KR100316153B1 - METHOD FOR MEASURING TOXICITY MATERIALS USING ELIMINATED LIGHT EMITTING MICROORGANISM AND BIOSENSOR KIT - Google Patents

Abstract

The present invention relates to a method for measuring a toxic substance and a biosensor kit for measuring toxic substances by measuring the difference in amount of light emitted by immobilized luminous microorganisms by a toxic substance, And provides a method and apparatus for analyzing toxic substances that are quick, easy to use and economical. The present invention also provides an effective apparatus and method for indicating biological toxicity to various trace toxic substances which are difficult to analyze even in physicochemical analysis.

Description

METHOD FOR MEASURING TOXICITY MATERIALS USING ELIMINATED LIGHT EMITTING MICROORGANISM AND BIOSENSOR KIT

The present invention relates to a method for measuring a substance by a biological method and an apparatus therefor, more particularly, a method for measuring a toxic substance using a light emitting microorganism, and a biosensor kit for the method. The method for measuring toxic substances according to the present invention and the biosensor kit for the same can be used for drinking water test, toxic test of cosmetics, medicines and medical devices. In particular, by detecting and warning toxic substances in water, It can be an important means to solve.

Recently, the technologies related to the water quality environment have been developed to improve the water pollution problem which is one of various environmental problems. However, the pollution degree of the water such as the water source, river water, lake, wastewater and sewage is still serious, The necessity of monitoring and preparation for environmental accidents is increasing day by day. Therefore, there is a great need to develop a water quality toxicity measuring device to prevent such damage.

Many researchers have developed a device for early detection and warning of wastewater effluent and other influx of toxic substances. In particular, bio-alarm devices are installed in river water, sewage treatment plants, and wastewater treatment plants. In the case of river water, it is installed for the purpose of prevention of damage to inflow of pollutants from wastewater discharge facility and accident prevention, and monitoring of river water. If there are many pollutants in the sewage treatment plant and there is industrial facility in the drainage area, More serious. In addition, since many wastewater treatment plants treat wastewater generated from wastewater or waste landfill together, there is a greater need to monitor the quality of water by installing biological alarm devices. In foreign countries, biological alarm systems are installed in wastewater discharge facilities.

Conventionally, there are physico-chemical methods and biological methods as methods for measuring the water-borne toxic substances. In the physico-chemical method, the wastewater to be measured is collected and transported to a laboratory, and then divided into organic pollutants, heavy metals, Analyze according to the ingredients. For the analysis of organic pollutants, carcinogenic substances such as chloromethane, chloroethane, 1,1-dichloromethane, methylene, and trichloroethane are mainly analyzed. The concentration of the organic pollutants is determined by gas chromatography (GC) . Chromium, arsenic, cadmium, lead, etc. are mainly used for heavy metal materials and they are measured by inductively coupled plasma (Inductively Coupled Plasma). The concentrations of diazinon, parathion, and malathion, which are organic phosphorus-based pesticides, in the sample are measured using gas chromatography. However, this method requires expensive analytical reagents and highly skilled analytical personnel such as GC-MS (gas chromatography-mass spectrometry) and ICP-MS (inductively coupled plasma-mass spectrometry) It is difficult to analyze if the analysis time is long and the composition of the inflow toxic substance is unknown. In addition, there is a problem that when the sampled sample is moved to a long distance, changes in components and concentration may occur due to reactions in the sample.

Representative of the conventional biological method is an analysis method Azur Environmental Inc. Microtox ^TM (trade name). It is a device for measuring toxicity using the bioluminescence phenomenon of a luminescent microorganism separated from the ocean. Referring to FIG. 1, the Microtox ^TM analysis method will be described in detail. The apparatus includes a pre-cooling tablet 21 for preparing a microorganism preparation using a lyophilized luminous microorganism (photobacterium phosphoreum), an optical meter A turret 23 having a built-in phototube 22, and a culture chamber 24 capable of gradually diluting the sample and adjusting the temperature. The method for measuring toxicity using this device is as follows

a) Add 1 ml of Microtox reconstitution solution to the cuvette in the pre-cooling tablet (1).

b) In culture (24), add 1 ml of Microtox Diluent to each of the four cuvettes from column A1 to A4.

c) B puts the micro-Tox dilution of 500 μ l of each cuvette in the row B1 to B5.

d) and then to a salt concentration adjustment of the sample into a 250 μ l Tox micro osmotic pressure fluid (Microtox osmotic adjustment solution) in a cuvette of A5, by mixing 2.5ml of the sample was added to this diluted sample is about 9%.

e) For the gradual dilution of A5 samples, transfer 1 ml of sample from A5 to A4, mix well, then transfer 1 ml of A4 sample from A3 and mix.

f) Leave for 5 minutes for temperature control.

g) The microtox reagent that has been stored frozen is quickly re-adjusted to the microtox regeneration liquid in the pre-cooling oven 21 and put back into the pre-cooling oven 21 for mixing.

h) 10 into a mixture of micro-Tox μ l of the reagent mixture to each cuvette and in column B, and then wait 5 to 15 minutes.

i) Each cuvette in row B is placed in a turret well (3), the turret is closed, the luminous intensity of the light emitted from the bacterial mixture is measured at a wavelength of 490 nm, recorded with a digital display and a chart recorder, Lt; / RTI >

j) after a quick column A into the diluted sample solution in the cuvette in the cuvette 500 μ l in each corresponding column B mix and record the light intensity after five minutes.

k) Measure and record the intensity again 15 minutes after the start of the analysis.

l) If the sample is excessively colored, perform color calibration experiments. Suspension samples should be centrifuged and supernatant should be used.

Through the above experiment process 0, 5, and obtaining the brightness (light level) of between 15 minutes and 5 minutes is calculated EC ₅₀ (effective concentration) 15 EC ₅₀ bun. Here, the concentration of the toxic substance in which 50% of the light emission amount is reduced is usually referred to as EC ₅₀ .

Since the microtox method of the present invention uses a lyophilized luminescent microorganism, the lyophilized luminescent microorganism must be purchased separately, and freeze-dried A separate reagent such as a microtox osmotic pressure regulating solution or a microtox regenerating solution should be added in order to reactivate the light emitting microorganisms and a separate temperature regulating device is required. Also, since the experimental method requires a series of preprocessing operations, it is troublesome and complicated.

In order to solve the above problems, the method and apparatus of the present invention provides a biosensor kit using immobilized light emitting microorganisms capable of measuring and analyzing a water toxic substance immediately at any position, and a method for measuring toxicity of a water system using the same, And a method and an apparatus for analyzing toxic substances which are simple and economical to use. The use of immobilized light emitting microorganisms also has the advantage of being able to maintain long-term activity and have a high reaction rate. In addition, the biological analysis method using such a light emitting microorganism is intended to provide an effective method and apparatus for indicating biological toxicity to various trace toxic substances which are difficult to analyze even in physicochemical analysis.

1 is a schematic diagram of a conventional Azur Environmental Inc. Microtox ^TM (TM) for measuring the toxic substance using a light-emitting microorganisms lyophilized.

FIG. 2 shows an embodiment of a conventional biosensor kit for measuring water quality toxicity according to the present invention.

Description of the Related Art

1. cap 2. network

3. Immobilized light emitting microorganisms 4. Glass

5. Band filter 6. Optical meter

7. Microprocessor 8. Keyboard and function keys

9. LCD screen (LED: using light-emitting diode)

10. Printer port 11. Communication port (RS232C)

12. Buzzer and alarm function 13. Power supply

14. Case 21. Previous well,

22. Photomultiplier tubes

23. turret 24. incubation well

25. Temperature set 26. 100% adjuster (100% adjust)

27. 0% adjuster (0% adjust)

28. Light-emitting diode

29. Operation panel

The present invention relates to a method for measuring a toxic substance in a water system using a luminescent microorganism and a biosensor kit for the same, and more particularly, to a method for measuring a toxic substance in a water system by measuring a change in the quantity of light of an immobilized luminescent microorganism caused by a toxic substance, To a biosensor kit.

Specifically, the present invention relates to a method for measuring a toxic substance, characterized by measuring a change in the amount of light of an immobilized light emitting microorganism due to a toxic substance.

The immobilized microorganism may be selected from recombinant strains obtained by introducing a gene encoding a photobacterium phosphoreum, Vibrio fischeri or a luminescent enzyme thereof into a host microorganism, can be immobilized on a porous substrate is selected from the last color (κ -carrageenan), polyacrylamide, cellulose, agarose-silver alginate, kappa. Alginate is preferable as the immobilization substrate, and the luminescent microorganism is a marine microorganism, and strontium alginate using the stromium ion is more preferable because the concentration of NaCl in the medium is high.

In addition, the measurement of toxic substances uses a photometer to convert the change in light quantity into a charge quantity and send it to the calculation means, and the calculation means analyzes and stores the amount of the sent charge. At this time, the concentration of the toxic substance can be determined by analyzing the change of the charge amount by the magic, the velocity method, and the bioluminescence method.

In addition, if the measured concentration of the toxic substance is equal to or higher than the set value, a method of alarming using an alarm device may be included.

The change of the amount of light is measured and analyzed. The change of the amount of light can be converted into the amount of charge by using an optical measuring device, and analyzed and stored by calculation means.

In addition, the present invention relates to a biosensor kit for measuring toxic substances using immobilized light emitting microorganisms, and a configuration diagram of the biosensor kit according to the present invention is shown in FIG. This biosensor kit can be composed of two parts. The first part is a part for measuring toxic substances using immobilized light emitting microorganisms (1-6 in FIG. 2), and the second part is a part for outputting the concentration of water toxicity 7-12).

The biosensor kit for measuring a toxic substance according to the present invention comprises a fixed light emitting microorganism supported by a net, a micro-organism supported by the network, a support for supporting the net and immobilized microorganisms, A measurement unit consisting of a band filter and an optical measuring unit for measuring a change in light quantity according to the water quality toxicity, an operation unit for converting the measurement value in the measurement unit into a water quality toxic concentration, and an input / output unit connected to the calculation unit , And a biosensor kit for measuring toxic substances using immobilized light emitting microorganisms.

In the present invention, a cap structure capable of supporting the immobilizing microorganism and desorbing the means for providing a darkroom condition is preferable. The optical measuring instrument may be selected from photomultiplier tubes (PMT) or photodiode.

In the present invention, the output device may be at least one device selected from a monitor, a printer port, a communication port, and an alarm alarm function device. The biosensor kit can also be used as a portable device.

The present invention provides a method for measuring a toxic substance in water by bringing a measuring part of a biosensor kit for measuring toxic substances into contact with a water system using the immobilized light emitting microorganism according to the present invention.

The measured values are analyzed by EC ₅₀ , specific rate method, bioluminescence intensity method and the like. These values are output through output means such as a liquid crystal screen, a printer port, or a communication port.

Here, the difference in the amount of change in the amount of emitted light is calculated through the specific velocity method, the bioluminescence amount method, the magic magic, etc., and the value calculated here has a value close to a straight line. The calculation method is as follows.

a) Method using specific rate

As a method for measuring the amount of bioluminescence occurring during a certain period of time,

μ = (ln L ₂ - ln L ₁ (t ₂ -t ₁ )

μ : Specific speed

t ₁ : Initial measurement time

t ₂ : second measuring time

L ₁ : First measured bioluminescence amount

L ₂ : the second measured bioluminescence amount

b) bioluminescence intensity < RTI ID = 0.0 >

I _o / I = I + Ks [Q]

I _o : Bioluminescence in the absence of toxic substances

I: Bioluminescence in the absence of toxic substances

Ks: constant

[Q]: concentration of toxic substance

c) Gamma value (gamma value)

The ratio of the amount of reduced light to the amount of remaining light

γ (t, T): Gamma effect for t time at T ° C

R (t) is the ratio of the bioluminescence amount of the sample (blank) to which the toxic substance has not flowed for the time t, and the bioluminescence amount of the sample to which the toxic substance is not introduced after the time t is divided by the value at the first measurement.

L (0): Bioluminescence of the sample at the first measurement

L (t) is the bioluminescence after t hours.

The γ value at each concentration can be obtained and the concentration and γ value of the toxic substance can be expressed in a log-log graph and linearized. At this time, the concentration of the toxic substance which becomes? = 1 becomes the EC ₅₀ value. The value at this time is different depending on the kind of each toxic substance. Using this characteristic, the value is obtained, and when the value exceeds EC ₅₀ , an alarm is issued or displayed.

In the production of the biosensor kit according to the present invention, the method of activating the existing free cells is complicated and uncomfortable to operate. Therefore, immobilized light emitting microorganisms are used to solve the problem. By using immobilized microorganisms, it is possible to measure toxic substances more sensitively and more rapidly than when water quality toxic substances are measured in free cells.

Microorganisms which can be used for the measurement of toxic substances according to the present invention are light-emitting microorganisms, specifically, light-emitting marine microorganisms. In this case, not only the light-emitting microorganism as a wild strain but also a recombinant strain in which a gene encoding a luminescent enzyme (lux gene) is introduced into a host microorganism such as Escherichia coli can be used, and a recombinant strain can be produced by a usual method. Photobacterium phosphoreum, Vibrio fischeri, and the like are examples of usable light emitting microorganisms.

The luminescence phenomenon is usually accomplished by the interaction of the luminescent enzymes luciferase, reduced type of fibrin and long chain aldehyde in the presence of oxygen, which constitutes a part of the cell's electron transport system. The fibrin mononucleotide (FMN) is reduced by NADH, which emits light while reacting with a luminescent enzyme or the like. Since the luminescence phenomenon depends on the flow of electrons, the change in luminescence represents a change in metabolic activity and the health state of the living body.

FMNH ₂ + O ₂ + RCHO → light emission + FMN + H ₂ O + RCOOH

The luminescence spectrum of the Photo Bacterium Forum 4, which is one of the luminescent microorganisms, is 421 to 630 nm and shows the maximum value at 490 nm which is the visible light region. The color actually emitted is the intermediate color between light blue and green. As mentioned above, this luminescence phenomenon occurs in connection with aerobic respiration metabolism, but ATP production does not occur because energy is emitted to light. If any toxic substance acts on this process, the quantity of emitted light decreases. This principle can be used to measure the toxicity of toxic substances. Unlike most other microorganisms that can not grow at low temperatures, the PhotoBacterium Force Forum (4) has the advantage of being able to grow at a low temperature of 4 ° C, allowing long-term storage at low temperatures and monitoring of relatively low- can do.

At this time, microorganisms were immobilized to add mobility and accuracy. The immobilization method can be carried out by any conventional immobilization method, but is particularly preferably a method for trapping microorganisms in a porous gel to limit free movement of cells. It can be maintained as a stable and active biocatalyst for a long time by immobilization, and the reaction rate is increased, and the productivity per unit volume is increased to increase the productivity.

As a method of immobilizing the light emitting microorganism, all commonly known immobilization methods can be used, and it is particularly preferable to immobilize the immobilized microorganism on a porous gel. For this, the choice of immobilized substrate is important, the immobilized substrate is insoluble, must not undergo chemical reactions with the toxic material or decompose, should be highly dispersible, and should be cheap and permeable. Examples of immobilizing substrates that satisfy these requirements include starch, alginate, carrageenan, and the like, with alginate being most preferred. Alginate has a relatively simple immobilization process, and cells can survive in immobilized materials and are harmless to the human body as it is used as a food additive, and the gel structure is safe as well as relatively inexpensive. It also helps to maintain the luminous metabolism of the luminous microorganisms without harming them.

Calcium ions, potassium ions, strontium ions and the like are used to cure the alginate. The principle of curing is that the alginate is composed of D-mannuronic acid and L-gluconic acid group, and L-gluconic acid is calcium ion , Strontium ions, and the like. The luminescent microorganism has been devised to use strontium alginate using a strontium ion since the concentration of NaCl in the culture medium as a marine bacteria is high and calcium ions are replaced by potassium ions.

Hereinafter, embodiments of the present invention will be described with reference to the drawings.

The water quality toxicity measurement biosensor kit of the present invention is shown in Fig. 2, in which strontium alginate is immobilized with a luminescent microorganism (3), and a net (2) is used to support immobilized luminescent microorganisms (3). This is combined using the cap (1). Here, the cap (1) also serves as a dark room for supporting immobilized cells and preventing other impure light from entering. The immobilized light-emitting microorganism 3 needs to be replaced after it is used once, so that it is preferable to have a cap shape that is easy to be detached.

To measure the toxic substances entering the water system, contact the measuring part (1-6) of the biosensor. Water is brought into contact with the immobilized light emitting microorganisms through the darkroom condition and the cap serving as a support for immobilized cells. At this time, the amount of emitted light changes depending on the kind and concentration of the toxic substance of the water quality. Such a change in the amount of emitted light is measured by an optical measuring device 6 which changes the light amount to the telephone amount. At this time, the net 2 serves to support the immobilized light emitting microorganisms 4 and to freely pass the water. The band filter 4 is necessary to narrow the range of the amount of light and to make an accurate measurement. The optical measuring device 6 converts the amount of light generated in the immobilized light emitting microorganism into the amount of charge, and sends a change in the amount of charge to the microprocessor. At this time, the change of the charge quantity is analyzed and calculated, and the communication port is mounted so that it can be displayed on the liquid crystal screen and used by using the printer port or connecting with a personal computer. In addition, toxic substances at risk levels may have a buzzer and an alarm when they enter. The power supply unit used in portable biosensor kits should be supplied between 4.5 and 9V for easy portability. Input operations required for use are entered through the keyboard and function keys. Before measurement of the toxic substance in the water system, the luminous intensity of the immobilized light emitting microorganism is inputted and used as reference data, and then immersed in the water system to measure a change in the amount of emitted light. Measured values can be measured within 5 minutes. These values are analyzed by EC ₅₀ , specific speed method, bioluminescence amount method, etc., and the microprocessor displays and performs the storing function and the arithmetic function. These values are outputted on a liquid crystal screen or the like, and are outputted through a printer port or a communication port.

The present invention provides a method for measuring toxic substances using an immobilized luminescent microorganism and a biosensor kit for the same, so that the present invention does not require a pretreatment step of a separate luminescent microorganism by using immobilized luminescent microorganisms, The present invention provides an apparatus and method for analyzing toxic substances. It is also an effective apparatus and method for indicating the biological toxicity of various trace toxic substances which are difficult to analyze even in physicochemical analysis.

Claims (9)

A photobacterium phosphoreum, a vibrio fischeri, or a recombinant strain in which a gene encoding a luminescent enzyme thereof (lux gene) is introduced into a host microorganism, and the immobilized light emitting microorganism is selected as a toxic substance And measuring and analyzing a change in light amount caused by the measurement of the amount of the toxic substance. 2. The method of claim 1, further comprising alarming when the further measured toxicant concentration is above a predetermined value. The method of claim 1, wherein the immobilized light emitting microorganism is immobilized on a porous substrate selected from the group consisting of alginate, strontium alginate, kappa -carrageenan, polyacrylamide, cellulose, and agarose. By weight. The method for measuring the concentration of a toxic substance according to claim 1, wherein the change of the quantity of light is converted into a quantity of charge by using an optical measuring device, and the quantity of the quantity of the toxic substance is analyzed by a calculation means. The method for measuring the concentration of a toxic substance according to claim 4, wherein the change of the amount of charge is analyzed by a magic magic, a specific velocity method, and a bioluminescence method. Means for supporting the immobilized microorganism supported by the net and supporting the immobilized microorganism which is in direct contact with the water system and supported by the net, and for providing a darkroom condition; a band filter and water quality For measuring toxic substances using immobilized light emitting microorganisms, comprising a measuring unit consisting of an optical measuring instrument for measuring a change in light quantity according to toxicity, computing means for converting the measurement value in the measuring unit into a water toxic concentration and input / output means connected to the computing means Biosensor kits. The biosensor kit for measuring toxic substances using immobilized light emitting microorganisms according to claim 6, wherein the means for supporting the immobilizing microorganisms and providing a dark room condition is a removable cap structure. The biosensor kit according to claim 6, wherein the optical measuring instrument is PMT (Photomultiplier tubes) or a photodiode. The biosensor kit according to claim 6, wherein the output device is at least one device selected from a monitor, a printer port, a communication port, and an alarm or an alarm device.