JPS6190047A - Method for automatic measurement of biochemical reaction - Google Patents

Abstract

PURPOSE:To obtain a signal to monitor and control momentarily the progressing condition of biochemical reaction in real time by measuring the change of the electrical conductivity of the living material in a reaction vessel. CONSTITUTION:Electrodes 9, 10 for measuring conductivity are inserted in the reaction vessel 7 and are connected to an oscillator 8. AC current from the oscillator 8 passes through the material in the vessel 7, the electrode 10 and a detecting resistor 11 and returns to the oscillator. The value proportional to the electrical conductivity of the material in the vessel 7 is obtd. from the AC voltage at both terminals of the resistor 11 when the measurement is made by maintaining the specified voltage of an AC power source. The signal taken from the resistor 11 is passed through an amplifying detector 12 and the change of the electrical conductivity with time is recorded in a recorder 13. The output of the detector 12 is compared with a set value in a comparator 14 which delivers a control signal through signal transmission lines 15, 16. The C9, C10 of the equiv. circuit for the current flowing between the electrodes 9 and 10 are he static capacity between the electrodes 9, 10 and the material in the vessel 7a. The progressing condition of the chemical reaction in the vessel 7 is monitored and discriminated by measuring momentarily the electric resistance R of the material in the vessel 7.

Description

[Detailed Description of the Invention] (a) Industrial Application Field This invention relates to a method that makes it possible to detect the progress of a reaction in a chemical reaction tank by measuring electrical conductivity and to control the reaction. .

(b) Conventional technology When measuring and controlling the progress of a reaction in a reaction tank, a sample is taken out from the reaction tank and subjected to chemical analysis, and judgments are made based on measured values such as component ratios. □ This method lacks speed because chemical analysis requires time. In addition to chemical analysis, direct reading using a dissolved oxygen meter or PH meter can also be used, but these methods have the disadvantage that they can only measure specific elements and cannot be measured unless they are in a liquid state.

Chemical analysis is also carried out for the measurement of AUTOLYS (autolysis, autolysis) in yeast and the like, but it has the same shortcomings as mentioned above, such as lack of rapidity.

Judgments have also been made based on the softening of the appearance with the naked eye, but this cannot be said to be a quantitative measurement.

When producing peptide seasonings by autolyzing meat or fish, it is necessary to stop the reaction within a tolerance of only 30 minutes out of several days of reaction time, but measurement methods such as chemical analysis require less time. It is inappropriate because it takes a lot of time.

The results of an experiment conducted by Masayoshi Takakuwa et al., who measured changes over time in compressed baker's yeast using conventional techniques, are shown below. (Reference 1) The results of measuring the ultraviolet absorption of a yeast washing solution prepared from yeast stored at 30°C and the pH of the washing solution are as follows.

Table 1 Changes in the amount of ultraviolet rays absorbed by compressed yeast stored at 30°C at 260.280 nm Absorption 0 3, 7 2.4 5.33
6.0 -'3.9 6.06
10.2 6.4 7.48 1
3.1 8.0? , 610 5
7.1 36.0 8.711 70
．． 3 43.0 8.5* Softening of the yeast was observed after 10 days of storage, at which time the absorbance of the washing solution at 260 nm and 280 nm rapidly increased. Furthermore, when the yeast softens, the cell wall ruptures and the contents leak out, which was observed in electron micrographs.C A typical measurement cell with a commercially available conductive needle used for water quality testing, etc. is shown in Figure 1.

In FIG. 1, (1) and (2) are measuring electrodes, which are usually made of platinum, and the inner surfaces are treated with platinum black to increase the effective surface area.

The cross-sectional area of measurement electrodes (1) and (2) is usually 1 cr+? (
Diameter 1.13 crr? ), measuring electrode (1) and (
2), and by measuring the conductance between the electrodes, the conductive dust in the solution can be determined by Siemens/cTn.

(3) is a glass cylinder, and the glass is the measuring electrode (1), (
It also covers the outer surface of 2). (4) is a hole made in the outer peripheral surface of the glass cylinder (3). The solution enters the cylinder (3) through this hole and the conductive dust is measured. Two to three holes (4) are usually formed on the outer peripheral surface of the cylinder. (5) and (6) are coated lead wires to the electrodes, which are connected to the measuring electrodes (1) and (2), respectively. Apply an AC electric field (approximately 100H2 for measurement of low conductivity solutions, approximately 1KH2 for measurement of high conductivity solutions) from an AC power supply to the measurement electrodes (1) and (2) through lead wires (5) and (6). hand,
Calculate the amount of conductive dust in the solution from the applied voltage and flowing current.

Measurements using such a conductive dust cell can only measure liquid conductive dust, and cannot measure mushy substances such as fermented animal and fish meat, or solid substances such as compressed yeast. .

(c) Problems to be solved by the invention Conventional systems still have various problems as mentioned above. The present invention attempts to solve these problems by a new method.

B) Means for solving the problem The present invention makes it possible to monitor the progress of biochemical reactions in real time and obtain signals to control the reactions by measuring electrical conductivity. It is.

A method for measuring electrical conductivity (hereinafter sometimes abbreviated as electrical conductivity) according to the present invention will be explained based on the drawings.

Figure 2 is an explanatory diagram showing the method for measuring electrical conductivity in a biochemical reaction tank. Two electrodes (9), α0) for measuring conductive dust are inserted into the reaction tank (7), and an oscillator ( 8), and the alternating current returns to the oscillator (8) from the oscillator (8) through the electrode (9), the substance in the reaction tank, the electrode α0), and the detection resistor (1υ).

The output impedance of the oscillator (8) of the AC power source and the detection resistor (11) are kept sufficiently lower than the resistance value of the substance in the reaction tank (7), and when the voltage of the AC power source is kept constant, the measurement is performed. The alternating current voltage across the detection resistor 01) has a value proportional to the electrical conductivity of the substance in the reaction tank.
- The signal taken in from the detection resistor passes through the amplification detector (12) and records the change in electrical conductivity over time on the recorder 03). Also, the amplified detector (the output of the suppressor is comparator 04)
The signal transmission line 051 (16
) to send a control signal through O If we attempt to measure conductive dust using direct current, we will not be able to obtain accurate values due to polarization effects in the electrodes, so we use alternating current for measurement.

Furthermore, even if the substance in the reaction tank (7) is a solid, non-flowing substance such as yeast, in the case of alternating current, the capacitance (
CAPACITIVE) connection allows current to flow and conductive dust to be measured.

FIG. 3 shows an equivalent circuit of the current flowing between the electrode (9) and aO+ in FIG. 2. (C9), (C1o' in Figure 3)
) is the capacitance between electrode (9) and electrode (10) in FIG. 2 and the substance in the reaction vessel. (2) is the electrical resistance of the substance in the reaction tank.

(E) Function The present invention monitors the progress of the chemical reaction within the reaction tank by measuring this electrical resistance (2) from time to time.

The positive and negative ions of the substances in the reaction tank carry the current, and the third
This produces electrical resistance (2) in the figure.

In the case of the compressed yeast mentioned in Reference 1, while the yeast is fresh, the number of ions that participate in electrical conduction is small, but when the cell wall ruptures and the contents of the yeast leak out, the number of ions that participate in electrical conduction increases. The number of resistors increases and the electrical resistance type becomes smaller.

In the present invention, which attempts to monitor the progress of a chemical reaction from the change over time in the measured value of resistance (R) (or electrical conductivity σ, which is the reciprocal of (2)), the measurement circuit is equipped with electrical resistance ( 2) and the impedance (C9ωΣ1
, (C1oωΣ1, and the detection resistor (1
The resistance value T of 1) needs to be set to a sufficiently smaller value than the electrical resistance (2).

If ω is the angular frequency of the AC power source used for measurement, it is given by: Therefore, V(t) is the electrical conductivity of the substance in the reaction tank σ(t) −CR(t))−1 In order to be considered to be approximately proportional to T dry (1), (C9ω
)-1<<R(t), (C1oω)-'<<R(t)
, must be.

Here, the electrical resistance (2) of the substance in the reaction tank and the voltage V developed across the detection resistor T are functions of time t to emphasize that they change over time according to the progress of the chemical reaction. To make this clear, they were written as R(t) and V(t).

The type and number of ions that contribute to electrical conduction change according to changes in the state of substances and chemical composition in the reaction tank, so the progress of chemical and biological reactions can be monitored by measuring electrically conductive dust. It is possible.

Unlike chemical analysis, which does not require time to measure, it is characterized by rapid measurement, and since measured values are still obtained in the form of electrical signals, it has the advantage that control signals can be automatically extracted.

(f) Example The present invention will be explained in detail below.

FIG. 4 is an explanatory diagram of an embodiment of the method of measuring the electrical conductivity of a reaction vessel according to the present invention.

The electrode (
19) and (20) have been inserted. The filling depth of baker's yeast is 10 mm, the diameter of the electrode is 2 mm, the insertion depth into the baker's yeast is 5 mm, and the distance between the electrodes is 2 mm.
FIG. 5 shows the results of measuring conductive dust over time by applying an alternating current electric field of 500 H2 between the electrodes.

The horizontal axis shows elapsed time in units of time, and the vertical axis shows conductive dust in units of microSiemens←壬=Gomojiao. In the case of baker's yeast for which this measurement was performed, approximately 80
A whitish color was observed over time, and softening was observed after about 130 hours. It can be seen in FIG. 5 that the conductive dust begins to change prior to this change in appearance.

The oscillator (8) shown in Figure 2 for measuring conductive dust, the detection resistor (11) shown in Figure 2, and the amplification detector (12)
For 1, a commercially available conductivity gauge may be used, or a circuit suitable for measurement may be manufactured.

FIG. 6 shows a block diagram of the prototype measurement circuit. In the figure, (21) is a clock pulse oscillator, for example 1.
Generates a 2MH2 pulse, and uses a frequency divider to generate a pulse of 1/2, for example.
When divided by 30, the output from frequency divider 128) generates one pulse approximately every 15 minutes. (g9+ is a timing circuit and a relay, and the relay is closed for 10 seconds, for example, every time a pulse is received from the frequency divider □□□). (Country) and examples are terminals to measurement electrodes.

is a resistor for detecting the current flowing in the circuit. Tsuyoshi and (2
71 are an amplification detector and a recorder, respectively. (22) is an oscillator, (eg 500) (Z, 1 volt, output impedance 10 ohms).

When measuring using this circuit, a current approximately proportional to the conductivity inside the reaction tank flows for 10 seconds every 15 minutes, and the recording on the recorder +Z7) is as shown in Figure 7, for example. .

1 In this way, if the current is passed for a short period of time and the measurement is carried out, the disturbance caused by passing the current through the reactant can be reduced. Output voltage to recorder (recorder) and terminal -
) and the resistance value between the terminal example and make a calibration table.
From the records recorded by the recorder, changes in the electrical conductivity of the material over time can be read.

Looking at the example in FIG. 7, it can be seen that the conductivity is constant immediately after the start of measurement, but after 80 hours has passed, the conductivity increases with time.

The electrodes mainly used in the present invention are cylindrical electrodes. This has the advantage that it can be used to penetrate not only liquid substances but also solid substances (for example, pressed yeast).

The relationship between the electrical resistance (8) measured using such an electrode and the electrical chamber conductivity of the material (←) is R-±・l! There is a relationship of nD-' πσ a . Here, In is a natural number, ■) is the distance between the two electrodes, and a is the radius of the electrode. Concentration of aqueous solution of magnesium chloride 5 x 10-4 motv/1 to i
Conductivity measured with a standard conductivity cell (as shown in Figure 1) and with a cylindrical electrode (as shown in Figure 4) in the range x i o'-2 mol/l. is plotted on a log-logarithmic scale as a function of the concentration of the electrolyte solution in FIG.

At each concentration, the conductivity measured with a cylindrical electrode is the same as the conductivity measured with a conductivity cell (specific conductivity microSiemens/cI
r+), which shows that measurements with cylindrical electrodes are sufficient for relative measurements of conductivity.

As described in the detailed description of the invention, conductivity measurements are made with alternating current. As expressed in the equivalent circuit shown in Figure 3, the exchange of current between the electrode and the substance to be measured takes place through the capacitance (C1), and its impedance (Cω
)-1 is such that the electrical resistance of the material is also much smaller (it is desirable to set C1 large).

To this end, it is effective to make the microscopic surface area much larger than the macroscopic surface area by using platinum electrodes treated with platinum black.

Further, when the resistance value of the material is low, as a method of further reducing (Cω)-1, ω is set to a large value of several tens of KH2. However, if a relatively high frequency is used, the fact that current flows through stray capacitance cannot be ignored, so the upper limit of the frequency used is about IMH2. Although it is easier to measure at lower frequencies, the lower limit of low frequencies is about 100H2 since (Cω)-1 can be ignored compared to the electrical resistance (8).

(g) Effects of the invention According to the present invention, the following effects can be obtained.

(1) It is possible to measure biochemical reactions of liquid and solid substances in real time, and thereby the reactions can be automatically controlled.

In order to monitor the progress of a chemical reaction, taking a sample and conducting a chemical analysis causes a time delay.
However, according to the present invention, the measured values can be displayed in real time, and since the measurements are output in the form of electrical signals, this signal can be used to automatically control the reaction tank. It becomes possible.

(2) Disturbance due to measurement can be reduced.

Compared to the disturbance caused to the system to be measured by taking out a sample for measurement, the disturbance caused is small because only a minute electrode is inserted.

(3) Quantitative measurement becomes possible.

In cases such as changes in yeast in bread over time, the method of the present invention allows for quantitative control, compared to the method in which experts judge and control based on observation.

[Brief explanation of drawings]

Figure 1 shows a cell for measuring electrical conductivity. FIG. 2 is a block diagram of conductive dust measurement in a biochemical reaction tank. Figure 3 is an equivalent circuit for measuring conductive dust. FIG. 4 is an example of measuring the electrical conductivity of a reaction tank. FIG. 5 is a diagram showing changes in conductive dust in baker's yeast over time. FIG. 6 is a block diagram of an embodiment of a circuit for measuring conductive dust. Figure 7 is an example of measurement of changes in conductive dust over time. FIG. 8 is a diagram showing the conductive dust measured by the conductive dust cell and the conductive dust measured by the cylindrical electrode as a function of the concentration of the electrolytic value solution.

Claims (1)

[Claims] Biochemistry characterized by measuring the change in electrical conductivity of the biological material in the biochemical reaction tank by applying an alternating current electric field, and determining the chemical reaction state of the biological material based on the change in the measured value. Automatic reaction measurement method.