. These short-term tests are based on the fact that when a specific bacteria is reacted with a chemical substance, the chemical substance is released inside the bacteria.
β induces NA injury and suppresses the accompanying SO8 reaction.
-D-galactosidase activity. Therefore, if it is possible to measure the activity of β-D-galactosidase generated within the bacterial cells, it will be possible to determine whether a chemical substance is a carcinogenic substance or not.

In order to measure the substances present or produced within a cell, in many cases the cell is subjected to some kind of treatment to destroy the cell membrane and release the substance outside the cell, a so-called extraction operation. There is a need. Therefore, methods using extractants such as surfactants, organic acids, inorganic acids, or organic solvents are being considered as a method for destroying cell membranes. For example, to briefly explain the method using an organic solvent, first, 1 m of a sample (2) is mixed with ethanol (99°5), which is an organic solvent.
%) 5xQ and shaken for 1 minute while boiling (78°C) this mixture to extract the target substance.

Next, a blow drying process is performed for 10 minutes to remove the organic solvent, the extract is diluted by dissolving it in 5zC distilled water, and the target substance in the sample is measured.

D1 Problems to be Solved by the Invention However, since the method using an extractant involves reagent treatment, there are the following problems.

■Since these reagents affect the final detection system and reduce detection sensitivity, operations such as neutralization, extraction, centrifugation, and dilution are required during the processing process to avoid their inhibition. Analysis takes time.

■Membrane destruction efficiency is greatly affected by the concentration of the reagent used, so adjustment of the reagent is required, which takes time and effort.

(2) Since the analysis process requires several operations such as neutralization, extraction, centrifugation, and dilution, a complicated mechanism is required to automate these operations.

An object of the present invention is to provide a method for measuring a target substance with simple processing steps, short time required for analysis, and high analytical precision for a target substance.

Means for Solving Problem E1 Ultrasound has long been known to have various applications such as breaking chemical bonds in polymers. Since membranes such as cells are mainly composed of proteins and lipids, we thought that this method could also be applied to the destruction of membranes made of higher-order structures of polymers.

Specifically, the present invention is characterized in that a solution containing cells is irradiated with ultrasound to destroy cell membranes and a substance to be measured inside the cells is extracted, and then this substance to be measured is measured.

F. Example (Example 1) Thaw a cryopreserved sample solution containing E. coli, and make the sample solution 0.
, 6 mQ into a test tube using a manual dispenser such as a pipette. Next, add 0°1i+ to this test tube! #! After adding 5', 7 mQ of phosphate buffer (pH 7.0), 30 W ultrasonic waves are applied for 1 to 2 minutes to destroy the cell membrane of E. coli and extract ATP. Next, 0,5 mQ of this solution was pipetted into a vial dedicated to the measuring device, and the reaction reagent containing the luminescent reagent was added to 0,5 mQ.
mQ is automatically dispensed and the luminescence amount is measured for 30 seconds immediately after dispensing.

The luminescence method described above is called the bioluminescence method, and as shown below, it is a simple method for determining the amount of ATP based on the amount of light generated by the reaction between ATP, luciferin, and luciferase. It is a highly sensitive measurement method. The light of fireflies corresponds to this.

Adenylluciferin + O*
>7denyloN silcipherin+ATPffi was measured in this way at various concentrations of E. coli, and a calibration curve representing the relationship is shown in FIG. For a sample solution in which the E. coli concentration is unknown, the ATPffl can be measured and the E. coli concentration can be determined based on that value and a calibration curve.

(Example 2) Salmonella bacterium was added to LB double base with various nutrients)
The culture was cultured overnight, and the culture solution was diluted to LB times so that the absorbance at a wavelength of 600 nm was 0.1. Test solution 3.0. thus obtained. Chemicals that cause DNA damage to vρ (4-nitroquinoline-N-oxide (4-nitroquinoline-N-oxide)
Add NOQ)) 0.051 and incubate at 37°C for 2 hours. This produces β-D-galactosidase in E. coli.

Next, 5.71 of O, l mQ/Q of phosphate buffer (pH 7, 0) was added to the cultured test solution 0°6 mQ, and then 30
Apply W ultrasonic waves for 1 to 2 minutes. After that, add 0.25z12 of this reaction solution, 2.25mf2.0 of Na-PBS (phosphate buffer) of 0.1mQ/Q, 30.5mQ of NaN of shoulder Q/Q, and lactose of 0.25iof2/12. .

Add OmQ and 6U/xQ (U is a unit representing enzyme activity) of glucose oxidase IN, Q, and add 3
Enzyme reaction was performed at 7°C for 2 hours. In addition, Na-PBS2.2
The reason why 5 mQ was added was to keep the ratio of lactose, xirecose okindase, and reaction solution constant. Thereafter, 0.2 m (1 m) of this enzyme reaction solution was dispensed into a vial dedicated to a chemiluminescence measurement device (Meidensha Luminometer UPD-8000), and 2
Luminol of /Q 0,5 m (l and 6 x 10-3 no
(Add 1/Q potassium ferricyanide 051,
After 30 seconds, the amount of light emitted was measured.

Here, the enzymatic reaction and chemiluminescence reaction in the above process are as follows.

<Enzyme reaction> β-D-Calactosodase lactose 10HzO> a-D-gluco-human-10β-
D-Nuctose a-D-Glucose 〈□〉 β-
D-glucose glucodoxide oxidase β-D-glucone 10t+HtO-> D-Gelconte 11202 Chemiluminescent reaction> 7 Potassium eryzoanide N20. + Luminol □〉 Aminophthal 1N2 + N
20+ Figure 2 is a calibration curve showing the relationship between a known amount of β-D-galactosidase activity and the amount of luminescence, and the results of quantifying 4-nitroquinoline-N-oxyto using this calibration curve. It is shown in Figure 3. Note that the β-D-galactosidase activity concentration and the 4-nitroquinoline-N-oxide concentration are in a proportional relationship, and the constant is determined by the type of enzyme reaction, reaction time, etc.

(Example 3) The same operation as in Example 2 was performed up to the step of irradiating ultrasonic waves, and after that, 0 nitrophenyl-β-D-galactopyranoside (ONPC) was added to the reaction solution 2, 171 (l). 0.
Add 2 mN and incubate at 37°C for 10 minutes. 1λ0ρ/Q of Na as a reaction stop solution after culturing.

lπg of CO3 is added and the absorbance is measured at 420 nm. The measurement results are shown in FIG. 4, and the amount of 4NQO can be determined by examining the calibration curve shown in this figure and the absorbance.

(Example 4) Salmonella sinensis was cultured overnight in LB leased land (trusted land equipped with various nutrients), and the culture solution was diluted with LB times so that the absorbance at a wavelength of 600 nm was 0.1. .

A chemical substance (4-nitroquinoline-N-oxide (4NQO)) 00511 (!) that causes DNA damage is added to the test solution 3°0tttQ obtained in this way, and the mixture is incubated at 37°C for 2 hours. D-galactosidase is produced.

Next, 0.61 of the cultured test solution was dispensed into a vial, and 0.61 was added to the vial. Imo (1/Q phosphate buffer (pH 7,
Add 1.9 mg of 0). Prepare 12 vials containing the sample solution obtained in this way, and 6 of them
The remaining 6 pieces will be irradiated with IOW ultrasound and the remaining 6 pieces will be irradiated with IOW ultrasound.
For each piece, 30W ultrasonic waves were irradiated and tJ was applied for 6 minutes.
The irradiation time for each sample was set at 0°1.2, 3.5, and 10 minutes. Next, add 0NPC0.2d to each vial and incubate at 37°C for 20 minutes. After that, 1 m (i) of 1/12 Na 2 CO3 was added as a reaction stop solution, and 4
Measure the absorbance at 20 nm. The measurement results are shown in FIG. In the figure, the black circles are for the case where the ultrasonic output is 30W, and the white circles are for the case of IOW. As can be seen from this figure, the absorbance is highest when the ultrasonic output is 30 W and the irradiation time is 1 to 2 minutes.

In the above, the cells targeted by the present invention may be either microorganisms or somatic cells, and examples of the microorganisms include bacteria such as Escherichia coli and Salmonella, filamentous fungi, yeast, amphiphiles,
Examples include unicellular algae and protozoa.

Substances to be extracted include both substances originally existing within cells and substances produced by external stimulation. The former substances originally existing in cells include metabolites, proteins, nucleic acids, lipids, etc. Among these, metabolites include nucleotides such as adenosine triphosphate, nucleosides, amino acids, hormones, and the like. The latter substance is an enzyme such as β-D-galactosidase.

In order to qualitatively or quantitatively quantify a target substance, an absorption method, a fluorescence method, a bioluminescence method, a chemiluminescence method, or the like can be used.

I-I. Effects of the Invention According to the present invention, since the cell membrane is destroyed by ultrasonic irradiation to extract intracellular substances, the processing process is simple, and therefore it is easy to automate and does not require complicated processing. No processing mechanism required. In addition, the time required for preparing and handling reagents can be shortened, and the target substance can be measured with high accuracy.

[Brief explanation of the drawing]

Figure 1 is a graph showing the relationship between ATP concentration and bacterial cell concentration.
Figure 2 is a graph showing the relationship between luminescence amount and β-D-galactosidase activity; Figure 3 is a graph showing the relationship between luminescence amount and 4NQOit; Figure 4 is a graph showing the relationship between absorbance and 4NQO; FIG. 5 is a graph showing the relationship between absorbance and ultrasound irradiation time. Figure 1 Relationship diagram between ATPa level and bacterial cell concentration Figure 4 Relationship diagram between luminescence amount and 4 N Q O Relationship diagram with amount 4 NQO amount (ng/cell)

Claims (1)

[Claims] (1) A method for measuring a substance in a cell, which comprises irradiating a solution containing cells with ultrasound to destroy the cell membrane and extracting a substance to be measured inside the cell, and then measuring the substance to be measured.