JP2006042655A - Detector and detection method for harmful substance - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a detector and a detection method for a harmful substance in which a microorganism capable of expressing a fluorescent protein in the presence of harmful substances is used, thereby promptly and simply detecting the harmful substances from the fluorescent intensity of the microorganismic membrane prepared by immobilizing the microorganism in a film, further enables the kinds of the harmful substances to be identified. <P>SOLUTION: The harmful substance detector comprises the microorganism membrane 6 that is prepared by immobilizing a gene recombination microorganism 1 that expresses the fluorescent protein in the presence of harmful substances in a thin film between a fine porous membrane 2 and a light-permeable membrane 3, a sample vessel 7 for allowing a sample 5 to come into contact with the microorganism membrane 6, an exciting light radiator part for radiating exciting light to the microorganism membrane 6 and the fluorescent light measuring part, and the fluorescent intensity of the microorganism membrane 6 is measured whereby harmful substances are detected. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention is intended for effluents in water and sewage treatment processes, environmental waters such as rivers and lakes, leachate from waste landfill final disposal sites, industrial waste elution water, industrial wastewater, etc. The present invention relates to a detection device and a detection method for harmful substances for the purpose of monitoring.

  In addition to 60,000 to 100,000 artificially synthesized chemical substances in the environment, unintentional products such as disinfection by-products such as trihalomethane, combustion by-products such as dioxin, or environmental changes Many chemical substances exist. Therefore, various chemical substances are mixed in environmental waters such as rivers and lakes. Although its concentration is considered to be very small, for example, in a water purification plant that uses such environmental water as raw water, it is required to continuously monitor water quality at a higher level from the viewpoint of health effects on the human body. It has been.

  In addition, wastewater containing chemical substances is discharged from the sewage treatment plant, waste final disposal site, and factories that use chemicals into rivers after ozone treatment, biological treatment, activated carbon treatment, and the like. For this reason, it is monitored whether such treatment processes are operating properly and harmful substances in the water are removed, evaluation results of each treatment process are fed back to the treatment processes, and measures such as improvement of treatment facilities are taken. It is necessary to take. For this purpose, it is necessary to quickly and easily detect harmful substances in the treated water, and to identify what kind of harmful substances they are.

Until now, in the evaluation test of harmful substances using microorganisms such as bacteria, an agar plate method, liquid culture method using microorganism growth inhibition as an index, or a method using physiological activity inhibition as an index have been performed.
In addition, many tests such as Ames test and umu test using microorganisms have been reported as short-term identification methods for carcinogens. These methods are methods for detecting DNA damage by a coloring reaction of a dye.

However, the above-described method requires a long time to obtain a result because it is necessary to cultivate microorganisms in advance and to grow an amount necessary for the test. In addition, in the test to detect by dispensing and dilution of the culture solution, coloring reaction of the dye, the operation of dispensing the coloring reagent and extraction of the dye is complicated, and it is difficult to process a large amount of sample, There was a problem in terms of simplicity.
In order to solve such a problem, a proposal has been made to detect toxic substances more simply and quickly using movement inhibition of microorganisms as an index (Patent Document 1). However, even in this proposal, the conventional problems described above have not been solved.
JP 2001-161393 A

  In view of the above-mentioned problems, the present invention uses a microorganism that expresses a fluorescent protein in the presence of a harmful substance, and can quickly and easily detect a harmful substance from the fluorescence intensity of a microorganism film in which the microorganism is immobilized in a film. Furthermore, it aims at providing the detection apparatus and detection method of a hazardous | toxic substance which enabled identification of the kind of hazardous | toxic substance itself.

  In order to achieve the above-mentioned object, the harmful substance detection apparatus according to the present invention is a thin film between a microporous film and a light-transmitting film. A microbial membrane immobilized in a shape, a sample container in which the microbial membrane is placed to bring the sample into contact with the microbial membrane, an excitation light irradiation unit for irradiating the microbial membrane with excitation light, a fluorescence measurement unit, And a harmful substance is detected by measuring the fluorescence intensity of the microbial membrane. The microporous membrane is preferably a microporous membrane that does not allow microorganisms to leak and at least allows the sample to pass therethrough, and the light transmissive membrane is preferably a light permeable membrane that can transmit fluorescence from the microorganism. . The microporous membrane has a pore size that can permeate a sample, nutrient components, oxygen, and the like, and is generally preferably hydrophilic. The sample is generally supplied as a sample solution and most commonly provided as sample water. The microporous membrane is made of a material that is transparent or nearly transparent and does not interfere with fluorescence transmission so that fluorescence from microorganisms can be transmitted.

  In a hazardous substance detection apparatus according to the present invention, in a preferred embodiment, the light permeable film is placed facing the outside of the sample container, and the microorganism film is irradiated with excitation light from the light permeable film side. In addition, the fluorescence of the microbial membrane can be measured. In general, the microporous membrane is placed on the bottom of the sample container with the top facing upward, and the light-transmitting membrane is directed downward so that it can be irradiated with excitation light.

  Furthermore, in another preferred embodiment, the hazardous substance detection apparatus according to the present invention uses two or more microbial membranes corresponding to each harmful substance in order to detect two or more different harmful substances. It is supposed to be installed in a container.

  In another aspect, the present invention is a method for detecting a harmful substance, which comprises a genetically modified microorganism that expresses a fluorescent protein in the presence of a harmful substance, a microporous membrane and a light-transmitting membrane. A microbial membrane is formed by immobilizing it in the form of a thin film, a sample is brought into contact with the microbial membrane, and a harmful substance is detected by measuring the fluorescence intensity of the microbial membrane. The microporous membrane is preferably a microporous membrane that does not allow microorganisms to leak and at least allows the sample to pass therethrough, and the light transmissive membrane is preferably a light permeable membrane that can transmit fluorescence from the microorganism. . The microporous membrane has a pore size that can permeate a sample, nutrient components, oxygen, and the like, and is generally preferably hydrophilic. The sample is generally supplied as a sample solution and most commonly provided as sample water. The microporous membrane is made of a material that is transparent or nearly transparent and does not interfere with fluorescence transmission so that fluorescence from microorganisms can be transmitted.

  In a method for detecting a harmful substance according to the present invention, in a preferred embodiment, the microporous membrane of the microbial membrane is brought into contact with a sample in a sample container, and the light permeable membrane is directed to the outside of the sample container. And irradiating the microbial membrane with excitation light from the light permeable membrane side to measure fluorescence of the microbial membrane. In general, the microporous membrane is placed on the bottom of the sample container with the top facing upward, and the light-transmitting membrane is directed downward so that it can be irradiated with excitation light.

  Furthermore, in another preferred embodiment, the method for detecting a hazardous substance according to the present invention includes installing two or more microbial membranes corresponding to each harmful substance in the sample container, and providing two or more different harmful substances. Trying to detect.

  ADVANTAGE OF THE INVENTION According to this invention, the hazardous | toxic substance detection apparatus and the detection method which can detect a hazardous | toxic substance rapidly and simply, and also enabled it to identify the kind of harmful substance itself are provided.

Hereinafter, an embodiment of a hazardous substance detection apparatus and detection method according to the present invention will be described with reference to the accompanying drawings.
First, a microbial membrane used in the hazardous substance detection apparatus according to the present invention will be described. FIG. 1 shows an embodiment of such a microbial membrane.
As shown in the figure, the microorganism membrane 6 according to the present embodiment has microorganisms 1 sandwiched between a microporous membrane 2 and a light-transmitting membrane 3 and is immobilized in a film shape. A double-sided tape 4 is interposed between the microporous film 2 and the light transmissive film 3. The left side of FIG. 1 shows a state in which the microporous membrane 2 is removed. Further, the right side of FIG. 1 shows a state in which the microporous film 2 is bonded.

The microorganism 1 is a genetically modified microorganism that expresses a fluorescent protein in the presence of a harmful substance.
Such a microorganism 1 can be produced by a gene recombination operation in which a plasmid DNA in which a control gene that functions in the presence of a harmful substance and a gene encoding a fluorescent protein are linked is introduced into a host microorganism and transformation is performed.
Here, examples of the control gene that functions depending on the presence of harmful substances include the umuDC gene involved in the repair of DNA damage caused by genetic damage substances. However, the control gene that can be used in the present invention is not limited to this, and a control gene that functions for each of various harmful substances can be used. The umuDC gene is a gene that functions to repair DNA damaged by an SOS response when microbial DNA is damaged by a gene-damaging substance. If a gene that expresses a fluorescent protein is linked downstream of the umuDC gene, the fluorescent protein is synthesized along with the SOS response.
Examples of the harmful substances include gene damaging substances, endocrine disrupting substances, heavy metals and the like.
Examples of the gene damaging substance include 4-nitroquinoline-N-oxide, benzo (a) pyrene, and 2- (2-furyl) -3- (5-nitro-2-furyl) acrylamide. .
Examples of the endocrine disrupting substance include 17β-estradiol, bisphenol A, and nonylphenol.
Examples of the heavy metal include mercury, cadmium, hexavalent chromium, and arsenic.

  GFP (green fluorescent protein) which emits green fluorescence is generally known as the fluorescent protein in the present invention, and GFP emits fluorescence at around 507 nm when irradiated with excitation light having a wavelength around 488 nm. Expression vectors encoding GFP are commercially available from Invitrogen and Clontech. In addition to the green fluorescent protein, an expression vector in which the fluorescent protein to be expressed is also commercially available. In addition to the green fluorescent protein, red fluorescent protein (excitation light near 558 nm, fluorescent light 583 nm) or yellow fluorescent protein (excitation light near 513 nm, fluorescent light 527 nm). Vicinity), blue fluorescent protein (excitation light near 433 nm, fluorescence near 475 nm) can also be used. When the aforementioned control gene is linked to these expression vectors and introduced into microorganisms, a fluorescent protein is expressed with a response to the harmful substance when exposed to the harmful substance.

Examples of host microorganisms that express fluorescent proteins include Salmonella bacteria such as Esherichia coli and Salmonella typhimurium , and Pseudomonas aeruginosa . By introducing a plasmid vector in which a control gene and a fluorescent protein are linked to these host microorganisms, a microorganism 1 that expresses the fluorescent protein used in the present invention can be produced.

  As the microporous membrane 2, a material having hydrophilicity and a pore size that prevents the microorganism 1 from leaking out after the microorganism 1 is immobilized is preferable. The pore size is preferably 0.2 to 1.0 μm. Further, it is desirable that the material is a hydrophilic material that can transmit a sample, a nutrient component, oxygen, and the like. The sample is generally supplied as a sample solution, most commonly as sample water.

  Examples of suitable materials constituting the microporous membrane 2 include nitrocellulose, hydrophilic polyvinylidene chloride, and polycarbonate. From the viewpoint of strength and the amount of cells that can be retained, nitrocellulose is most preferred.

  In general, when bacteria are used as the microorganism 1, for example, the pore size of the membrane material used for capturing bacteria is 0.2 μm, 0.45 μm, or 1.0 μm. Here, as will be described later, when the microbial membrane 6 is produced, the bacterial culture is suction filtered. The pore size is optimally 0.45 μm when bacteria are used. If it is at least this pore size, it is difficult to cause clogging and a large number of cells can be fixed.

  Next, as the light transmissive film 3, an inorganic material such as glass or quartz, or an organic material such as transparent soft vinyl chloride or polycarbonate can be used. An organic material is preferable from the viewpoint that it is difficult to break during film formation or when attached to a sample container. Furthermore, the light-transmitting film 3 needs to be cut into a circular shape with a predetermined size using a punch or the like by punching the light-transmitting material. For this reason, a soft material is suitable, and from this point of view, transparent soft vinyl chloride is one of the most suitable as the light transmissive film 3.

  In the present embodiment, as described above, the microorganism 1 is sandwiched between the microporous film 2 and the light transmissive film 3 and immobilized in a film shape. In order to immobilize the microorganism 1 in the form of a film, it is preferable to use a polymer gelling agent. As such a polymer gelling agent, sodium alginate, polysaccharide carrageenan, agar, protein collagen, gelatin or the like can be used, and a photocuring resin, polyacrylamide or the like of a polymer material can also be used.

  Among the above, sodium alginate gels only by adding a calcium chloride solution and is suitable as a gelling material. When sodium alginate is used, if the viscosity of the 1% solution is 500 to 600 cP and the molecular weight is about 100,000, the cells can be well maintained while maintaining good permeability of oxygen and nutrients.

Next, an embodiment of a method for producing a microbial membrane will be described with reference to FIG.
First, as shown in FIG. 2 (a), a microporous membrane 2 is pre-adhered to one side of a double-sided tape 4 and, for example, a bacterial culture solution is dropped as microorganism 1 while sucking from the microporous membrane 2 side. The bacteria in the culture solution are retained on the microporous membrane 2.

  Subsequently, as shown in FIG. 2 (b), a sodium alginate solution 21 as a polymer gelling agent is dropped to prevent bacterial flow.

Subsequently, as shown in FIG. 2C, the light transmissive film 3 is bonded to the opposite side of the double-sided tape 4. And in order to gelatinize sodium alginate, the microbial membrane 6 is immersed in a calcium chloride solution.
As the double-sided tape 4, a material such as polyimide can be used. For example, a double-sided adhesive tape manufactured by Nitto Denko Corporation can be used.

Next, an embodiment of a harmful substance detection apparatus using the microbial membrane 6 as described above will be described with reference to FIG.
The harmful substance detection apparatus according to the present embodiment includes a microbial film 6, a sample container 7, an excitation light irradiation unit, a fluorescence measurement unit, and a calculation unit 17 as main components.
The sample container 7 is a cylindrical container for injecting a sample (sample water) 5, and a microbial membrane 6 is attached to the bottom. The microbial membrane 6 is mounted by adhering the microporous membrane 2 to the sample container 7 with the microporous membrane 2 facing upward. However, the sample container 7 and the microbial membrane 6 form a container so that the sample does not leak. For example, the fixing method is not limited.

  The excitation light irradiating unit mainly includes an objective lens 8, an excitation side filter 9, a condensing lens 10, an excitation light source 11, and a dichroic mirror 12. The objective lens 8 and the condenser lens 10 are ordinary optical lenses. The excitation light source 11 is generally a mercury lamp, and 254 nm, 313 nm, 405 nm, 436 nm, etc. are efficiently emitted as main wavelengths. The excitation side filter 9 is a filter for extracting light necessary for excitation of the fluorescent substance from the excitation light source. The dichroic mirror 12 is a mirror for reflecting the excitation light and directing it toward the condenser lens 10 and transmitting the fluorescence.

The fluorescence measuring unit includes the objective lens 8, the fluorescence side filter 13, the imaging lens 14, and the image sensor 15 as main components. The fluorescence side filter 13 is a filter for transmitting only the fluorescence from the fluorescent protein of the microorganism membrane. The imaging lens 14 is a normal optical lens. The imaging element 15 is configured by an optical sensor such as a CCD camera, for example.
The harmful substance detection apparatus according to the present embodiment further includes a calculation unit 17 for controlling the excitation light irradiation unit and the fluorescence measurement unit. It should be noted that the present invention also includes a form in which this calculation unit is configured as a part of the excitation light irradiation unit or the fluorescence measurement unit.

Next, a harmful substance detection method using the harmful substance detection apparatus according to the embodiment of FIG. 3 will be described.
First, in a non-measurement state, the culture solution is injected into the sample container 7, and the nutrient 1 and oxygen in the culture solution are permeated through the microporous membrane 2 and the microorganism 1 ( Bacteria).
When measuring harmful substances, the culture solution is removed and the sample 5 is injected into the sample container 7. As a result, the sample 5 contacts the microbial membrane 6. The sample 5 is generally injected as a sample liquid (sample water). Sample 5 permeates through microporous membrane 2. Here, when a harmful substance such as hydrocyanic acid is present in the sample 5, for example, an SOS reaction occurs and a fluorescent protein is expressed.

In order to excite such a fluorescent protein, excitation light is emitted from the excitation light source 11 according to an instruction from the calculation unit 17, and the excitation light is converted into parallel light by the condenser lens 10. The parallel light passes through the excitation side filter 9, is reflected by the dichroic mirror 12, and is irradiated onto the microorganism film 6 through the objective lens 8. The fluorescence from the excited fluorescent protein is converted into parallel light by the objective lens 8. The parallel light passes through the dichroic mirror 12 and is captured by the image sensor 15 via the fluorescent side filter 13 and the imaging lens 14.
Measurement information about the fluorescence captured by the image sensor 15 is transmitted to the calculation unit 17.
Thereby, the fluorescence intensity can be measured by receiving the fluorescence from the microorganism film 6, and the harmful substance mixed in the sample 5 can be detected.

FIG. 4 shows another embodiment of the harmful substance detection apparatus according to the present invention.
As a control gene, a gene that is different for each harmful substance, such as a gene that responds to a gene-damaging substance, a gene that responds to an endocrine disruptor, a gene that responds to heavy metals, etc. Can be adopted. If such a different gene is linked downstream with a gene that expresses a fluorescent protein, a response can be generated for each harmful substance. That is, a plurality of types of genetically modified microorganisms that express fluorescent proteins according to different harmful substances can be used corresponding to such different harmful substances.
As described above, a plurality of harmful substances contained in a sample can be simultaneously detected from the fluorescence intensity of a sample containing a plurality of harmful substances using a plurality of microbial films.

FIG. 4 shows an embodiment of such a harmful substance detection apparatus. In this harmful substance detection apparatus, five microbial films 32 to 36 are attached to the bottom of a sample container 31 into which a sample 30 is injected. And the measurement unit 37 which consists of an excitation light irradiation part and a fluorescence measurement part is installed so that the movement under the sample container 31 is possible. The measurement unit 37 has the same configuration as that of the detection device of FIG. 3 and measures the fluorescence intensity of each of the microorganism films 32 to 36 while being controlled by the calculation unit 38. The measurement operation is basically the same as that described for the detection apparatus of FIG.
According to the detection method using the harmful substance detection apparatus according to this embodiment, the fluorescence intensity of any of the five kinds of harmful substances in the sample 30 can be detected almost simultaneously.

It is a perspective view explaining one embodiment of a microbial membrane which can be used for a hazardous substance detection device according to the present invention. It is a conceptual diagram explaining the production method of the microbial membrane which can be used for the detection apparatus of the harmful | toxic substance based on this invention. 1 is a conceptual diagram illustrating an embodiment of a hazardous substance detection device according to the present invention. It is a conceptual diagram explaining other embodiment of the hazardous | toxic substance detection apparatus which concerns on this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Microorganism 2 Microporous film 3 Light permeable film 4 Double-sided tape 5 Sample 6 Microorganism film 7 Sample container 8 Objective lens 9 Excitation side filter 10 Condensing lens 11 Excitation light source 12 Dichroic mirror 13 Fluorescence side filter 14 Imaging Lens 15 Image sensor 16 Fluorescence filter 17 Calculation unit 30 Sample 31 Sample container 32, 33, 34, 35, 36 Microbial membrane 37 Measurement unit 38 Calculation unit

Claims (8)

In order to bring a genetically modified microorganism expressing a fluorescent protein in the presence of a harmful substance into a thin film between a microporous membrane and a light-transmitting membrane, and a sample in contact with the microorganism membrane A sample container in which the microbial film is installed, an excitation light irradiation unit for irradiating the microbial film with excitation light, and a fluorescence measurement unit, and detects harmful substances by measuring the fluorescence intensity of the microbial film. A device for detecting harmful substances characterized in that The microporous membrane is a microporous membrane that does not allow microorganisms to leak and at least allows a sample to pass therethrough, and the light transmissive membrane is a light permeable membrane that can transmit fluorescence from the microorganism. The hazardous substance detection device according to claim 1. The light-transmitting film is placed toward the outside of the sample container, and the microorganism film is irradiated with excitation light from the light-transmitting film side to measure fluorescence of the microorganism film. Item 3. The hazardous substance detection device according to Item 1 or 2. The hazardous substance according to any one of claims 1 to 3, wherein two or more microbial membranes corresponding to each harmful substance are installed in the sample container in order to detect two or more different harmful substances. Detection device. A microbial membrane is formed by immobilizing a genetically modified microorganism that expresses a fluorescent protein in the presence of harmful substances in a thin film between a microporous membrane and a light-transmitting membrane, and a sample is placed on the microbial membrane. A method for detecting a toxic substance, wherein the toxic substance is detected by contacting and measuring the fluorescence intensity of the microbial membrane. The microporous membrane is a microporous membrane that does not allow microorganisms to leak and at least allows the sample to permeate. The light transmissive membrane is a light permeable membrane that can transmit fluorescence from the microorganism, and the fluorescence of the microbial membrane. 6. The method for detecting a harmful substance according to claim 5, wherein the harmful substance is detected by measuring the intensity. The microporous membrane of the microbial membrane is brought into contact with a sample in a sample container, the light permeable membrane is placed toward the outside of the sample container, and excitation light is applied to the microbial membrane from the light permeable membrane side. The method for detecting a harmful substance according to claim 5 or 6, wherein the fluorescence of the microbial membrane is measured by irradiation. The detection of harmful substances according to any one of claims 5 to 7, wherein two or more microbial membranes corresponding to each harmful substance are installed in the sample container, and two or more different harmful substances are detected. Method.

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