JPH10276796A - Physiological activity measuring method and device therefor - Google Patents

Abstract

(57) [Summary] [Problem] To accurately measure a physiological activity such as drug sensitivity in a short time. SOLUTION: A medium to which a physiological activity measurement target substance is added and a medium to which a physiological activity measurement target substance and a reagent for physiological activity measurement are added are prepared, and the amount of dissolved oxygen in both mediums is measured using an oxygen electrode 4. In measuring the physiological activity by comparing the measured dissolved oxygen amounts, the oxygen electrode 4
After applying a bias voltage for measurement to
The physiological activity is measured based on the change rate of the dissolved oxygen amount within the time range from when the predetermined time elapses to when the second predetermined time elapses.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method and apparatus for measuring biological activity, and more particularly, to drug sensitivity to bacteria, cells, etc., and BOD (Biologic).
The present invention relates to a method and an apparatus for measuring a physiological activity such as environmental measurement such as al Oxygen Demand), allergen measurement, DNA amount measurement, and detection of vascular disorder.

[0002]

2. Description of the Related Art Conventionally, a KB disk method has been known as a standard method for measuring drug sensitivity to bacteria, cells, and the like. The KB disk method is a method for visually determining the extent to which the growth of bacteria and cells is suppressed around a filter paper to which a drug has been added in advance. Therefore, it is necessary to grow bacteria and cells to such an extent that they can be visually judged, and there is an inconvenience that the required time becomes extremely long. In particular, such as rapid administration of antibiotics and intraoperative administration of anticancer drugs,
If the drug sensitivity needs to be measured quickly, it cannot be dealt with at all.

In order to solve such inconvenience, bacteria, cells, etc. are inoculated in a liquid medium containing a drug, and the respiratory activity (= oxygen consumption) is measured using an oxygen electrode. A method for measuring sensitivity has been proposed (see JP-A-56-140898).
If this method is adopted, it seems that drug sensitivity can be measured quickly.

[0004]

However, Japanese Patent Application Laid-Open No.
The method described in Japanese Patent No. 140898 simply measures the amount of dissolved oxygen (dissolved oxygen concentration) in a liquid medium. However, unless the drug concentration is extremely high and the activities of bacteria, cells, etc. are completely stopped, As the dissolved oxygen amount decreases with time, and the initial dissolved oxygen amount in the liquid medium is not constant, for example, even if the dissolved oxygen amount is measured after a certain time has elapsed from the start of the measurement, It is almost impossible to accurately measure sensitivity.

[0005]

SUMMARY OF THE INVENTION The present invention has been made in view of the above-mentioned problems, and an object of the present invention is to provide a method and an apparatus for measuring a physiological activity which can accurately measure a physiological activity such as drug sensitivity in a short time. The purpose is.

[0006]

According to a first aspect of the present invention, there is provided a method for measuring a physiological activity, comprising preparing a medium containing a substance to be measured and a medium containing a substance to be measured and a reagent for measuring a physiological activity. The amount of dissolved oxygen in both media was measured using an oxygen electrode, and in measuring the physiological activity by comparing the measured amount of dissolved oxygen, a bias voltage for measurement was applied to the oxygen electrode. Thereafter, a physiological activity is measured based on a change rate of a dissolved oxygen amount within a time range from when a first predetermined time elapses to when a second predetermined time elapses.

According to a second aspect of the present invention, after a bias voltage for measurement is applied to the oxygen electrode as the first predetermined time, the ratio of the transient current which does not depend on the amount of dissolved oxygen is changed. The time until the amount of dissolved oxygen in the medium becomes equal to or less than the second predetermined ratio with respect to the amount of dissolved oxygen in the medium is used as the second predetermined time. It is a method of adopting.

The physiological activity measuring apparatus according to claim 3 measures the amount of dissolved oxygen in both the medium containing the substance to be measured and the medium containing the substance to be measured and the reagent for measuring the physiological activity. An oxygen electrode, and a bioactivity measurement result output means for outputting a bioactivity by comparing the measured dissolved oxygen amount, as a bioactivity measurement result output means, a bias voltage for measurement with respect to the oxygen electrode Is applied, the physiological activity is measured based on the change rate of the dissolved oxygen amount within the time range from when the first predetermined time elapses to when the second predetermined time elapses, and an output is used. ing.

According to a fourth aspect of the present invention, after applying a bias voltage for measurement to the oxygen electrode as the first predetermined time, the ratio of the transient current which does not depend on the dissolved oxygen amount is determined. The time until the ratio becomes equal to or less than the ratio is adopted, and as the second predetermined time, the time until the dissolved oxygen amount in the medium becomes equal to or less than the predetermined ratio with respect to the initial dissolved oxygen amount is adopted.

[0010]

According to the method of the first aspect, a medium containing a substance to be measured and a medium containing a substance to be measured and a reagent for measuring a physiological activity are prepared. Measure the amount of dissolved oxygen using an oxygen electrode,
In measuring the physiological activity by comparing the measured dissolved oxygen amounts, after applying a bias voltage for measurement to the oxygen electrode, a second predetermined time has passed after a first predetermined time has elapsed. Since the physiological activity is measured based on the change rate of the dissolved oxygen amount in the time range until the time elapses, based on whether there is a significant difference in the change rate of the dissolved oxygen amount in both culture media. Physiological activity can be measured. As for the required time, the change rate of the amount of dissolved oxygen in the time range from when the first predetermined time elapses to when the second predetermined time elapses may be measured. In comparison, the required time can be significantly reduced. Furthermore, regarding the accuracy of the physiological activity measurement, the change rate of the dissolved oxygen amount is used, and it is determined whether or not there is a significant difference in the change rate of the dissolved oxygen amount in both culture media. JP-A-56-1
The accuracy can be significantly increased as compared to the method described in US Pat.

According to the physiological activity measuring method of the present invention, after applying a bias voltage for measurement to the oxygen electrode as the first predetermined time, the ratio of the transient current not depending on the dissolved oxygen amount is determined. Adopts the time until the amount becomes less than or equal to the first predetermined ratio, and as the second predetermined time, the time until the amount of dissolved oxygen in the medium becomes equal to or less than the second predetermined ratio with respect to the initial amount of dissolved oxygen or Since the fixed time is employed, the same operation as the first aspect can be achieved.

According to the third aspect of the present invention, a medium to which a substance to be measured for physiological activity is added and a medium to which a substance to be measured and a reagent for measuring physiological activity are added are prepared. Measure the amount of dissolved oxygen with an oxygen electrode,
In outputting the physiological activity by comparing the measured dissolved oxygen amount by the physiological activity measurement result output means,
In the physiological activity measurement result output means, after applying a bias voltage for measurement to the oxygen electrode, the dissolved oxygen within a time range from when a first predetermined time elapses to when a second predetermined time elapses. The physiological activity can be measured and output based on the rate of change of the amount. Therefore, the physiological activity can be measured based on whether or not there is a significant difference in the rate of change in the amount of dissolved oxygen in both media. As for the required time, the change rate of the amount of dissolved oxygen in the time range from when the first predetermined time elapses to when the second predetermined time elapses may be measured. In comparison, the required time can be significantly reduced. Furthermore, regarding the accuracy of the physiological activity measurement, the change rate of the dissolved oxygen amount is used, and it is determined whether or not there is a significant difference in the change rate of the dissolved oxygen amount in both culture media. The accuracy can be significantly improved as compared with the method described in JP-A-56-140898.

According to the physiological activity measuring device of the present invention, after applying a bias voltage for measurement to the oxygen electrode as the first predetermined time, the ratio of the transient current which does not depend on the amount of dissolved oxygen. Since the time until the amount becomes equal to or less than the predetermined ratio is adopted, and as the second predetermined time, the time until the amount of dissolved oxygen in the medium becomes equal to or less than the predetermined ratio with respect to the initial amount of dissolved oxygen is adopted, The same operation as the third aspect can be achieved.

[0014]

Embodiments of the present invention will be described below in detail with reference to the accompanying drawings. FIG. 1 is a block diagram schematically showing one embodiment of the physiological activity measuring device of the present invention, and FIG. 2 is a schematic diagram showing an example of a configuration and an attached state of an oxygen electrode. This embodiment is for measuring drug sensitivity among the physiological activities of bacteria.

In this embodiment, a first measuring cell 1 containing only a liquid medium (for example, a conventionally known liquid medium) and a second measuring cell 2 containing a liquid medium and bacteria.
And a third measurement cell 3 containing a liquid medium, bacteria and a drug are prepared in advance, and an oxygen electrode 4 is provided for each cell. In the oxygen electrode 4, for example, a working electrode 43, a reference electrode 42, and a counter electrode 44 are formed on a ceramic substrate 40 by printing and baking, and unnecessary portions are covered with a resist 41 so that the exposed area of each electrode becomes a predetermined area. It is. And a lead terminal 4 electrically connected to each pole.
5, 46 and 47 are formed. However, an oxygen electrode having a configuration in which the reference electrode 42 is omitted may be employed.

The first, second, and third measuring cells 1, 2,
A bias voltage is applied to the oxygen electrodes 4 provided on the reference numeral 3 by potentiostats 11, 21, and 31, respectively. This bias voltage is, for example, the reference electrode 42
The bias voltage is applied to the working electrode 43. In the state where the bias voltage is applied to the working electrode 43 in this manner, the current output from the working electrode 43 (a current corresponding to the amount of dissolved oxygen, hereinafter referred to as a measurement current) is supplied to the current-voltage converter 12, The A / D converter 51 converts the voltage signal output from the current-voltage converters 12, 22, 32 into a digital voltage signal, and converts the voltage signal output from the current-voltage converters 12, 22, 32 into a digital voltage signal. And the data processing device 52 measures the drug sensitivity.

However, the first measuring cell 1 may be omitted. The operation of the physiological activity measuring device having the above-described configuration will be described in detail with reference to the change over time of the measured current shown in FIG. 3 and the flowchart shown in FIG. In step SP1, the measurement of drug sensitivity is started by applying a bias voltage to each oxygen electrode 4. In step SP2,
It is determined whether a first predetermined time t1 (see FIG. 3) has elapsed since the start of the measurement. If it is determined that the first predetermined time t1 has not elapsed, the determination in step SP2 is performed again. Conversely, when it is determined that the first predetermined time t1 has elapsed, in step SP3, it is determined whether or not a second predetermined time t2 (see FIG. 3) has elapsed since the start of the measurement.
When it is determined that the second predetermined time t2 has not elapsed, in step SP4, the measured current from each oxygen electrode 4 is converted into a voltage signal and converted into a digital voltage signal, and the converted current signal is transmitted to the data processing device 52. Capture, again step SP3
Is determined. Conversely, if it is determined in step SP3 that the second predetermined time t2 has elapsed, the process proceeds to step SP3.
5, two digital voltage signals whose measurement timing is shifted by a predetermined time (for example, 10 minutes) are extracted from the taken digital voltage signals, and in step SP 6, the two digital voltage signals are extracted. The difference is calculated, and in step SP7, the average value (difference average value) of all the calculated differences is calculated.
In, the presence or absence of drug sensitivity is determined by comparing the difference average values corresponding to the first, second, and third measurement cells 1, 2, and 3, and a series of processing ends.

Note that the first predetermined time is a time during which a state in which a transient current is dominant immediately after a bias voltage is applied to the oxygen electrode 4 is eliminated. In other words, the charging current to the electric double layer on the surface of the oxygen electrode 4 and the current for oxygen consumption until the formation of a steady diffusion layer of oxygen near the electrode are reduced, and do not depend on the dissolved oxygen amount. Time (for example, 1 to 30 minutes) until the ratio of the transient current becomes equal to or less than the first predetermined ratio (for example, 20 to 25%), and 5 to 15 when measuring drug sensitivity of bacteria. Minute). However, a time until the current becomes smaller than a predetermined current (for example, a predetermined current empirically determined) may be used.

During the second predetermined time, a stable diffusion layer is formed in the vicinity of the oxygen electrode 4, a current proportional to the oxygen concentration in the solution flows, and the amount of dissolved oxygen in the solution makes it impossible for bacteria to breathe. It is time to decrease to a certain level. In other words, the time until the amount of dissolved oxygen in the solution becomes equal to or less than the second predetermined ratio (for example, 50 to 3%) with respect to the initial amount of dissolved oxygen (for example, one minute to several days, A few hours). However, the time until the oxygen in the solution is almost consumed by the respiration of the bacteria and almost disappears and the oxygen concentration decreasing speed becomes lower than a predetermined speed (for example, a predetermined speed close to zero) may be used.

The first and second predetermined times are the amount of the solution, the area of the electrode, the temperature, the viscosity of the solution, the composition of the solution (medium type), the roughness of the electrode surface, the bacterial species and their concentration, the air in the solution. It is affected by the contact area, pressure, applied voltage, electrode material, type and concentration of the drug, and the like. Therefore, it is preferable to determine in advance empirically based on these factors. FIG. 5 shows that E. coli as bacteria. E.coli, the bacteria concentration is 10E8 cells / cc, the bacteria concentration is 10E7 cells / cc, and the bacteria-free one are prepared, and the measurement result is based only on the measured current value output from the oxygen electrode. It is possible to discriminate between those having a bacterial concentration of 10E8 cells / cc and those having no bacteria, but having a bacterial concentration of 10E7 cells / cc.
It can be seen that it is not possible to distinguish between those without bacteria and those without bacteria.

FIG. 6 shows E. coli as bacteria. co
li and a bacterial concentration of 10E7 / cc,
It is a diagram showing a measurement result based on the rate of change of the measured current value output from the oxygen electrode (the difference between the measured current values at the time when the measurement time is shifted by 20 minutes), prepared without the bacteria, It can be seen that the concentration of 10E7 cells / cc can be distinguished from that without bacteria.

In each of the figures, 0 is a sample without bacteria, 7 is a sample with a bacterial concentration of 10E7 cells / cc,
Reference numeral 8 indicates a bacterial concentration of 10E8 cells / cc. The numbers following 0, 7, and 8 indicate the identification numbers of the oxygen electrodes. A comparison between FIG. 5 and FIG. 6 shows that measurement sensitivity can be increased by performing measurement based on the rate of change of the measured current value.

FIG. 7 is a diagram showing the time-dependent change of the measured current when IPM (imipenem) is adopted as the drug and the concentration (mcg / ml) of IPM is changed. In this state, the presence or absence of drug sensitivity is determined. Can not do. However, as shown in FIG. 8, if the change with time of the change rate of the measured current value is employed, the presence or absence of drug sensitivity can be easily and reliably determined.

FIG. 9 is a diagram showing the change over time of the measured current for measuring the toxicity of 1% ethanol to cancer cells, and FIG. 10 is the change rate of the measured current output from the oxygen electrode (measurement time is It is a figure which shows a temporal change of the difference (measurement value between measured current values) at the time point shifted by 1 minute. In both figures, the closed circles indicate the medium only, the closed triangles indicate the medium and 1% ethanol, and the open circles indicate the medium and the cancer cells (myeloma).
3.3E6 / ml), open triangles indicate medium and cancer cells (my
elloma 3.3E6 / ml) and 1% ethanol
Each is shown.

In this case, whether the change in the measured current with time or the change in the rate of change in the measured current value with time is used.
It seems that the toxicity of 1% ethanol to cancer cells can be determined, but depending on the initial dissolved oxygen concentration, the toxicity of 1% ethanol to cancer cells can be determined by using the time course of the measured current. May not be determined. However, if the time-dependent change in the rate of change of the measured current value is employed, the toxicity of 1% ethanol on cancer cells can be determined regardless of the initial dissolved oxygen concentration.

In the embodiment shown in FIGS. 1 and 2, the number of the third measuring cells 3 is increased, and a drug having a different concentration is added to each of the third measuring cells 3 so that the number of the third measuring cells 3 is increased. Not only the presence or absence of sensitivity but also the concentration of a drug that can be expected to have a therapeutic effect can be measured. As is clear from the above, the time required for measurement can be reduced to 1 hour or less, so that it can be suitably applied to uses such as rapid administration of antibiotics and intraoperative administration of anticancer agents.

Further, alternative to animal experiments, measurement of BOD,
Environmental indicators Measurement of organism respiration, detection of vascular disorders, DN
A. It can be applied to the detection of allergy and the like. When detecting DNA, the following (1) to (6)
May be performed. (1) The oxygen electrode is modified with a metal surface treating agent (for example, N-hydroxysuccinimide).

(2) DNA complementary to DNA to be detected
(Probe) with a linker (for example, amino linker 2)
Attached. (3) Add (1) and (2) to make a detection electrode. (4) The sample DNA is added to the detection electrode to perform DNA hybridization.

(5) removing unreacted sample DNA;
A non-specific adsorbent for DNA (eg, polyamine, divalent metal ion) is allowed to act. (6) Measure the oxygen output (output indicating the amount of dissolved oxygen) in the solution, and compare with the control (output not affected by the sample DNA) to calculate the sample DNA amount.

When detecting an allergen, the following processes (1) to (5) may be performed. (1) The oxygen electrode is modified with a metal surface treating agent (for example, N-hydroxysuccinimide). (2) adsorb the antibody against the allergen on the electrode surface,
This is a detection electrode. (3) A sample allergen is added to the detection electrode, and an antigen-antibody reaction is performed.

(4) The unreacted sample allergen is removed, and a non-specific adsorbent for the protein (eg, polyamine, divalent metal ion) is allowed to act. (5) The oxygen output (output indicating the amount of dissolved oxygen) in the solution is measured and compared with the control (output not affected by the sample allergen) to calculate the sample allergen amount.

Incidentally, it is possible to measure other proteins (such as chemical mediators) by employing a method for detecting allergens. Furthermore, animal cells (human cancer cells, alternative to animal experiments) and BOD can be measured by employing the same method as that for measuring drug sensitivity of bacteria. Furthermore, since nitric oxide can be measured directly with an oxygen electrode, it can be applied to the detection of vascular disorders.

[0033]

According to the first aspect of the present invention, the physiological activity can be measured based on whether or not there is a significant difference in the rate of change of the dissolved oxygen amount between the two media, and the required time is significantly reduced. And the specific effect that accuracy can be significantly increased.

The second aspect of the invention has the same effect as the first aspect. The invention of claim 3 can measure the physiological activity based on whether or not there is a significant difference in the rate of change of the dissolved oxygen amount in both culture media, and can significantly reduce the required time, This has a unique effect that accuracy can be significantly increased.

The fourth aspect of the invention has the same effect as the third aspect.

[Brief description of the drawings]

FIG. 1 is a block diagram schematically showing one embodiment of a physiological activity measuring device of the present invention.

FIG. 2 is a schematic diagram showing an example of a configuration and a mounted state of an oxygen electrode.

FIG. 3 is a diagram showing a change with time of a measured current.

FIG. 4 is a flowchart illustrating a drug sensitivity measurement process.

FIG. 5. E. coli as bacteria. E. coli and a bacterial concentration of 10E8 / cc and a bacterial concentration of 10E7 / cc
FIG. 9C is a diagram showing a measurement result based on only a measured current value output from an oxygen electrode, prepared for a sample having a sample c and a sample having no bacteria.

FIG. 6 shows E. coli as bacteria. FIG. 9 is a diagram showing a measurement result based on a change rate of a measurement current value output from an oxygen electrode, prepared by using E. coli and having a bacterial concentration of 10E7 cells / cc and a bacterial-free one.

FIG. 7 is a diagram showing a time change of a measured current when IPM is adopted as a drug and the concentration of IPM is varied.

FIG. 8 is a diagram showing a time-based change in the rate of change of the measured current when IPM is used as a drug and the concentration of IPM is varied.

FIG. 9 is a diagram showing a change over time of a measured current for measuring toxicity of 1% ethanol on cancer cells.

FIG. 10 is a diagram showing a change with time of a change rate of a measured current value output from an oxygen electrode.

[Explanation of symbols]

 4 Oxygen electrode 52 Data processing device

 ──────────────────────────────────────────────────続 き Continuing from the front page (72) Inventor Junichiro Arai 3 Miyukigaoka, Tsukuba, Ibaraki Pref. Daikin Industries, Ltd.

Claims (4)

[Claims] 1. A medium to which a substance to be measured for physiological activity is added and a medium to which a substance to be measured for physiological activity and a reagent for measuring physiological activity are added are prepared. The amount of dissolved oxygen in both mediums is measured by an oxygen electrode (4). A method for measuring a physiological activity by comparing the amount of dissolved oxygen measured using the method, wherein a bias voltage for measurement is applied to an oxygen electrode (4) for a first predetermined time A physiological activity measuring method characterized in that a physiological activity is measured based on a change rate of a dissolved oxygen amount within a time range from when a predetermined time elapses to when a second predetermined time elapses. 2. A method according to claim 1, wherein after applying a bias voltage for measurement to the oxygen electrode, a ratio of a transient current not depending on a dissolved oxygen amount is equal to or less than the first predetermined ratio. The second predetermined time is a time or a fixed time until the dissolved oxygen amount in the medium becomes equal to or less than a second predetermined ratio with respect to the initial dissolved oxygen amount. The method for measuring physiological activity according to the above. 3. A medium to which a substance to be measured for physiological activity is added, a medium to which a substance to be measured for physiological activity and a reagent for measuring physiological activity are added, and an oxygen electrode (4) for measuring the amount of dissolved oxygen in both the medium. A physiological activity measurement result output means (5) for outputting a physiological activity by comparing the measured dissolved oxygen amount.
2) wherein the physiological activity measurement result output means (52) comprises an oxygen electrode (4).
After applying a bias voltage for measurement to
A physiological activity is measured and output based on a change rate of a dissolved oxygen amount within a time range from when a predetermined time elapses to when a second predetermined time elapses. apparatus. 4. The method according to claim 1, wherein a bias voltage for measurement is applied to the oxygen electrode (4) for a first predetermined time until the ratio of a transient current that does not depend on the amount of dissolved oxygen becomes equal to or lower than a predetermined ratio. The physiological activity measuring device according to claim 3, wherein the second predetermined time is a time until the dissolved oxygen amount in the medium becomes a predetermined ratio or less with respect to the initial dissolved oxygen amount.