JP2004108972A - Harmfulness of liquid diagnostic method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To evaluate a harmfulness of a liquid under a certain criterion as well as shortening a processing time by an automatic diagnosis of a harmfulness. <P>SOLUTION: A diagnostic device 10 is constituted as follows: a vessel 12 is filled with a liquid and a microbe, and a cell immobilizing gel and a solution for cell dissolving/DNA rewinding is inpoured into the mixture; after the vessel 12 is set in a capillary electrophoretic device 22, a voltage is impressed by a voltage impressing device 28 so as to electrophorese a fragmented DNA in the vessel 12 into a capillary column 26; an absorbency measuring device 30 measures the light absorption of the electrophoresed fragmented DNA. Thereby, a defective degree of the DNA is analyzed and the harmfulness of the liquid can be diagnosed. <P>COPYRIGHT: (C)2004,JPO

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method for diagnosing harmfulness of a liquid, and more particularly to a method for diagnosing harmfulness of a liquid using a biological material to determine toxicity and harmfulness caused by contaminants in the liquid.
[0002]
[Prior art]
In the living environment, there are various chemical substances such as synthetic chemical substances produced artificially and by-products produced by processing. There are unknown harmful substances produced by biological metabolism and chemical conversion in the environment. In order to evaluate the harmfulness of a liquid containing these, it is effective to use a bioassay (Bioassay) for comprehensively evaluating the harmfulness of the liquid itself together with analytical methods such as BOD and COD. Has been developed and put into practical use.
[0003]
Alkaline single cell gel electrophoresis (comet assay), which is one of the bioassays, is a technique for evaluating the DNA damage of a sample by electrophoresing fragmented DNA and measuring the DNA that has flowed out. is there. Features include high detection sensitivity, quick assay, test with a small amount of sample, and quantification of DNA damage at the level of individual cells (for example, see Patent Document 1).
[0004]
In a conventional system using this comet assay, first, a eukaryotic microorganism (green algae, flagellate, algae, etc.) is mixed with a sample, and the cells are embedded in agar on a slide glass. Then, the cells are lysed using a surfactant, and then immersed in an alkaline solution to perform unwinding of the DNA and electrophoresis. Next, after neutralization and staining, observation and analysis are performed using a fluorescence microscope. This makes it possible to detect the harmfulness of water itself that contains pollutants that are difficult to predict.
[0005]
[Patent Document 1]
JP 2001-83120 A (pages 2-3)
[0006]
[Problems to be solved by the invention]
However, the conventional system has a problem in that the toxicity evaluation of the liquid is a batch operation, so that it takes a long time for the diagnosis and it is difficult to automatically perform the harmfulness diagnosis. In addition, since the diagnosis is performed by observation, there is a problem that the evaluation by the observer varies.
[0007]
The present invention has been made in view of such circumstances, shortens the processing time, enables automatic diagnosis of harmfulness, and can evaluate the harmfulness of a liquid based on a certain standard. It is an object of the present invention to provide a method for diagnosing the harmfulness of a liquid which can improve the harmfulness.
[0008]
[Means for Solving the Problems]
In order to achieve the object, the invention according to claim 1 is a method for diagnosing liquid harm, which analyzes harmfulness of a liquid by single-cell gel electrophoresis, wherein a cell mixing step of mixing cells with the liquid, Dissolving the immobilized cells by injecting an immobilization gel into a liquid mixture of the cells and the cells and immobilizing the cells, and injecting a lysis / rewind solution into the mixture; Then, a lysis / rewinding step of unwinding the DNA of the lysed cells, an electrophoresis step of electrophoresing the unwound DNA in a capillary column, and irradiating test DNA to the DNA to be electrophoresed in the capillary column, And a damage analysis step of analyzing a degree of damage to the DNA.
[0009]
According to the present invention, the DNA to be electrophoresed in the capillary column is irradiated with the inspection light to analyze the degree of DNA damage, so that the electrophoresis step and the damage analysis step can be performed continuously. Therefore, the measurement time can be significantly reduced as compared with the conventional case where the measurement is performed by the batch method. Further, according to the configuration of the present invention, the electrophoresis step and the damage analysis step, which were considered to be difficult to automate in single-cell gel electrophoresis, can be automated, so that automatic diagnosis can be performed.
[0010]
Furthermore, according to the present invention, the degree of DNA damage is analyzed based on the result of irradiation with the inspection light, so that the evaluation criterion is always constant, and the analysis accuracy can be improved.
[0011]
According to the second aspect of the present invention, the fixing step and the dissolving / unwinding step are performed in a microreactor. According to the present invention, since the microreactor is used, the size of the device can be reduced.
[0012]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, preferred embodiments of the method for diagnosing harmfulness of wastewater according to the present invention will be described in detail with reference to the accompanying drawings.
[0013]
FIG. 1 is a configuration diagram showing a first embodiment of a diagnostic apparatus to which a hazard diagnosis method according to the present invention is applied.
[0014]
As shown in FIG. 1, the diagnostic apparatus 10 includes a container 12, and a drainage supply pipe 14 is connected to the container 12. Waste water as a sample is supplied to the container 12 through the drain supply pipe 14.
[0015]
The drainage supply pipe 14 is connected to a microorganism supply pipe 16 from which microorganisms (e.g., Euglena gracilis) are supplied. As a result, a mixture in which the microorganisms are mixed with the wastewater is supplied to the container 12.
[0016]
Further, a gel injection tube 18 is connected to the drainage supply tube 14, and a gel for fixing cells is injected from the gel injection tube 18. Thereby, the gel for cell immobilization is injected into the mixture, and the cells of the microorganism are immobilized in about 2 to 5 minutes. As a result, since the DNA contained in the cells is retained, it is possible to prevent the DNA from flowing out when the cells are lysed.
[0017]
Further, a dissolution / rewind solution injection tube 20 is connected to the container 12, and the dissolution / rewind solution is injected from the injection tube 20. Thereby, the fixed cells are lysed, and the DNA contained in the lysed cells is unwound. As a result, the DNA in the container 12 becomes only the fragmented single-stranded DNA (hereinafter, referred to as fragmented DNA) in 5 to 20 minutes. The lysis / rewind solution may be divided into two types of solutions, namely, a cell lysis (Lysis) solution and a DNA unwind (unwinding) solution.
[0018]
The container 12 is designed to be portable, and is set in the capillary electrophoresis apparatus 22 after injecting the dissolution / rewind solution. The capillary electrophoresis device 22 mainly includes a container 12, a collection container 24, a capillary column 26, a voltage application device 28, and an absorbance measurement device 30. The recovery container 24 stores the same solution as the solution in the container 12.
[0019]
The capillary column 26 is a tube having an inner diameter of 100 μm or less, and one end is immersed in the solution in the container 12 and the other end is immersed in the solution in the collection container 24. The inside of the capillary column 26 is filled with the same solution as in the container 12 and the collection container 24.
[0020]
The voltage application device 28 has electrodes 28A and 28B in the container 12 and the collection container 24, and can apply a predetermined voltage (for example, 10 to 30 kv) between the two. When a voltage is applied by the voltage applying device 28, the fragmented DNA contained in the solution is electrophoresed and moves in the capillary column 26. At this time, the difference in size of the fragmented DNA appears as a difference in moving speed, and the smaller the DNA, the faster it moves. Then, the fragmented DNA passes through the position of the absorbance measuring device 30 in about 2 to 5 minutes.
[0021]
The absorbance measurement device 30 includes a light source 30A and a detection unit 30B that are arranged to face each other with the capillary column 26 interposed therebetween, emits inspection light from the light source 30A to the detection unit 30B, and receives the detection light at the detection unit 30B. Then, the amount of DNA moving in the capillary column 26 is detected by detecting the ultraviolet / visible absorption spectrum of the sample component. At that time, since the DNA moving in the capillary column has a different moving speed depending on the size, the DNA chain length can be confirmed by measuring the retention time. Therefore, the amounts of DNA having different chain lengths can be detected based on the detection peak areas obtained by the detection. Thereby, the degree of DNA damage can be analyzed, and as a result, the harmfulness of water itself can be diagnosed.
[0022]
Next, a method for diagnosing the harmfulness of water from the detection results of the absorbance will be described with reference to FIGS. FIGS. 2 and 3 show the results of tests performed using the above-described diagnostic device 10 as described below.
[0023]
In this test, (1) a sample wastewater and a sample microorganism Euglena are mixed and exposed. Next, (2) after removing the waste water, a cell lysis / DNA unwinding solution (pH 13) is added to make only single-stranded DNA. Next, (3) the sample solution is injected into the capillary column by applying a voltage. Then, (4) electrophoresis is performed under predetermined conditions (applied voltage: -10 kV, current: 2 mA, capillary temperature: 25 ° C., capillary inner diameter: 75 μm, capillary effective length: 380 mm, detection wavelength: 260 nm) to separate DNAs of various molecular weights. I do. (5) The absorbance is measured at the detector site to detect and evaluate DNA.
[0024]
Such a test was performed on raw water (TOC concentration: 500 mg / L), 10-fold diluted water (TOC concentration: 50 mg / L), and 100-fold diluted water (TOC concentration: 5 mg / L). The results are shown in FIGS. 2 (a) to 2 (c), respectively. FIG. 2 (d) shows the results of testing untreated wastewater that can be discharged (TOC concentration: 0 mg / L). In addition, as a comparative example, unfragmented DNA (hereinafter, referred to as non-fragmented DNA) was tested in each sample, and the results were displayed alongside the test results of the fragmented DNA. Further, the results obtained from FIGS. 2A to 2D are summarized in a table and shown in FIG.
[0025]
As shown in FIG. 2A, in the raw water of leachate leachate, as a result of measuring the absorbance of the fragmented DNA, 12 large detection peaks were detected. In addition, as shown in FIG. 3, 26 ng / μL of the fragmented DNA was confirmed. Thereby, it can be confirmed that the raw water of leachate leachate is harmful.
[0026]
Further, as shown in FIG. 2 (b), when the leachate leachate was diluted 10-fold, the number of detection peaks was smaller than that in the case of raw water, but five were detected, and fragmented DNA was present at 8 ng / μL. Thereby, harmfulness was confirmed even when diluted 10-fold.
[0027]
As shown in FIG. 2 (c), when the leachate leachate was diluted 100 times, the number of detection peaks of fragmented DNA was reduced to one, which was similar to the untreated condition shown in FIG. 2 (d). Almost gone. In addition, only 2 ng / μL of fragmented DNA was confirmed. From the above results, it can be seen that the leachate leachate used in the test should be discharged after diluting it 100 times or more.
[0028]
The harmfulness of water itself can be diagnosed by measuring the absorbance while electrophoresing the fragmented DNA in this manner.
[0029]
On the other hand, the detection peak of non-fragmented DNA does not change at all even when diluted. Therefore, in order to diagnose the harmfulness of water, it is necessary to fragment DNA for measurement.
[0030]
Next, the operation of the diagnostic device 10 configured as described above will be described.
[0031]
While electrophoresing the fragmented DNA, the capillary electrophoresis device 22 measures the absorbance of the DNA during the electrophoresis. That is, the electrophoresis step of electrophoresing DNA and the damage analysis step of analyzing the degree of DNA damage are performed simultaneously. Therefore, the measurement time can be significantly reduced.
[0032]
Further, according to the diagnostic device 10, the electrophoresis step and the damage analysis step are performed using the capillary electrophoresis apparatus 22, so that the electrophoresis step and the damage analysis step can be newly automated. Other steps (ie, a mixing step of mixing wastewater and cells (or a microorganism treatment step), an immobilization step of immobilizing cells, and a lysis / rewinding step of lysing cells and unwinding DNA) Can be automated than before. Thereby, all the steps can be automated.
[0033]
Furthermore, according to the diagnostic apparatus 10, since the analysis of the degree of DNA damage is performed based on the measurement result of the absorbance, the diagnostic apparatus 10 always has a fixed criterion and can suppress an analysis error.
[0034]
FIG. 4 is a configuration diagram showing a second embodiment of a diagnostic apparatus to which the hazard diagnostic method according to the present invention is applied.
[0035]
As shown in the figure, the diagnostic device 32 is a microchip and has a microreactor 34. The microreactor 34 is connected to a mixed solution injection tube 36, a cell lysis solution injection tube 38, a DNA rewind solution injection tube 40, a capillary column 42, and a drainage tube 44. The microreactor 34 includes pipes 34a to 34f for flowing the injection pipes from the respective injection pipes 36, 38, and 40 and for mixing the injection liquid. In addition, a gel for fixing cells is injected into the microreactor 34 in advance.
[0036]
In the diagnostic device 32 configured as described above, first, a mixed solution of drainage and cells is injected into the microreactor 34 from the mixed solution injection pipe 36. Thus, the cells are fixed by the cell fixing gel that has been injected in advance. Next, the cell lysate is injected from the cell lysate injection tube 38. As a result, the cells are lysed. Then, the DNA unwinding solution is injected from the DNA unwinding solution injection tube 40 to fragment the DNA. In this case, it is preferable to automate the injection after the mixing, the injection of the cell lysate, and the injection of the DNA unwinding solution by using an injection pump and a timer mechanism provided in each injection tube.
[0037]
Next, after the treatment solution is removed by the drain pipe 44, the gel is dissolved again. Then, the fragmented DNA is electrophoresed into the capillary column 42 by applying a voltage using a voltage application device (not shown). At the same time, the absorbance is measured by an absorbance measuring device 46 provided on the capillary column 42. Thereby, the degree of damage of the fragmented DNA can be analyzed.
[0038]
According to the diagnostic device 32 described above, since the entire process can be formed into a microchip structure, the size of the device can be significantly reduced. For example, one side d of the microchip can be reduced to 50 mm.
[0039]
Further, according to the diagnostic device 32, all the steps can be performed continuously, so that automatic diagnosis can be performed and the processing capability can be greatly improved.
[0040]
In the diagnostic device 32, if a plurality of capillary columns 42 are arranged on a chip, a plurality of samples can be measured at one time.
[0041]
【The invention's effect】
As described above, according to the method for diagnosing liquid harm according to the present invention, the DNA to be electrophoresed in the capillary column is irradiated with test light to analyze the degree of damage to the DNA. This can be performed continuously, and as a result, the process can be automated and the measurement time can be reduced.
[Brief description of the drawings]
FIG. 1 is a configuration diagram showing a first embodiment of a diagnostic apparatus using a hazard diagnosis method according to the present invention; FIG. 2 is a diagram showing an example of test results; FIG. 3 is a table showing results of FIG. 2; 4 is a block diagram showing a second embodiment of the diagnostic apparatus using the hazard diagnostic method according to the present invention. [Description of References] 10 ... diagnostic apparatus, 12 ... container, 14 ... drain supply pipe, 16 ... microorganism supply pipe, 18: Gel injection tube, 20: Dissolution / rewind solution injection tube, 22: Capillary electrophoresis device, 24: Recovery container, 26: Capillary column, 28: Voltage application device, 30: Absorbance measurement device, 32: Diagnostic device (micro Chip), 34: microreactor, 36: mixed solution injection tube, 38: cell lysis solution injection tube, 40: DNA unwinding solution injection tube, 42: capillary column, 44: drain tube, 46: absorbance measuring device

Claims (2)

In a liquid hazard diagnosis method for analyzing the hazard of liquid by single-cell gel electrophoresis,
A cell mixing step of mixing cells with the liquid,
Injecting an immobilization gel into a liquid mixture of the liquid and the cells, an immobilization step of immobilizing the cells,
A lysis / rewind step of lysing the fixed cells by injecting a lysis / rewind solution into the mixture, and rewinding the DNA of the lysed cells;
An electrophoresis step of electrophoresing the unwound DNA in a capillary column,
A damage analysis step of irradiating the DNA to be electrophoresed in the capillary column with test light and analyzing a degree of damage of the DNA,
A harmful diagnosis method comprising: The harmful diagnosis method according to claim 1, wherein the fixing step and the dissolving / unwinding step are performed in a microreactor.

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