JP4602018B2 - Hazardous substance detection method and apparatus - Google Patents

Description

  The present invention relates to a hazardous substance detection method and apparatus for detecting contamination of water from water purification plants or tap water sources such as rivers, lakes, dams, and groundwater.

  Rivers, lakes, dams, groundwater, etc. are used as sources of raw water for water supply, but accidents may occur in which chemical substances, oils, etc. flow into these raw waters. These chemical substances may contain harmful substances that have a significant impact on human health. If water containing harmful substances is supplied, a wide range of people will be involved in water accidents such as health injuries. In order to avoid such a situation and supply safe water, it is important to monitor the quality of raw water.

  In water purification plants, fish are raised in raw water as a means for monitoring contamination with harmful substances, and abnormal behavior of fish is also detected by observation or image analysis by the observer. However, this method requires troubles such as feeding fish and maintaining the aquarium, and has problems such as being influenced by the health condition of the fish itself and sensitivity depending on individual differences.

  Although it is conceivable to apply instrumental analysis to detect chemical substances contained in water intake, it is difficult to perform instrumental analysis for all chemical substances that may flow in, and practically limiting the subject of analysis. Will do. The disadvantage in this case is that it is impossible to detect chemical substances that are not supposed to flow. Moreover, pretreatment of sample water is necessary for measurement, and it is not suitable for continuous monitoring.

  Under these circumstances, as a method for continuously detecting contamination of harmful substances, a method for comprehensively evaluating the harmfulness of sample water by measuring the load of harmful substances on the activity of organisms has been proposed. In particular, by using a biosensor that combines an oxygen electrode and a microbial membrane, the method for monitoring the respiratory activity of microorganisms from the oxygen consumption of the microbial membrane is a continuous and simple method of mixing harmful substances into sample water. (For example, refer to Patent Document 1).

  In this document, the respiratory activity of iron-oxidizing bacteria is monitored. Respiratory activity of microorganisms is detected by chemical substances that cause direct inhibition of respiratory response, inhibition of other metabolic reactions, destruction of cell walls and cell membranes, etc., so that a relatively wide range of harmful substances can be detected. . However, since toxicity to single cells of single-cell organisms is basically an object of evaluation, it may not always correspond to human toxicity. In particular, it is difficult for biosensors to directly measure inhibition of information transmission between multiple cells, chronic toxicity that accumulates in tissues for a long time and develops toxicity.

Note that a technique for detecting an interaction between a chemical substance and a living tissue in order to detect contamination of harmful substances in water has already been disclosed (see, for example, Patent Document 2). This method includes a pseudo cell membrane that interacts with a chemical substance, and the physical information of the pseudo cell membrane, such as membrane potential, electrical properties, etc., which changes the interaction of the chemical substance to the pseudo cell membrane by reaction with the chemical substance. Capacitance, ion permeability, luminescence, exotherm, endotherm, etc. are measured, and chemical contamination is determined from the difference in output values before and after the reaction.
JP-A-11-37969 JP 11-56389 A

  The present invention has been made to solve the above-described problems, and provides a hazardous substance detection method and apparatus capable of detecting a wider range of harmful substances, including harmful substances that are difficult for biosensors to deal with. It is intended to do.

In order to achieve the above object, the harmful substance detection apparatus according to the first aspect of the present invention immobilizes microorganisms on the electrode and monitors changes in the production amount or consumption of the electrode active material by the microorganisms contained in the sample water. A biosensor that detects the entry of harmful substances into the sample water, and phospholipids or artificial polymer membranes to replace them as the main components, and various functional molecules that interact with the harmful substances contained in the sample water. And a pseudo cell membrane sensor that captures the presence of harmful substances in the sample water as a change in membrane potential of the pseudo cell membrane .

  In the detection of harmful substances by a biosensor using microorganisms, it is used that when a harmful substance is mixed in sample water, some change appears in the activity of microorganisms due to the influence of the harmful substances. The change in the production amount or consumption of the electrode active material by the microorganism based on the activity change due to the harmful substance is monitored. For example, by immobilizing microorganisms on a dissolved oxygen electrode and monitoring oxygen consumption due to respiration of microorganisms, contamination of harmful substances can be detected from a decrease in oxygen consumption due to harmful substances.

  At this time, it is difficult to detect the inflow of a chemical substance that does not affect the activity of the microorganism used, or a chemical substance that does not cause a change in activity that can be detected at the electrode. Among these chemical substances, harmful substances that may affect the human body do not affect the activity of microorganisms, but still interact with the human body, in other words, living tissue.

  Pseudo-cell membranes have a structure that mimics biological cell membranes, and are mainly composed of lipid bilayers composed mainly of phospholipids or artificial polymer membranes that replace them. Functions such as various proteins and sugars that act on poisons It is made by blending molecules. In order to make toxic substances that are not subject to measurement by the biosensor into the measurement object of the pseudo cell membrane sensor, the biosensor does not allow measurement by including functional molecules such as proteins and sugars that interact with the toxic substance in the pseudo cell membrane. Possible harmful substances can be detected.

  A characteristic of a biosensor is that a single biosensor element (microorganism) can comprehensively evaluate the load that can be applied to various types of harmful substances. However, it is difficult to obtain information on the type of harmful substance from the change in respiratory activity, which is an evaluation index, and it is difficult to identify the type of harmful substance from the sensor response.

The invention according to claim 2 is the harmful substance detection apparatus according to claim 1, wherein the pseudo cell membrane sensor has a plurality of types of pseudo cell membranes having different compositions that exhibit different membrane potential changes with respect to a plurality of types of harmful substances. A membrane potential measuring device for measuring a membrane potential change of a pseudo cell membrane sensor, and a measurement signal from the membrane potential measuring device being input, and pattern recognition of the measurement signal indicating the membrane potential change of each pseudo cell membrane And an arithmetic unit for determining the type of harmful substance .

  In this case, the pseudo cell membrane sensor includes a plurality of types of pseudo cell membranes having different compositions, and how the output value changes according to the response of each pseudo cell membrane sensor to a harmful substance, that is, the type and amount of the toxic substance. Based on these response patterns, it is possible to roughly classify the types of harmful substances from the output values from a plurality of sensors.

  By combining a biosensor and multiple types of pseudo cell membrane sensors, it is possible to estimate the type of chemical substance flowing in while monitoring the load on the organism with the biosensor, further improving the accuracy of water quality monitoring be able to.

The invention according to claim 3 is the harmful substance detection apparatus according to claim 1 or 2, wherein the pseudo cell membrane includes a biological functional element that irreversibly binds to a chronic toxic substance, and the toxicity is manifested by long-term accumulation. It is characterized by detecting chronic toxic substances.

  Especially for chronic toxic substances that are difficult to measure with a biosensor, a functional element capable of capturing chronic toxic substances is embedded in the pseudo cell membrane, and among the response patterns obtained from multiple types of pseudo cell membranes, chronic toxicity Chronic toxic substances can be detected by identifying patterns characteristic to the substance. In this case, it is possible to cover the acute toxic substance by the biosensor and the chronic toxic substance by the pseudo cell membrane sensor.

  The invention according to claim 4 is characterized in that, in the harmful substance detection device according to any one of claims 1 to 3, iron-oxidizing bacteria are used as microorganisms immobilized on the biosensor.

  Many types of microorganisms can be applied as microorganisms to be immobilized on biosensors. However, in order to achieve stable measurement, chemical synthesis autotrophs that selectively use specific chemical substances as substrates. Bacteria are desirable. Furthermore, stability can be improved by utilizing iron-oxidizing bacteria that are active in the acidic region.

The harmful substance detection method of the invention according to claim 5 is to monitor the change in the production amount or consumption of the electrode active material by the microorganisms contained in the sample water when the harmful substance contained in the sample water is normally monitored. The biosensor that detects the contamination of harmful substances in the sample water continuously with a biosensor, and when a biosensor detects a water quality abnormality, it has a composition that shows different membrane potential changes for multiple types of harmful substances. This is a method of identifying the type and amount of harmful substances that caused water quality abnormalities by supplying sample water to a pseudo cell membrane sensor having a plurality of different pseudo cell membranes and recognizing patterns obtained from the pseudo cell membrane sensors. Pseudo cell membrane sensors are mainly composed of phospholipids or artificial polymer membranes instead of various functional molecules that interact with harmful substances contained in sample water. Has a pseudo cell membrane is created by coupling, in which capture the presence of harmful substances in the water sample as a membrane potential change of the pseudo cell membrane, characterized in that.

  Rather than operating two sensors at all times, the biological load is continuously monitored by the biosensor during normal times. When a harmful substance is detected by the biosensor, sample water is supplied to the pseudo cell membrane sensor. It is configured to operate. The biosensor can be configured with a relatively simple device, can easily perform continuous measurement, and can quickly detect an abnormality. Therefore, monitoring of harmful substances is always performed with a biosensor that can be operated continuously at low cost for a long period of time, and if any harmful substance is mixed with the biosensor, the pseudo cell membrane sensor is operated to determine the type of harmful substance. It becomes effective to specify.

The hazardous substance detection method of the invention according to claim 6 is a method for monitoring a harmful substance contained in a sample water in a normal state, and for a plurality of types of pseudo substances having different compositions that show different membrane potential changes for a plurality of kinds of harmful substances. Electrode by microorganisms contained in sample water when it is continuously performed by supplying sample water to a pseudo cell membrane sensor having a cell membrane , and the pseudo cell membrane sensor obtains an output signal pattern in which the harmful substance is assumed to be an acute toxic substance A method of confirming the contamination of an acute toxic substance by a biosensor that detects the contamination of the harmful substance in the sample water by monitoring the change in the production amount or consumption of the active material. An artificial polymer film that is an alternative to this, and is created by blending various functional molecules that interact with harmful substances contained in the sample water. Have membranes, but to capture the presence of harmful substances in the water sample as a membrane potential change of the pseudo cell membrane, characterized in that.

  In this case, monitoring is normally performed by a pseudo cell membrane sensor, and when a response pattern corresponding to a harmful substance exhibiting acute toxicity is detected, sample water is supplied to the biosensor to operate. Pseudo cell membrane sensor can detect harmful substances with relatively high sensitivity, but even if a response pattern peculiar to harmful substances is obtained, it contains chemical substances that are not toxic and similar to harmful substances There is also a possibility. For this reason, it becomes possible to evaluate the substantial harmfulness of the chemical substance contained in the sample water by actually confirming the load on the organism with the biosensor.

  ADVANTAGE OF THE INVENTION According to this invention, while being able to aim at the expansion of the detection target substance of a biosensor, the hazardous substance detection method and apparatus which can identify a chemical substance can be provided.

  FIG. 1 shows an example of the configuration of a hazardous substance detection apparatus according to the present invention. A feature of this apparatus is that it includes both a biosensor 1 and a pseudo cell membrane sensor 2 as means for detecting contamination of harmful substances in sample water. The sample water is supplied from the sample water supply means 3 and supplied to the biosensor 1 by the pump 5 through the biosensor sample water supply valve 4. The same sample water from the same sample water supply means 3 is also supplied to the pseudo cell membrane sensor 2 by the pump 7 via the pseudo cell membrane sensor sample water supply valve 6.

The biosensor 1 includes a dissolved oxygen electrode 8 and a microbial membrane 9, and the respiratory activity of microorganisms immobilized on the microbial membrane 9 is detected by the dissolved oxygen electrode 8. The micro detection output of the biosensor 1 is measured by the output processing unit 10. In order to activate the microbial membrane 9 , a nutrient source of microorganisms is supplied from the substrate solution tank 11 through the valve 13 a and the pump 14. Further, since it is necessary to perform cleaning in order to maintain long-term stability, the cleaning liquid is periodically supplied from the cleaning liquid tank 12 via the valve 13b and the pump 14. Switching of these liquids is controlled by opening and closing valves 13 a and 13 b, fed by a pump 14, mixed with sample water supplied from the pump 5, and supplied to the biosensor 1.

The pseudo cell membrane sensor 2 includes a pseudo cell membrane 15, an electrode 16, and a membrane potential measuring tank 17. A pseudo cell membrane 15 is arranged at the center of the membrane potential measuring tank 17, and a pair of electrodes 16 are arranged so as to sandwich it. The output of the pseudo cell membrane sensor 2 taken out from the electrode 16 is measured by the membrane potential measuring device 18, and the presence of harmful substances in the pseudo cell membrane 15 is captured by capturing the change of the membrane potential with respect to the initial value. That is, the pseudo cell membrane sensor 2 captures physical changes such as electricity, light, and heat caused by the action of harmful substances on the pseudo cell membrane 15 as changes in membrane potential. As shown in FIG. 1, when a plurality of (three in the illustrated example) pseudo cell membrane sensors are applied, each sensor output is measured.

The detection outputs of the biosensor 1 and the pseudo cell membrane sensor 2 are transmitted to the arithmetic unit 19, where calculation processing such as determination of the amount of harmful substances mixed and estimation of the type of harmful substances is performed based on both detection outputs. Further, the configuration example of FIG. 1 includes a calibration water supply means 20 for supplying calibration water to the biosensor 1 and a water collection tank 21 for collecting water for analysis when detecting harmful substances. The calibration water supply means 20 is connected to the inlet of the pump 5 through the valve 13c. The water sampling tank 21 is connected to the inlet of the valve 6 through the valve 13d.

  FIG. 2 shows an example of the response of the hazardous substance detection by the biosensor 1. At a few minutes after the start of measurement, the detected current value is temporarily reduced by adding ferrous sulfate as a substrate solution. That is, by adding a solution containing a substrate of microorganisms to the biosensor 1, the microorganisms consume oxygen by respiration. When an iron-oxidizing bacterium (Thiobacilus ferroxidans) is used as a microorganism, respiration is activated by adding a solution containing iron ions as a nutrient source. As a result, since oxygen is consumed by the microbial membrane 9, the amount of oxygen reaching the oxygen electrode is reduced and the current value is lowered. If no harmful substances are added as they are, the transition will be at a low level as shown by the characteristic line 21, but if some harmful substances are added after a few minutes have passed since the detection current value has decreased, the characteristic line 22 shows. In general, the detected current value increases. In the characteristic line 22, when the harmful substance is added, the microorganisms are inhibited from breathing, the current value increases, and when the warning line A is exceeded, the contamination of the harmful substance is detected.

  FIG. 3 shows changes in membrane potential when various pseudo cell membranes are used in the case where cyanide is added as a harmful substance after a few minutes after the substrate solution is added. The characteristic line 31 shows the case where the pseudo cell membrane 1 is used, the characteristic line 32 shows the case where the pseudo cell membrane 2 is used, and the characteristic line 33 shows the case where the pseudo cell membrane 3 is used.

  The pseudo cell membrane is composed of an artificially produced lipid bimolecular membrane or the like, and contains various functional molecules in the membrane. As the lipid membrane, synthetic surfactants such as monoolein and triolein + cholesterol, various phospholipids, glycolipids and the like are applicable, but those having a bilayer structure are more preferable. As functional molecules, various proteins, sugars, and artificially synthesized molecules that interact with harmful substances separated from biological cell membrane components are applied. Examples of functional molecules applicable to the detection of various substances include ionophores for heavy metal and other ionic chemical substances, pesticides, antibodies produced by chemical substances that become antigens, and chemical substances that have endocrine disrupting properties. Examples thereof include a receptor, a microorganism having a property of assimilating a specific chemical substance, for example, a membrane-constituting lipid collected from cyanogen-assimilating bacteria.

  Under normal conditions, the output membrane potential of the pseudo cell membrane sensor 2 changes at a constant value. However, when a harmful substance (cyanide in the example of FIG. 3) is mixed in the sample water, the harmful substance acts on the functional substance or the pseudo cell membrane in the pseudo cell membrane 15, and the membrane potential changes. In the example of FIG. 3, the response when cyanide and an agricultural chemical are added as harmful substances to several types of pseudo cell membranes is shown. In the pseudo cell membrane 1 indicated by the characteristic line 31, the potential changes with respect to cyanide, in the pseudo cell membrane 2 indicated by the characteristic line 32, the potential changes with respect to the pesticide, and in the pseudo cell membrane 3 indicated by the characteristic line 33, the cyanide and the pesticide. The potential changes for both. Using this characteristic, it is possible to identify the harmful substance species from the potential change pattern of each pseudo cell membrane.

  As described above, it is difficult for biosensors to detect harmful substances that do not affect the activity of microorganisms. In biosensors using iron-oxidizing bacteria, the effects on respiratory activity are less likely to appear for certain pesticides and heavy metals. Here, by detecting the output change of the pseudo cell membrane 2 or the pseudo cell membrane 3 shown in FIG. 3, it is possible to detect an agricultural chemical that is difficult to detect by the biosensor 1.

  In addition, a chronic toxic substance that has low acute toxicity but develops toxicity by accumulating and acting on a living tissue for a long period of time is a substance that is difficult to detect with a biosensor. As for chronic toxic substances, response patterns of a plurality of pseudo cell membranes against chronic toxic substances are measured in advance and stored in a database, and when a similar response pattern is shown, it is possible to detect contamination of chronic toxic substances.

  Thus, when aiming at the expansion of the detection object of a biosensor, the biosensor 1 and the pseudo cell membrane sensor 2 are operated in parallel and simultaneously to perform water quality monitoring. In addition, taking advantage of each feature, it is possible to take a method of operating one of the sensors at all times. Hereinafter, each operation method will be described in more detail.

FIG. 4 is a flowchart for explaining an operation example of the harmful substance detection apparatus shown in FIG. In this operation example, monitoring by the biosensor 1 is always performed (step S1). It is monitored whether or not the output of the biosensor 1 has exceeded the alarm value A (step S2). If it exceeds, it is determined that a harmful substance has been mixed, and an alarm is given to the effect that the harmful substance has been mixed (step S3). Subsequently, harmful substance identification, that is, pattern recognition is performed by the pseudo cell membrane sensor 2 (step S4). When the pseudo-cell membrane sensor 2 estimates the type of rough harmful substance, and determines whether or not it is an acute toxic substance that may have a great influence on the human body (step S5). In addition, a warning that an acute toxic substance has been mixed is issued (step S6), and the water intake is temporarily stopped (step S7). In step S5, if the substance is not an acutely toxic substance, that is, if the type of the toxic substance is a substance that can be removed by water purification treatment, or if it is not a substance that has a significant impact immediately, the type of the toxic substance is changed. The display is stopped (step S8), and after water sampling / detailed analysis (step S9), appropriate measures are taken separately.

FIG. 5 is a flowchart for explaining another operation example of the harmful substance detection apparatus shown in FIG. In this operation example, monitoring by the pseudo cell membrane sensor 2 is always performed to determine whether or not a harmful substance is mixed (steps S1 and S2), and if it is determined that a harmful substance is mixed, the harmful substance is mixed. Is issued (step S3). Further, the pseudo cell membrane sensor 2 estimates whether the mixed harmful substance is an acute toxic substance (step S5). If it is estimated that the harmful substance is an acute toxic substance , the biosensor 1 is activated (step S10). Measurement by the sensor 1 is performed. Here, the biosensor 1 is checked for an output increase exceeding the alarm value (step S11), and if the alarm value is exceeded, an alarm is given that an acute toxic substance has been mixed (step S6), and water is temporarily taken. Is taken (step S7). If it is not an acute toxic substance in step S5, that is, if the type of the toxic substance is a substance that can be removed by water purification treatment, or if it is not a substance that has a significant impact immediately, and in step S11, an alarm value If it does not exceed, after performing water sampling and detailed analysis (step S9), appropriate measures can be taken separately.

The block diagram which shows an example of the hazardous | toxic substance detection apparatus by this invention. The characteristic diagram which shows the example of a response of the hazardous | toxic substance detection by a biosensor. The characteristic line figure which shows the example of a response of the hazardous | toxic substance detection by a pseudo cell membrane sensor. The flowchart which shows an example of the procedure at the time of always monitoring with a biosensor using the hazardous | toxic substance detection apparatus of FIG. The flowchart which shows an example of the procedure at the time of always monitoring with a pseudo cell membrane sensor using the harmful substance detection apparatus of FIG.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Biosensor 2 Pseudo cell membrane sensor 3 Sample water supply means 4 Biosensor sample water supply valve 5, 7, 14 Pump 6 Pseudo cell membrane sensor sample water supply valve 8 Dissolved oxygen electrode 9 Microbial membrane 10 Output processing part 11 Substrate solution tank 12 Washing liquid Tanks 13a to 13d Valve 15 Pseudo cell membrane 16 Electrode 17 Membrane potential measurement tank 18 Membrane potential measurement device 19 Arithmetic device 20 Calibration water supply means 21 Water collection tank

Claims (6)

A biosensor that immobilizes microorganisms on the electrode and detects contamination of harmful substances in the sample water by monitoring changes in the production amount or consumption of the electrode active material by the microorganisms contained in the sample water;
It has a pseudo cell membrane made by blending various functional molecules that interact with harmful substances contained in the sample water, the main component of which is a phospholipid or an artificial polymer membrane instead of the phospholipid, and the sample water A pseudo cell membrane sensor that captures the presence of harmful substances as a change in membrane potential of the pseudo cell membrane;
Hazardous substance detection apparatus comprising the. The pseudo cell membrane sensor has a plurality of types of pseudo cell membranes having different compositions that exhibit different membrane potential changes for a plurality of types of harmful substances,
A membrane potential measuring device for measuring a membrane potential change of the pseudo cell membrane sensor;
An arithmetic device that inputs a measurement signal from the membrane potential measuring device and determines the type of harmful substance by recognizing a pattern of the measurement signal indicating the membrane potential change of each pseudo cell membrane;
Hazardous substance detection apparatus according to claim 1, further comprising a. The pseudo cell membrane containing the biological function element for irreversibly binding and chronic toxic substances harmful according to claim 1 or 2, characterized in that the detection of chronic toxic substances toxicity expressed by long-term storage Substance detection device.   The harmful substance detection apparatus according to any one of claims 1 to 3, wherein iron-oxidizing bacteria are used as microorganisms immobilized on the biosensor. When monitoring harmful substances contained in the sample water, the contamination of the sample water is detected by monitoring changes in the production or consumption of the electrode active material by microorganisms contained in the sample water. When continuously performed by a biosensor and an abnormality in water quality is detected by the biosensor,
By supplying sample water to a pseudo cell membrane sensor having a plurality of pseudo cell membranes having different compositions that exhibit different membrane potential changes for a plurality of types of harmful substances, and pattern recognition of signals obtained from the pseudo cell membrane sensor A method for identifying the type and amount of harmful substances that cause water quality abnormalities ,
The pseudo cell membrane sensor has a pseudo cell membrane created by blending various functional molecules that interact with harmful substances contained in the sample water, the main component of which is a phospholipid or an artificial polymer membrane instead of the phospholipid. And the presence of harmful substances in the sample water is regarded as a change in membrane potential of the pseudo cell membrane,
A method for detecting a harmful substance. When monitoring harmful substances contained in sample water, supply sample water to a pseudo cell membrane sensor having multiple pseudo cell membranes with different compositions that show different membrane potential changes for multiple types of harmful substances When the output signal pattern in which the harmful substance is estimated to be an acute toxic substance is obtained by the pseudo cell membrane sensor,
This is a method for confirming the contamination of acute toxic substances by a biosensor that detects the contamination of harmful substances in the sample water by monitoring changes in the production or consumption of the electrode active material by microorganisms contained in the sample water. And
The pseudo cell membrane sensor has a pseudo cell membrane created by blending various functional molecules that interact with harmful substances contained in the sample water, the main component of which is a phospholipid or an artificial polymer membrane instead of the phospholipid. And the presence of harmful substances in the sample water is regarded as a change in membrane potential of the pseudo cell membrane,
A method for detecting a harmful substance.

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