JPH03123481A - Apparatus for automatic simultaneous determination of cell concentration and cell activity - Google Patents

Abstract

PURPOSE:To determine the cell concentration and cell activity in a cell suspension liquid at the same time by placing a chemiluminescence detector in a detection channel and an absorbance detector in a sample supplying channel, etc., mixing a sample supplied through a sample channel and a reagent supplied through a reagent channel in a mixer and transferring the mixture through the detection channel. CONSTITUTION:A sample liquid S containing suspended cells in injected into a carrier liquid C1 and an ATP extraction liquid R is injected into the carrier liquid before a mixer 11. A luminescent reagent E containing luciferin and luciferase is injected into a carrier liquid C2 and the sample liquid is mixed to the reagent liquid with a mixer 12. A sample channel 2 is joined to a reagent channel 4 and the mixture is passed through the mixer 13 to a chemiluminescence detector 6 and an absorbance detector 7 to determine the ATP concentration and turbidity. The ATP concentration in the cell and the concentration outside of the cell can be separately determined by injecting the ATP extraction liquid R and without using the injection of the liquid.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to an apparatus for simultaneously measuring cell concentration and cell activity of a cell suspension.

[Conventional technology]

Conventionally, fermentation processes and the like have been monitored by measuring cell concentration and cell activity in cell suspensions.

The cell concentration of the cell suspension is measured by microscopy, colony counting, dry weight measurement, turbidity measurement, etc.
On the other hand, cell activity in cell suspensions can be measured by measuring the cell respiration rate.
It is determined by methods such as measuring enzyme activity and measuring cellular adenosine triphosphate (ATP) concentration.

However, since manual analysis methods have traditionally been mainly used to measure cell concentration and cell activity, they require a lot of time, are complicated to operate, and are susceptible to human error. there were. In particular, when measuring both cell concentration and cell activity, it is necessary to measure them continuously using the same sample because it is not possible to obtain a correlation between the two using separate samples. Because they change over time due to life activities, there was a problem that while one was being measured, the other would change.

Furthermore, analysis of ATP concentration is effective and widely used for measuring cell activity. This analysis shows that luciferin and M
A by catalysis of luciferase in the presence of g2+
This method decomposes TP into adenosine monophosphine (AMP) and measures the ATP concentration from the amount of light emitted by the following reaction: g AMP + orluciferin + Co + ppi + hν
However, in the conventional manual analysis method, a luminescent reagent containing luciferin and luciferase and an ATP extract are quickly added to the sample solution collected in a container almost simultaneously, and the whole container is placed in a photometer. Addition takes time, and it is difficult to accurately determine the intracellular ATP concentration, which fluctuates over time due to life activities. Furthermore, in the conventional method described above, the total concentration of ATP released from the inside of the cell and extracellular ATP is determined by the ATP extract, and it is easy to calculate the intracellular ATP concentration and the extracellular ATP concentration separately. It was difficult to measure.

[Problem to be solved by the invention]

The present invention provides an apparatus that almost simultaneously and automatically measures cell concentration and cell activity in a cell suspension, which is a sample liquid, by turbidity measurement and ATP concentration measurement, respectively. Intracellular ATP concentration and extracellular A
It is an object of the present invention to provide an apparatus that can measure the TP concentration and the TP concentration separately.

[Means to solve the problem]

In order to achieve the above object, the automatic cell concentration and cell activity measurement device of the present invention has a flow analyzer that includes a sample liquid supply path for injecting a sample liquid in which cells are suspended into a carrier liquid, and a sample liquid supply path for injecting a sample liquid in which cells are suspended into a carrier liquid. A sample flow path that transfers the diluted sample liquid through a mixer, a reagent flow path that injects a luminescent reagent containing luciferin and luciferase into a carrier liquid and transfers it through the mixer, and a merging of the sample flow path and the reagent flow path. and a chemiluminescent detector provided in the detection channel, and an absorbance detector provided in any of the sample liquid supply channel, the sample channel, or the detection channel, Furthermore, the present invention is characterized in that an extract supply path is provided for injecting an ATP extract into the sample flow path before the mixer, if necessary.

[Effect]

In the present invention, by adopting a flow analysis method and implementing the device, it is possible to quickly collect sample liquid, quickly inject ATP extract and luminescence reagent, measure luminescence amount, and measure turbidity in parallel with these. In addition, intracellular and extracellular ATP concentrations can be measured separately and accurately as necessary.

To explain the apparatus of the present invention with reference to FIG. 1, this measuring apparatus includes a sample liquid supply path 1 for injecting a sample liquid S in which cells are suspended into a carrier liquid C1, and a sample liquid S injected into the carrier liquid C1 to be mixed. A sample flow path 2 for transferring the sample through the mixer 11, an extract supply path 3 for introducing the ATP extract into the sample flow path 2 before the mixer 11, and a luminescent reagent E containing luciferin and luciferase in the carrier liquid C2. A reagent flow path 4 that is injected, mixed in a mixer 12 and transferred, and a detection flow that merges the sample flow path 2 and the reagent flow path 4 and is transferred to the chemiluminescence detector 6 and absorbance detector 7 via the mixer 13. Road 5
It is equipped with.

When measuring extracellular ATP concentration and turbidity using the above measurement device, the sample liquid S in which cells are suspended is supplied to the injector 9 by the pump 15, and the injector 9 is rotated to form a flow path for the carrier liquid c1. By switching to , the cut sample liquid S is sent sandwiched between the carrier liquid C1 pumped by the pump 14 and mixed in the mixer 11.

On the other hand, the luminescent reagent E is supplied to another injector 10 by the pump 18, and the luminescent reagent E cut out by the injector 10 in the same way as above and F is sandwiched between the carrier liquid c2 pumped by the pump 17 and is transferred to the mixer 12. sent and mixed. The carrier liquid 01 containing the sample liquid S that has passed through the mixer 11 and the carrier liquid C2 containing the luminescent reagent E from the mixer 12 merge and flow through the detection channel 5, and are mixed in the mixer 13 and react. Chemiluminescence detector 6 and absorbance detector 7
It passes through and is discharged. In the chemiluminescence detector 6, the above AT
The amount of luminescence accompanying the decomposition reaction of P is detected, and the absorbance detector 7 detects the sample solution S mixed and diluted in the mixer 11.13.
The absorbance of is detected and both are sent as electrical signals to the processing device 8 for data processing, and the difference between the obtained value and the blank value is displayed or printed out as the extracellular ATP concentration and turbidity.

In addition, when measuring the intracellular ATP concentration, before the sample liquid S injected into the carrier liquid C1 by the injector 9 reaches the mixer 11, it is extracted from the extract liquid supply path 3 by the ATP extraction liquid pump 16. By injecting the sample into the sample channel 2, the cells are destroyed to release ATP within the cells, and the amount of light emitted by the reaction with the luminescence reagent E is measured in the same manner as described above to determine the total ATP concentration inside and outside the cells. This whole A
The intracellular ATP concentration can be determined by subtracting the extracellular ATP concentration measured as described above from the TP concentration.

In this case, turbidity is not measured.

In this way, in the device of the present invention, each pump and injector can be combined depending on the object to be measured, for example, the pump 15.18 and the injector 9.10 can be operated at the appropriate timing and/or the length and inner diameter of the flow path can be adjusted. As a result, the sample Is and the luminescent reagent E can be combined in the detection channel 5 and reacted, so that the blank value, extracellular ATP concentration, turbidity, total ATP concentration, and intracellular ATP−@=@+ concentration are short. It is measured continuously within a period of time (for example, within 5 minutes).

The absorbance detector 7 for measuring turbidity may be installed in any of the sample liquid supply path 1, sample flow path 2, or detection flow path 5 depending on the concentration of the sample liquid S, and the measurement wavelength is usually It is 500-700 nm.

〔Example〕

Example 1 The ATP concentration and turbidity of a dry yeast suspension were measured using the measuring device shown in FIG.

The dry yeast suspensions used were samples prepared by suspending dry yeast in distilled water and diluting it to various concentrations. Carrier liquid C1 was pH 7 with 1 mM KDTA added.
,75 HEPES buffer, carrier solution C2 is 1mM
pH 7,75 with EDTA and 10mM Mg2+
HIIGPES buffer, luminescence reagent E was pH 7 containing 0.2mM luciferin and 1% luciferase.
, 75 Hz PIICS buffer, ATP extraction extract surfactant.

The ATP concentration and turbidity measurement procedure is as described above, but injectors 9 and 10 are linked to inject the same sample liquid twice in succession to increase the measurement accuracy, and the length of the flow path is By adjusting each sample solution S and luminescent reagent E
and were reacted by merging at the same timing in the detection channel 5, and the same sample was measured twice in succession. For the measurement of extracellular ATP concentration and turbidity, the flow rate of carrier liquid C1 was
．． 5ml/min % carrier liquid C2 flow rate 0.9
6 ml/min, and for the measurement of total ATP concentration, the flow rate of carrier liquid C1 was 0.5 ml/min.
The flow rate of the carrier liquid C2 was set to 0.35 m1/min, the ATP extraction liquid boundary 50- was injected, and the absorbance was set at wavelength 6.
Measured at 00 nm.

Figure 2 shows the response curves of the outputs of the chemiluminescence detector 6 and the absorbance detector 7, which were measured twice consecutively.
Beak B corresponds to the total ATP concentration of the dry yeast suspension in g/l, Beak C corresponds to the extracellular ATP concentration of the same suspension, Beak C corresponds to the turbidity of the same suspension, and Beaks D, E, and F correspond to the total ATP concentration of the dry yeast suspension. 5g/
Total ATP concentration of 1 dry yeast suspension, extracellular AT
Corresponding to P concentration and turbidity, beak GSH, 2g/l
The total ATP concentration, the extracellular ATP concentration, and the turbidity of the Dryist suspension were determined for each sample in about 5 minutes.

FIG. 3 shows the relationship between the dry yeast concentration obtained in each of the above measurements, intracellular ATP concentration (curve A), extracellular ATP concentration (curve B)%, and turbidity (curve C).

Example 2 Using the measuring device shown in Figure 1, A'T was measured for the yeast culture solution at regular intervals after the start of culture under the same conditions as in Example 1.
P concentration and turbidity were measured.

In addition, yeast culture is carried out using S. cerevisi as bacterial cells.
A solution of 4% glucose, 1% polypeptone, 0.5% yeast extract, and 0.2% potassium hydrogen phosphate and magnesium sulfate dissolved in pure water (
pH 6.0).

Figure 4 shows the yeast culture time and the turbidity of the culture solution (curve A)1.
The relationship between viable bacterial count (curve B), intracellular ATP concentration (curve C), and glucose concentration (curve D) is shown. In addition, the number of viable bacteria is determined by diluting the culture solution with sterilized distilled water to an appropriate concentration, and inoculating 1- into an agar medium using the above medium.
The number of colonies produced after culturing for 30 hours or more was determined by colony counting method. Glucose concentration was measured with a glucose meter using a Glutosum electrode.

As can be seen from Figure 4, until the glucose concentration reaches zero and cell growth stops, turbidity, viable cell count, intracellular AT
The P concentration also increases with the same tendency.

When the growth stops and the cell becomes stationary, the turbidity and the number of viable bacteria remain almost constant, but the intracellular ATP concentration gradually decreases. This indicates that turbidity well represents cell concentration and intracellular ATP concentration well represents cell activity.

Example 3 As in Example 2, the ATP concentration and turbidity of the E. coli culture solution were measured at regular intervals after the start of culture.

In addition, E. coli was cultured using E and Co11 as bacterial cells.
Meat extract 1%, polypeptone 1% and NaC as medium
This was carried out using a solution (pH 7.2) in which 0.2% of 1 was dissolved in pure water.

Figure 5 shows the culture time of E. coli and the turbidity of the culture solution (curve A).
, viable bacterial count (curve B) N and intracellular ATP concentration (curve C)
showed the relationship between The number of viable bacteria was determined by colony counting method G described in Example 2. It can be seen from FIG. 5 that the turbidity well represents the cell concentration and the intracellular ATP concentration well represents the cell activity.

〔Effect of the invention〕

According to the present invention, the turbidity, extracellular ATP concentration, and intracellular A
TP concentration and total ATP concentration can be measured separately, almost simultaneously, and automatically.

Therefore, the present invention can be applied to, for example, a fermentation process. r') Very effective for real-time monitoring.

[Brief explanation of drawings]

FIG. 1 is a schematic diagram showing a specific example of the apparatus of the present invention. Figure 2 shows the response curve of ATP concentration and turbidity output in Example 1, and Figure 3 shows the dry yeast concentration of the dry yeast culture solution in Example 1 and the intracellular ATP concentration (curve A). , extracellular ATP concentration (curve B), and turbidity (curve C). Figures 4 and 5 show the turbidity (curve A), viable cell count (curve B), and intracellular ATP of the yeast culture solution and E. coli culture solution in Example 2 and Example 3, respectively.
It is a graph showing the relationship between concentration (curve C) and glucose concentration (curve D). 1. Sample liquid supply path 2. Sample flow path 3. Extract liquid supply path 4. Reagent flow path 5. Detection flow path 6. Chemiluminescence detector 7
・・Absorbance detector 8・・Processing device 9.10・・
Injector 11.12.13... Mixer 14.15.16.17.18... Pump Figure 1 Figure 2 A Figure 3 1 0 00 Dry Ist Concentration (g/L) 0 ? 0 0 Time (minutes) Figure 4 Procedural amendment (spontaneous) 1゜Indication of the case Heisei Patent Application No. 261709 3. Relationship with the person making the amendment case

Claims (2)

[Claims] (1) In the flow analyzer, there is a sample liquid supply path that injects a sample liquid in which cells are suspended into a carrier liquid, a sample flow path that transfers the sample liquid injected into the carrier liquid via a mixer, and luciferin and luciferase. A reagent flow path that injects a luminescent reagent containing a carrier liquid into a carrier liquid and transfers it through a mixer, a detection flow path that merges the sample flow path and the reagent flow path and transfers it through the mixer, and a chemical reagent provided in the detection flow path. A simultaneous automatic measurement device for cell concentration and cell activity, comprising a luminescence detector and an absorbance detector provided in any one of the sample liquid supply path, the sample flow path, or the detection flow path. (2) The flow analyzer according to claim (1), further comprising an extract supply path for injecting an ATP extract into the sample flow path before the mixer, and simultaneous automatic measurement of cell concentration and cell activity. measuring device.