JP2004357544A - Method for measuring concentration of solution with live species and apparatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for measuring the concentration of a solution with a live species, by which the concentration can highly selectively be measured with the live species such as yeast without needing a special experiment device, and to provide an apparatus therefor. <P>SOLUTION: This method for measuring the concentration of the solution with the live species comprises measuring the change in the growth of a cell or microbial cell transformant which changes its activity or proliferation degree in response to the concentration of a substance to be measured, thereby indirectly measuring the substance to be measured. For example, a yeast transformant which has a compensating factor for compensating one or more defected conditions by the expression of genes under an environment having the defected conditions among conditions essential for the living and proliferation is used to determine the concentration of the substance to be measured, while using the change in the proliferation degree of the yeast as a signal. In an example, a measuring apparatus using a combination of a Clark type oxygen electrode with an immobilized yeast membrane disposed on the cathode side of the oxygen electrode is used. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method and an apparatus for measuring the concentration of a chemical substance using a biological species. The present invention relates to a method and an apparatus for measuring the concentration of a solution using a biological species, which converts the concentration of a substance into the cell activity / proliferation of the cell / bacteria for measurement.
[0002]
[Prior art]
In the measurement of environmental samples, the so-called bioassay method, which uses the biological reaction of cells and cells, measures the effects on living organisms directly and in a complex manner, compared to analytical chemistry methods that use instrumental analysis. There is an advantage that the measurement system can be simplified by utilizing the specificity of the biological substance, and there are several practical examples. For example, the BOD method (biochemical oxygen demand) standardized in JIS K0400-21-10 as an indicator of the nutrient amount of water quality is based on the identification of the amount of oxygen consumed in decomposing organic matter of a specific bacterium, thereby identifying the water quality. This is a kind of bioassay method for evaluation.
[0003]
In such a bioassay method using cells or cells, a measurement method utilizing the high specificity of a living body is known. In such a selective bioassay method using cells and cells, measurement is usually performed using the expression of a specific enzyme in a cell as an index. For example, Non-Patent Document 1 discloses a response element in which an allyl hydrocarbon receptor (AhR) -allyl hydrocarbon nuclear translocator (Arnt) complex bound to dioxins was introduced into cells as a reporter gene using Hepa1c1c7 cells. Discloses a method for estimating a dioxin concentration from the amount of Luciferase generated by activating the transcription of a Luciferase gene having a foreign substance response element (XRE).
[0004]
In addition, using a similar technique, the budding yeast Saccharomyces cerevisiae was arranged with a human estrogen receptor gene downstream of a promoter having transcription activity in the yeast and a β-galactosidase gene as a reporter gene downstream of an estrogen response element. It has been shown that the expression level of β-galactosidase in the transformant using the gene changes in an estradiol concentration-dependent manner.
[0005]
These reporter gene assays require the use of a substrate reaction to detect the amount of expressed enzyme as a measurement method, and require steps such as centrifugation to elute proteins in biological species, addition of detergent, and freeze-thawing. It is always the case. Usually, a luminescence method, a fluorescence method, an absorption method, or the like is often used for the measurement of the substrate reaction, and therefore, a dedicated measuring device is usually required. For this reason, such a bioassay method requires a place equipped with equipment, and it is not easy to perform, for example, portable measurement.
[0006]
On the other hand, as a method for solving such a problem, a measurement method using an electrode is known. For example, there is known a method based on JIS K3602 in which a sensor chip is formed by, for example, combining an immobilized microorganism membrane and an electrode for measuring dissolved oxygen in BOD measurement. Further, there is known a glucose measuring device using an enzyme glucose oxidase constructed by utilizing only a specific catalytic reaction by extracting only an enzyme from a biological species, as defined in JIS K0701, for example.
[0007]
[Non-patent document 1]
Organohalogen Compounds Vol. 40 (1999), p. 27-30
[0008]
[Problems to be solved by the invention]
The present invention has been made in view of the above circumstances of the prior art, and has as its object to perform highly selective measurement using a biological species such as a cell with a relatively simple apparatus and method. It is an object of the present invention to provide a method and an apparatus for measuring the concentration of a solution using the method.
[0009]
[Means for Solving the Problems]
That is, the invention according to claim 1 is a method for measuring the concentration of a test substance in a solution using a biological species composed of cells or cells, wherein the cells or cells are indispensable for their survival and growth. A transformant which is placed in an environment having a deficiency condition that lacks one or more of the following conditions, and has a compensation factor for compensating the deficiency condition by gene expression in accordance with the amount of the test substance, wherein the cell or the bacterial cell is used. There is provided a method for measuring the concentration of a solution using a biological species, which comprises determining the concentration of a substance to be measured using a change in the cell activity or the degree of proliferation of the cells or the cells as an index.
[0010]
The invention according to claim 2 is the method according to claim 1, wherein the deficiency condition is determined by auxotrophy.
[0011]
The invention according to claim 3 uses the cell according to claim 1, wherein the cell or bacterial body contains at least a part of a ligand-binding transcription factor or a ligand-binding region of the regulator, or a mutant thereof. It is characterized by being transformed by a gene encoding a fusion protein and a gene obtained by connecting a gene encoding the compensation factor downstream of a recognition sequence of the transcription factor and a base sequence required for transcription initiation. This is a method for measuring the concentration of a solution using a living species to be used.
[0012]
The invention according to claim 4 is a method for measuring the concentration of a test substance in a solution using a biological species consisting of cells or cells, wherein the cells or cells are capable of expressing a specific enzyme. It is placed in a drug-added environment containing a drug that exhibits toxicity and inhibits the survival and growth of the cells or cells, and the cells or cells express the enzyme in accordance with the amount of the test substance. A transformant having an enzyme-producing factor produced by the method, wherein the concentration of the analyte is determined using the change in the cell activity or proliferation of the cells or cells as an index, and the solution concentration using a biological species. Is a measuring method.
[0013]
The invention according to claim 5 uses the cell according to claim 4, wherein the cell or bacterial body contains at least a part of a ligand-responsive transcription factor or a ligand binding region of the regulator, or a mutant thereof. A gene encoding the fusion protein and a gene obtained by connecting the gene encoding the enzyme-producing factor downstream of a recognition sequence of the transcription factor and a base sequence required for transcription initiation. It is a method for measuring the concentration of a solution using a biological species described as a feature.
[0014]
The invention according to claim 6 is the method for measuring a solution concentration using a biological species according to any one of claims 1 to 5, characterized in that yeast is used as the cells or cells.
[0015]
The invention according to claim 7 is an apparatus for measuring the concentration of a test substance in a solution using a biological species consisting of cells or cells, wherein the cells or cells are essential for survival and growth. A transformant which is placed in an environment having a deficiency condition lacking one or more of the above, and has a compensating factor for compensating the deficiency condition by gene expression in accordance with the abundance of the test substance, wherein the cells or cells are provided, Determining the concentration of the analyte using the change in the cell activity or proliferation of the cells or cells as an index, and immobilizing the cells or cells on a sensor for measuring the change in the activity or proliferation. And measuring the concentration of the test substance in the solution by a change in the output of the sensor.
[0016]
The invention according to claim 8 is an apparatus for measuring the concentration of a test substance in a solution using a biological species consisting of cells or cells, wherein the cells or cells are capable of expressing a specific enzyme. It is placed in a drug-added environment containing a drug that exhibits toxicity and inhibits the survival and growth of the cells or cells, and the cells or cells express the enzyme in accordance with the amount of the test substance. A transformant having an enzyme-producing factor produced by the method, comprising determining the concentration of the analyte using the change in the cell activity or proliferation of the cells or cells as an index, wherein the cells or cells are A solution concentration measuring apparatus using a biological species, which is immobilized on a sensor for measuring a change in activity or proliferation degree, and measures a concentration of a test substance in a solution by an output change of the sensor. .
[0017]
A ninth aspect of the present invention is the apparatus for measuring a solution concentration using a biological species according to the seventh or eighth aspect, wherein yeast is used as the cells or cells.
[0018]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, embodiments of the present invention will be described.
The present invention uses a cell / bacterium transformant whose cell activity / proliferation degree changes depending on the concentration of a test substance, and indirectly measures the concentration of the test substance by measuring the change in growth. It is assumed that.
[0019]
In the present invention, the mechanism of transcriptional activation by the analyte is not specified. For example, an operon site on DNA and a fusion protein of an operon binding protein and a ligand-responsive transcriptional regulator, a foreign substance response element on DNA and an allyl hydrocarbon receptor , Estrogen responsive elements on DNA, estrogen receptors, etc., and their related proteins in combination or steroid hormones such as androgens, glucocorticoids, nuclear hormone receptors such as thyroid hormones, fat-soluble vitamins, prostanoids For example, a mechanism that adjusts the transcription in the positive or negative direction in response to any measurement target, such as a system using the above method, can be used.
[0020]
The environmental conditions compensated by gene expression in the present invention are not particularly specified, but nucleic acids such as adenine and its synthase, histidine, uracil, tryptophan, essential amino acids such as leucine and its synthase, ampicillin, Examples include antibiotics such as kanamycin and its degrading enzymes.
[0021]
Although there is no particular limitation on the environmental conditions under which an enzyme is produced by gene expression to exhibit toxicity, there is a condition in which the produced enzyme changes an added drug into a toxic substance, for example, 5-fluoroorotin. Any combination of acid and uracil synthase, α-aminoadipic acid and lysine synthase can be used.
[0022]
As the cells / cells to be used, any living species can be used as long as a transformant can be prepared, and human cells, mouse cells, yeast, nematodes, Escherichia coli, etc. can be used. Particularly, those capable of stably producing and culturing a transformant are particularly preferably used.
[0023]
In the present invention, as a method for measuring the cell activity / proliferation, any method is applicable as long as the measurement is possible. A method having rapid and high reproducibility is desirable. For example, turbidity measurement, oxygen consumption measurement, reporter gene production amount measurement (reporter gene assay) and the like can be used, but not limited thereto, weight change measurement, It is possible to use techniques such as total protein amount measurement, counting, sugar content change measurement, amino acid concentration change measurement, and alcohol concentration measurement.
[0024]
In particular, in the case of performing measurement by oxygen consumption, any known dissolved oxygen measurement method can be used by using the biological species, and any measurement method used for measurement of biochemical oxygen demand is preferably used. Can be done.
[0025]
This measurement can also be performed using a sensor. That is, it is possible to identify the concentration of the analyte by immobilizing the biological species on a sensor capable of measuring the change in the activity or proliferation of the cells or bacterial cells used for the measurement and performing the measurement. . In this case, any known sensor can be applied as long as it achieves the desired purpose, but the dissolved oxygen concentration, hydrogen peroxide concentration, carbon dioxide concentration, sugar content, alcohol concentration, amino acid concentration, A minute weight measurement using a probe or the like can be preferably used. In this case, it is desirable to form an immobilized membrane on which the cells or cells used for measurement are immobilized on the sensor surface.
[0026]
As the carrier used for the immobilization of the cells or the cells, for example, nitrocellulose, polyacrylonitrile, photocurable resin, polyurethane and the like can be used, and immobilization is performed using a conventionally known technique.
[0027]
【Example】
Hereinafter, the present invention will be described more specifically with reference to examples. However, the following examples should be regarded as helping to obtain a specific understanding of the present invention, and the scope of the present invention is not limited by the following examples. It is not done.
[0028]
(Example 1)
The following plasmids (a), (b) and (c) were introduced into a wild strain of Saccharomyces cerevisiae by the lithium acetate method. Specifically, the yeast in the logarithmic growth phase is spun down, the supernatant is discarded, and then suspended in a lithium acetate-Tris buffer solution. The denatured salmon sperm DNA and the following (a), (b), and (c) are shown. Agar free from histidine, leucine and tryptophan was prepared by mixing with DNA, adding polyethylene glycol, treating at 42 ° C., and adding a part of essential amino acids and glucose as a carbon source using Difco's Yeast Nitrogen base. The transformed yeast strain 1 was obtained by spreading on a medium.
[0029]
As a control, a yeast strain 2 transformed by the following (a), (b) and (d), which was prepared using the same method, was obtained.
[0030]
(A) Preparation of AhR Expression Plasmid AhR-encoding DNA is described, for example, in J. Am. It can be prepared according to Sambrook et al., Molecular Cloning, 2nd edition. Specifically, first, human HepG2 cells are homogenized in a solution containing guanidine hydrochloride, phenol and chloroform are added, centrifugation is performed, and the supernatant fraction is collected to remove the protein component. Thereafter, total RNA is extracted by the SDS-phenol method. Using the obtained total RNA as a template, a single-stranded cDNA is synthesized using oligo dT primer and reverse transcriptase.
[0031]
Furthermore, PCR was performed using the obtained single-stranded cDNA as a template and the following oligonucleotides as primers to obtain a double-stranded DNA fragment of AhR. This is inserted into the cloning site of pRS313, a plasmid having a known histidine marker and a centromere and usable in S. cerevisiae, by joining a transcription activation region of alcohol dehydrogenase and a terminator according to ordinary genetic engineering techniques. Further, a plasmid that expresses AhR in yeast was prepared by further insertion during this period.
[0032]
(B) Preparation of Arnt Expression Plasmid A double-stranded DNA fragment of Arnt was obtained in the same manner as in AhR, and this was cloned into a cloning site of pRS315, a plasmid having a known leucine marker and centromere and usable in budding yeast. Further, a transcription-activating region of alcohol dehydrogenase and a terminator were further inserted while being connected and inserted in accordance with ordinary genetic engineering techniques, thereby producing a plasmid expressing Arnt in yeast.
[0033]
(C) Preparation of uracil expression plasmid In the cloning site of pRS314, a plasmid having a tryptophan marker and a centromere and usable in budding yeast, a human foreign body response element (XRE) recognized by the AhR-Arnt complex and yeast A functional tk core promoter sequence and a region encoding a uracil synthase excised from pRS316, a plasmid having a uracil marker and a centromere downstream thereof and usable in Saccharomyces cerevisiae, can be obtained by conventional genetic engineering. It was built according to the standard method.
[0034]
(D) Preparation of control of uracil expression plasmid Human foreign body response element (XRE) recognized by AhR-Arnt complex and yeast in pRS314 which is a plasmid having tryptophan marker and centromere and usable in budding yeast. Only the tk core promoter sequence that was able to function therein was incorporated and produced.
[0035]
These cell lines are cultured in a liquid medium lacking the markers histidine, leucine, and tryptophan until the logarithmic growth phase, and the medium is replaced with a liquid medium lacking histidine, leucine, tryptophan, and uracil, and further cultured for 1 hour. After adjusting the turbidity (OD 600 nm) to 0.2 using the same medium, 100 μl of the mixture was dispensed into a 96-well plate, and beta-naphthoflavone, a ligand of AhR, was added to each plate. Was added at each concentration, and the mixture was allowed to stand at 30 ° C. for 20 hours and cultured.
[0036]
Thereafter, the absorbance (wavelength: 600 nm) of each well of the plate was measured, and the value obtained by subtracting the turbidity in yeast strain 2 from the turbidity in yeast strain 1 was recorded as the response to each beta naphthoflavone. As a result, as shown in FIG. 1, a change in turbidity according to the ligand concentration could be observed.
In FIG. 1, the horizontal axis represents the logarithm of the substrate concentration (beta naphthoflavone concentration) expressed in molar concentration, and the vertical axis represents the absorbance (relative value).
[0037]
(Example 2)
The following measurements were performed using the yeast strains 1 and 2 prepared in Example 1.
The cell line was cultured in a liquid medium lacking the marker until the logarithmic growth phase, and the medium was replaced with a liquid medium lacking histidine, leucine, and tryptophan, and further containing 1 mg / L of 5-fluoroorotic acid, and then further cultured for 1 hour. After culturing for an hour and adjusting the turbidity (OD 600 nm) to 0.2 using the same medium, 100 μl of the mixture was dispensed into a 96-well plate, and each of them was a beta, which is a ligand of AhR. Naphthoflavones were added at each concentration, and the cells were allowed to stand at 30 ° C. for 20 hours and cultured.
[0038]
Thereafter, the absorbance (wavelength: 600 nm) of each well of the plate was measured, and the value obtained by subtracting the turbidity in yeast strain 2 from the turbidity in yeast strain 1 was recorded as the response to each beta naphthoflavone. As a result, as shown in FIG. 2, a change in turbidity according to the ligand concentration could be observed.
In FIG. 2, the horizontal axis is the logarithm of the substrate concentration (beta-naphthoflavon concentration) expressed in molar concentration, and the vertical axis is the absorbance (relative value).
[0039]
(Example 3)
In the present embodiment, an example is shown in which yeast is immobilized on a sensor for measuring a change in the degree of proliferation, and the concentration of beta naphthoflavone in the solution is measured by a change in the output of the sensor.
FIG. 3 shows a measuring device used in this embodiment. This measuring device is composed of a combination of a commercially available Clark-type oxygen electrode and a yeast immobilized membrane disposed on the cathode side of the oxygen electrode. In FIG. 3, reference numeral 1 denotes a sensor, which comprises the oxygen electrode 2 and the yeast fixed membrane 3. Reference numeral 4 denotes a sample solution, reference numeral 5 denotes a stirrer, and reference numeral 6 denotes a measuring device.
[0040]
The yeast fixed membrane was prepared as follows. Cell line 1 was cultured in a liquid medium lacking the marker until the logarithmic growth phase, and the medium was replaced with a liquid medium lacking histidine, leucine, tryptophan and uracil, followed by further culturing for 1 hour, followed by centrifugation and precipitation. Approximately 50 microliters were dropped on a nitrocellulose membrane having a pore size of 0.4 micrometer, suction-filtered, and fixed with the same nitrocellulose membrane.
[0041]
Thereafter, an electrolyte is injected into the oxygen electrode, and the oxygen electrode is connected to a power source. Next, a solution (sample solution) was injected into the measuring section. The solution to be injected was prepared by mixing a sterilized 0.5 M Tris buffer adjusted to pH 7.5 with a liquid medium lacking histidine, leucine, tryptophan and uracil at a ratio of 1: 9.
[0042]
After the current value of the recording device was stabilized, beta naphthoflavone dissolved in dimethylsulfoxide (DMSO) was added to the solution on the measurement section side so that the final concentration of DMSO was 0.1% at each concentration, and after 6 hours. The change in the measured value was recorded. When this was compared with the case where only DMSO was added, a change in response value according to the concentration could be observed as shown in FIG.
[0043]
【The invention's effect】
As described in detail above, according to the present invention, highly selective measurement using a biological species such as yeast can be performed without requiring special experimental equipment. The present invention provides a measurement method and a device therefor, and particularly enables measurement of many measurement objects by using a relatively simple device and operation in the same system.
[Brief description of the drawings]
FIG. 1 is a graph showing the relationship between the beta naphthoflavone concentration (substrate concentration) and the turbidity change (absorbance change) of a sample solution obtained in Example 1 of the present invention.
FIG. 2 is a graph showing the relationship between the beta naphthoflavone concentration (substrate concentration) and the turbidity change (absorbance change) of a sample solution obtained in Example 2 of the present invention.
FIG. 3 is an explanatory diagram of a measuring device used in Embodiment 3 of the present invention.
FIG. 4 is a graph showing the relationship between the beta naphthoflavone concentration (substrate concentration) of the sample solution and the current change amount obtained in Example 3 of the present invention.
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1 ... Sensor, 2 ... Oxygen electrode, 3 ... Yeast fixed membrane, 4 ... Sample solution, 5 ... Stirrer, 6 ... Measuring device.

Claims (9)

A method of measuring the concentration of a test substance in a solution using a biological species consisting of cells or bacterial cells,
The cells or cells are placed in an environment having a deficient condition that lacks one or more of the conditions essential for their survival and growth, and the cells or cells are subjected to the deficiency condition in accordance with the amount of the test substance. A transformant having a compensation factor that compensates by gene expression, wherein a solution using a biological species, wherein the concentration of the analyte is determined using a change in the cell activity or growth rate of the cells or cells as an index How to measure the concentration. The method according to claim 1, wherein the deficiency condition is determined by auxotrophy. The cell or the bacterial cell may be a portion containing at least a part of a ligand-binding region of a ligand-responsive transcription factor or the regulatory factor, or a mutant thereof, a gene encoding a fusion protein using the same, and a recognition sequence of the transcription factor. 2. The method according to claim 1, wherein the gene is transformed by a gene obtained by connecting a gene encoding the compensation factor downstream of a base sequence required for initiation of transcription. Method for measuring solution concentration. A method of measuring the concentration of a test substance in a solution using a biological species consisting of cells or bacterial cells,
The cells or cells are placed in a drug-added environment containing a drug that exhibits toxicity to the expression of a specific enzyme and inhibits the survival and growth of the cells or cells, and Is a transformant having an enzyme-producing factor that produces the enzyme by gene expression in accordance with the abundance of the test substance, and the concentration of the test substance is determined by using the change in the cell activity or proliferation of the cells or bacterial cells as an index. A method for measuring the concentration of a solution using a biological species, characterized in that: The cell or the bacterial cell may be a portion containing at least a part of a ligand-binding region of a ligand-responsive transcription factor or the regulatory factor, or a mutant thereof, a gene encoding a fusion protein using the same, and a recognition sequence of the transcription factor. 5. The method according to claim 4, wherein the gene is transformed with a gene obtained by connecting the gene encoding the enzyme-producing factor downstream of a base sequence necessary for initiation of transcription. How to measure the concentration of the solution. The method according to any one of claims 1 to 5, wherein yeast is used as the cells or cells. An apparatus for measuring the concentration of a test substance in a solution using a biological species consisting of cells or bacterial cells,
The cells or cells are placed in an environment having a deficient condition that lacks one or more of the conditions essential for their survival and growth, and the cells or cells are subjected to the deficiency condition in accordance with the amount of the test substance. A transformant having a compensating factor that compensates by gene expression, comprising determining the concentration of the test substance using a change in the cell activity or proliferation of the cells or cells as an index,
The cell or the cell is immobilized on a sensor for measuring a change in the activity or the degree of proliferation, and a biological species characterized by measuring the concentration of a test substance in a solution by a change in the output of the sensor. Measuring device for solution concentration. An apparatus for measuring the concentration of a test substance in a solution using a biological species consisting of cells or bacterial cells,
The cells or cells are placed in a drug-added environment containing a drug that exhibits toxicity to the expression of a specific enzyme and inhibits the survival and growth of the cells or cells, and Is a transformant having an enzyme-producing factor that produces the enzyme by gene expression in accordance with the abundance of the test substance, and the concentration of the test substance is determined by using the change in the cell activity or proliferation of the cells or bacterial cells as an index. To determine
The cell or the cell is immobilized on a sensor for measuring a change in the activity or the degree of proliferation, and a biological species characterized by measuring the concentration of a test substance in a solution by a change in the output of the sensor. Measuring device for solution concentration. 9. The apparatus according to claim 7, wherein yeast is used as the cells or cells.

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