KR20110125510A - An instrument for detecting microbes in water with pre-treatment system and method thereof - Google Patents

Abstract

PURPOSE: A pretreatment and test apparatus for detecting bacteria in drinking water is provided to quickly isolate and concentrate bacteria from a liquid phase. CONSTITUTION: A pretreatment and test apparatus for detecting bacteria comprises: a pretreatment part(100) which generated photoreaction of bacteria by inputting measuring samples and reaction samples and concentrating bacteria contained in the measuring sample; and a measuring unit(200) which measures luminescence by the photoreaction of the bacteria and detecting the amount of the bacteria in the sample .

Description

An instrument for detecting microbes in water with pre-treatment system and method

The present invention relates to a bacterial metering device that allows the rapid separation of bacteria from a liquid sample and quantitative analysis of the bacteria. More specifically, the ATP in the cell is concentrated after filtering and filtering the bacteria in the water quality. The present invention relates to a pretreatment and metering device and method thereof for testing bacteria in drinking water that can be measured through a luminescence reaction and thereby measure the number of bacteria.

In general, the method of reading bacteria in the water based on the medium method is used to identify harmful strains. It takes at least two days to read, and slow growth of bacteria can extend the incubation period for one week or more, and requires a lot of expert skills to analyze the sample. In addition, this metering method can determine whether or not contamination by harmful strains present in the water quality is about 1 to 2 days after sampling, it is difficult to take immediate action and requires expensive samples when using the recently developed antibodies, There is a need for expert analysis.

Accordingly, a method of using ATP has been steadily studied as an alternative. The quantification of bacteria using ATP has been studied steadily since the 1960s, and the basic principle is ATP-Bioluminescence using Luciferin molecule and Luciferase enzyme possessed by Firefly. by measuring the luminescence showing a peak at 560 nm during the reaction. This method has the advantage of being very responsive and reading very little luminescence in real time through the photosensor. However, there is a possibility that ATP may exist outside the bacteria, which may lead to inaccurate measurements.

In addition, there is no bacteria (negative) in drinking water or food, or very small concentration (100 / ml), which requires very high sensitivity because it must be detected and distinguished. In addition, a large amount of samples (25ml-100ml) should be searched for reliability. Probing sensors based on conventional ATP bioluminescence reactions require a concentration technique that reduces the volume of the sample due to the small sensing area, making it difficult to measure large amounts of low concentration samples.

The technical problem to be solved by the present invention is to provide a pretreatment and metering device and method for testing bacteria in drinking water that can rapidly and continuously quantify a small amount of bacteria suspended in the water quality.

In one embodiment of the present invention for achieving the above object, a measurement sample and a reaction sample are introduced into the reaction chamber through a filter to concentrate and collect the bacteria contained in the measurement sample and to generate a photoreaction of the collected bacteria. The pre-treatment and metering apparatus for testing bacteria in drinking water, including a measuring unit for measuring the amount of bacteria in the sample by measuring the luminescence caused by the photoreaction of bacteria generated in the pre-treatment unit.

Another embodiment of the present invention for achieving the above object is a pre-treatment to inject a measurement sample and a reaction sample into the reaction chamber through a filter to concentrate and collect the bacteria contained in the measurement sample and to generate a photoreaction of the collected bacteria Step, pretreatment and metering method for testing the bacteria in drinking water, including a measuring step of detecting the amount of bacteria in the sample by measuring the luminescence by the photoreaction of the bacteria.

According to the bacteriological test apparatus according to the present invention, it is possible to collect bacteria while removing external ATP at the same time by concentrating by a membrane filter, thereby real-time quantitative measurement of bacteria in the water quality, and thus food and drinking water evaluation as well as food. It will be easily applicable to food safety inspection by measuring the pollution degree of.

In addition, the present invention can save professional manpower and metering time, and thus can easily contribute to public health and health as it enables constant monitoring, prophylaxis, and quick response of water quality by simply and precisely measuring the degree of contamination in the water within a short time. There is an advantage to that.

In addition, the present invention will be able to easily and quickly measure the floating bacteria in various groundwater and drinking water to investigate the source of pollutants or to continuously monitor most domestic water resources, and to provide electricity for the development of food and environmental biochip technology. .

1 is a block diagram illustrating the overall configuration of a pre-treatment and metering device for testing bacteria in drinking water according to the present invention.
2 is a conceptual diagram illustrating the operation principle of the bacterial meter reading apparatus according to the present invention.
3 is a reference picture illustrating an embodiment of a bacterial meter reading apparatus according to the present invention.
Figure 4 is a reference picture showing an example implemented by setting the bacterial meter reading apparatus according to the present invention for easy portability.
5 is a flowchart illustrating a bacterial meter reading method according to the present invention.

Hereinafter, with reference to the accompanying drawings the configuration and operation of the pre-treatment and meter reading apparatus for testing bacteria in drinking water according to an embodiment of the present invention will be described in detail.

The terms or words used in this specification and claims are not to be construed as limiting in their usual or dictionary meanings, and the inventors may properly define the concept of terms in order to best explain their invention in the best way possible. It should be interpreted as meaning and concept corresponding to the technical idea of the present invention. Therefore, since the embodiments shown in the specification and the configuration shown in the drawings is only one of the most preferred embodiment of the present invention, it is understood that there may be various equivalents and modifications that can replace them at the time of the present application shall.

Figure 1 is a block diagram schematically showing the overall configuration of the pre-treatment and metering apparatus for testing bacteria in drinking water according to an embodiment of the present invention, Figure 2 is a view for explaining the operating principle of the bacterial metering apparatus according to the present invention 3 is a conceptual view, and FIG. 3 is a cross-sectional view showing an embodiment of a bacterial meter reading apparatus according to the present invention.

As shown in FIG. 1, the bacterial meter reading device according to an embodiment of the present invention includes a pretreatment unit 100 for concentrating and collecting bacteria in a measurement sample and inducing a photoreaction of the collected bacteria, and a luminescence degree according to a photoreaction. It is configured to include a measuring unit 200 for detecting the amount of bacteria in the sample accordingly. The operating principle by the apparatus of the present invention made in the pre-processing unit 100 and the measuring unit 200 is illustrated in the conceptual diagram of FIG. 2, and the apparatus of the present invention injects the measurement sample 12 as illustrated in FIG. 3. Filter 102 which collects the bacteria contained in the sample and passes the external ATP and the medium, and induces a luminescence reaction of the bacteria collected by the filter 102 and the sample in a predetermined volume so as to measure its luminescence. The collecting reaction chamber 103 can be embodied by speaking up and down.

The pretreatment unit 100 collects the bacteria in the measurement sample using the filter 102 and extracts the ATP of the collected bacteria to cause a photoreaction. The bacterial collection unit 110 and the ATP extraction unit 120 And a photoreaction induction part 130.

The bacterial collection unit 110 may be configured using the filter 102, by allowing bacteria contained in the measurement sample to be filtered out of the filter 102 and allowing the external ATP and the medium to pass through the filter 102. Only bacteria can be separated from the media and concentrated and collected.

The ATP extractor 120 extracts bacteria's ATP by applying a cell lysate (ATP releasing agent: see 13a of FIG. 2) to the bacteria that are filtered and concentrated by the filter 102 in the bacteria collector 110. The ATP of the bacteria thus extracted is collected in the reaction chamber 103.

The photoreaction induction unit 130 is a portion that causes a photoreaction of the reaction sample (luciferin-luciferase reagent (see 13b of FIG. 2)) to the ATP of the bacteria in the reaction chamber 103. The reaction samples available for this photoreaction are luciferin molecules, or luciferase enzymes, which cause ATP-bio-luminescence reactions to measure luminescence by bacteria. .

The measuring unit 200 includes a light amount detecting unit 210, an amplifier 220, a key input unit 230, a main control unit 240, an LCD control unit 250, an LCD display unit 260, and a power supply unit 270. It is composed.

As illustrated in FIG. 3, the light quantity detector 210 may include a transparent glass film 211 having high light transmittance at the lower portion of the reaction chamber 103 and a light sensor 212 built in the lower portion of the transparent glass film. have. The transparent glass film 211 also serves to prevent the sample from directly touching the surface of the optical sensor. The power supply line 213 and the signal line 214 are respectively connected to the optical sensor 212 and drawn out to the outside, and the light quantity detecting unit 210 generates light generated inside the reaction chamber 103 through the optical sensor 212. The amount of light for the reaction is sensed and the voltage is varied according to the degree of light emission and output through the signal line 214. In this case, the connection part of the reaction chamber 103 and the optical sensor 212 may be configured to be completely sealed with aluminum to prevent external light from leaking out or leaking.

The amplifier 220 amplifies the voltage output from the light amount detector to increase the light sensitivity.

The key input unit 230 includes a plurality of keys for the user interface.

The main controller 240 adjusts the overall operation of the device according to the key input value, and digitally processes the light sensing signal amplified by the amplifying unit to output a quantitative numerical value according to the degree of light emission and a display control signal thereof.

The LCD control unit 250 and the LCD display unit 260 control the operation of the LCD according to the quantitative numerical value provided from the main controller and the display control signal to display the quantitative numerical value according to the degree of light emission through the LCD.

The power supply unit 260 supplies operating power to each part.

Meanwhile, the filter 102 is fixedly installed in the filter holder 102a as shown in the cross-sectional view of FIG. 3, and the filter holder 102a allows the reaction chamber 103 and the internal space to communicate with each other to measure the measurement sample 12. ) Is concentrated and collected through the filter and the external ATP and the medium passing through the filter is sent to the outside through the reaction chamber (103). To this end, an injection hole 101 through which a sample can be injected is formed in the upper portion of the filter holder 102a, and an outlet 104 through which the tube 105 is connected is formed in the lower side of the reaction chamber 103. In addition, the filter 102 is composed of a membrane filter having a pore diameter (0.45㎛) of a size smaller than the diameter of the bacteria (0.8 ~ 1㎛) to filter and concentrate the bacteria contained in the measurement sample and to configure the sample The main medium and the external ATP allow for passage. This membrane filter continuously removes external ATP and media present in the water to be measured, allowing the device of the present invention to rapidly and continuously concentrate and collect low concentrations of bacteria in the water. Make it measureable. In particular, the connection between the reaction chamber 103 and the tube 105 is completely sealed with a component made of aluminum to prevent external light from leaking in or leaking out. The outer diameter of the reaction chamber 103 is chemically resistant and stable. As a Teflon material, it is preferable to use the tube of 1.6 mm and internal diameter 0.6 mm, for example.

4 is a reference picture illustrating an embodiment of a bacterial meter reading apparatus according to the present invention, and FIG. 5 is a reference picture showing an example of setting the bacterial meter reading apparatus according to the present invention for easy portability. .

As shown in FIG. 4, the bacterial meter reading apparatus according to the present invention increases the height of the discharge port 104 by installing four supports at the bottom of the reaction chamber 103 so that the measurement sample can be easily escaped. The media container of is always below the discharge port 104 of the reaction chamber 103 to prevent the medium (12a) to flow back.

Meanwhile, the apparatus of the present invention can also be configured as a portable set as illustrated in FIG. 5. This set bacteria test device can be made relatively compact as shown in Figure 5 150mm x 130mm x 190mm, it is possible to increase the portability by attaching a handle on the top. In addition, when the cover of the portable device is opened, the LCD display unit 260 is installed to be exposed to the outside, and a key input unit 230 including an injection hole 101 for injecting a measurement sample, a filter holder 102a, and a simple operation button is provided. In addition, the holder for storing the syringe (syringe) in which the washing and active buffer and the reaction sample is injected may be further provided. This holder basically requires four sample injections per measurement in the order of washing and activation buffer-measurement sample-response sample-wash buffer. Samples can be stored.

On the other hand, the bacteria metering method according to an embodiment of the present invention, as shown in Figure 6 through the filter 102, the measurement sample and the reaction sample is put into the reaction chamber 103 to concentrate the bacteria contained in the measurement sample and It comprises a pre-treatment step (S101-S103) for collecting and generating a photoreaction of the collected bacteria, and measuring steps (S104, S105) for detecting the amount of bacteria in the sample by measuring the luminescence caused by the photoreaction of the bacteria do. Although the bacterial test method according to the present invention is not shown in the drawings, a pre-washing step of pre-cleaning the inside of the reaction chamber by injecting a washing / activation buffer for washing and activation into the inside of the reaction chamber before the pretreatment step, After the measuring step, the washing buffer may be further injected into the reaction chamber to wash the inside of the reaction chamber, and then the washing step may be further included.

In the pretreatment step, a measurement sample is introduced into the reaction chamber through a filter to collect and collect bacteria contained in the measurement sample (S101), and a cell lysate is introduced into the reaction chamber to introduce bacteria ATP. ATP extraction step (S102) to extract, and a reaction reaction step (S103) for introducing a reaction sample into the reaction chamber to cause a photoreaction by ATP. In this pretreatment step, the measurement sample and the reaction sample are sequentially introduced into the reaction chamber 103 to concentrate and collect the bacteria in the measurement sample, and then extract ATP of the collected bacteria from the measurement sample and cause a photoreaction.

In the bacterial collection step (S101), in particular, only the bacteria are separated and concentrated and collected from the external ATP and the medium present in the measurement sample through the filtering of the measurement sample. Made by a filter.

When the cell lysate 13a and the reaction sample 13b are introduced through the inlet 101 of the filter holder 102a in the ATP extraction step S102 and the photoreaction induction step S103, the reaction chamber 103 is disposed inside the reaction chamber 103. By the mixture 13 of the cell lysate 13a and the reaction sample 13b, ATP inside the cells of the bacteria concentrated and collected in the bacterial collection step S101 is extracted, and the photoreaction by the ATP is caused at the same time. do. As the reaction sample used in the photoreaction inducing step, a luciferin molecule or luciferase, which causes a bioluminescence reaction with ATP, may be used.

The measuring step may include a step (S104) of quantitatively detecting the amount of bacteria in the sample by measuring the luminescence caused by the photoreaction of the bacteria and displaying a detection value according to the detected bacterial concentration (S105). have.

The bacterial meter reading apparatus and the metering operation of the apparatus according to the present invention having such a configuration will be described in detail.

First, the bacterial concentration standard of drinking water should be able to detect very low concentration of bacteria in order to measure in real time to 100 individuals / ml or less based on the culture method. Therefore, in order to test bacteria from drinking water, a process of concentration and collection in real time is essential.

In the inspection apparatus of the present invention, when the measurement sample is to be injected into the inlet 101 of the filter holder 102a installed in the pretreatment unit 100 when measuring bacteria such as drinking water, the membrane filter 102 is disposed therein. In the size of 0.45um), while continuously passing through the medium to continuously collect bacteria (12b: size 0.8 ~ 1um) it can generate the effect of rapid concentration. In addition, since the medium 12a is continuously washed in this process, the external ATP contained in the measurement sample can be removed at the same time. Afterwards, the cell lysate (13a) or the ATP releasing agent is used to extract and measure the ATP inside the cell, so that the particles can be distinguished even if they are trapped other than bacteria. That is, the membrane filter 102 having a pore size smaller than the diameter of the bacterium as shown in FIG. 2 is filtered through the medium 12a but not the bacterial particles 12b. Therefore, when the measurement sample including the bacteria is injected into the injection hole 101 as in step ① of FIG. 2, only bacteria 12b having a larger particle size than the pores of the filter are filtered as shown in step ② of FIG. 2. . In this process, since the medium 12a and the external ATP are continuously removed except for the bacteria suspended in the measurement sample, the concentration is simultaneously collected. Next, extract ATP with a cell lysing solution (13a: ATP Releasing Agent) as in step 3 of FIG. . Therefore, finally, as illustrated in step ④ of FIG. 2, the luminous reaction is generated by the extracted ATP and the injected chemicals, and the amount of light generated is an optical sensor in the light amount detector 210 of the measurement unit 200. It can be measured by.

The output voltage of the optical sensor is changed according to the luminescence, and this light quantity detection output is amplified by a predetermined level through the amplifier 220 to increase the sensitivity, and then is input to the main controller 230 (MCU) of the main board. . In particular, the device of the present invention is optimized to measure the amount of light of the 560nm wavelength generated during the ATP emission reaction using a photodiode with the maximum output value in the wavelength range of 500nm to 600nm, the signal input from the optical sensor to the MCU is digital It is converted into a signal and calculated. These signal values are output in decimal to the LCD display unit 260 via the LCD control unit 250, so that the user can show the output value and the quantitative value (number of bacteria) that the output value means. The above-described apparatus of the present invention can measure in real time after the reaction occurs from the measurement sample, so that it can be quantified faster than the conventional culture-based measurement.

The power supply unit 270 supplies power required for each module, and the key input unit 230 enables device control by a user as a user interface portion.

As described above, the present invention has been described by way of limited embodiments and drawings, but the present invention is not limited to the above-described embodiments, which can be variously modified and modified by those skilled in the art to which the present invention pertains. Modifications are possible. Accordingly, the spirit of the present invention should be understood only by the claims set forth below, and all equivalent or equivalent modifications thereof will belong to the scope of the present invention.

12: measurement sample 12a: medium
12b: Bacteria 13: Mixture
13a: Cell Lysis Solution 13b: Reaction Sample
100: pretreatment unit 101: injection hole
102 filter 102a filter holder
103: reaction chamber 104: discharge port
105: tube 110: bacteria detection unit
120: ATP extraction unit 130: photo reaction induction unit
200: measuring unit 210: light amount detecting unit
211: transparent glass film 212: light sensor
213: power supply line 214: signal line
220: amplification unit 230: key input unit
240: main controller 250: LCD control unit
260: LCD display 270: power supply

Claims (17)

A pretreatment unit which inputs a measurement sample and a reaction sample to a reaction chamber through a filter to concentrate and collect bacteria included in the measurement sample and to generate a photoreaction of the collected bacteria;
And a measuring unit for measuring the amount of bacteria in the sample by measuring the luminescence caused by the photoreaction of the bacteria generated in the pretreatment unit. The method of claim 1, wherein the preprocessing unit,
A bacterial collecting part for separating and concentrating and collecting bacteria from external ATP and a medium included in the measurement sample introduced into the reaction chamber;
An ATP extracting unit extracting ATP in the cell by adding a cell lysing solution to the bacteria concentrated and collected in the reaction chamber by the bacterial collecting unit;
Pre-treatment and metering apparatus for testing bacteria in drinking water, comprising: a photo reaction inducing unit for causing a photo reaction by ATP by inputting the reaction sample to the extracted ATP. The method of claim 2, wherein the photoreaction induction unit,
Pretreatment and metering for bacterial testing in drinking water characterized by causing ATP-bioluminescence reaction which can measure the luminescence by bacteria using luciferin molecule or luciferase enzyme Device. The method of claim 1, wherein the preprocessing unit,
A filter for collecting bacteria and passing external ATP included in the measurement sample is installed at the front end of the reaction chamber, and an inlet and an outlet for injecting and discharging the sample are formed to communicate with the inner space of the filter and the chamber. Pre-treatment and metering device for testing bacteria in drinking water, characterized in that to separate the bacteria in the measurement sample from the external ATP and the medium. The method of claim 4, wherein the filter,
A pretreatment and metering device for testing bacteria in drinking water, characterized in that the membrane filter having a pore diameter smaller than the diameter of the bacteria. The method of claim 1, wherein the preprocessing unit,
For testing the bacteria in drinking water, characterized in that for the measurement sample and the reaction sample sequentially introduced into the reaction chamber through the filter, the concentration and collection of bacteria in the measurement sample, the photoreaction of the collected bacteria by the reaction sample is generated. Pretreatment and Metering Device. The method of claim 1, wherein the measuring unit,
A light amount detecting unit having a light sensor to detect a light amount and output a variable voltage according to the degree of light emission;
An amplifier for amplifying a voltage output from the light amount detector to increase light sensitivity;
A key input unit having a plurality of keys for a user interface;
A main control unit for controlling the overall operation of the apparatus according to a key input value, and digitally processing the light sensing signal amplified by the amplifying unit to output a quantitative numerical value according to the degree of light emission and a display control signal thereof;
An LCD control unit and an LCD display unit for controlling the operation of the LCD according to the quantitative numerical value provided by the main controller and the display control signal to display the quantitative numerical value according to the degree of light emission through the LCD; And,
A pretreatment and metering device for testing bacteria in drinking water, characterized in that it comprises a power supply for supplying the operating power to each of the parts. A pretreatment step of introducing a measurement sample and a reaction sample into the reaction chamber through a filter to concentrate and collect bacteria included in the measurement sample and to generate a photoreaction of the collected bacteria;
Measuring step of detecting the amount of bacteria in the sample by measuring the luminescence by the photoreaction of the bacteria generated in the pre-treatment step; Pre-treatment and metering method for testing bacteria in drinking water, characterized in that it comprises a. The method of claim 8, wherein the pretreatment step,
A bacterial collection step of concentrating and collecting bacteria contained in the measurement sample by inserting the measurement sample into the reaction chamber through the filter;
An ATP extraction step of extracting ATP in the cells of the concentrated and collected bacteria in the bacterial collection step by injecting the cell lysate into the reaction chamber;
Pre-treatment and metering method for testing the bacteria in drinking water, comprising; a photo reaction inducing step of causing a photo reaction by ATP by introducing a reaction sample into the reaction chamber. The method of claim 9, wherein the bacterial collection step,
A pretreatment and metering method for testing bacteria in drinking water, characterized in that the bacteria are separated, concentrated and collected only from the external ATP and the medium present in the measurement sample by filtering the measurement sample. The method of claim 9, wherein the bacterial collection step,
A pretreatment and metering method for testing bacteria in drinking water, characterized in that the membrane filter having a pore diameter of less than the diameter of the bacteria. The method of claim 8, wherein the pretreatment step,
Through the filter, the measurement sample and the reaction sample are sequentially introduced into the reaction chamber to concentrate and collect the bacteria in the measurement sample, and then generate a photoreaction of the collecting bacteria for the reaction sample. Pretreatment and Metering Methods. The method of claim 9, wherein the photo-induction step,
A method of pretreatment and metering for testing bacteria in drinking water, characterized by using a luciferin molecule or luciferase as a reaction sample which causes a bioluminescence reaction with ATP extracted from collected bacteria. The method of claim 8, wherein the measuring step,
Displaying a detection value according to the detected concentration of bacteria; Pre-treatment and metering method for testing bacteria in drinking water comprising a. The method of claim 8,
The pre-cleaning step of washing the interior of the reaction chamber in advance by injecting a washing / activation buffer for cleaning and activation (activation) in the reaction chamber before the pre-treatment step further comprises a bacterial test in drinking water Pretreatment and metering methods. The method according to any one of claims 8 to 15,
After the measuring step again injecting a washing buffer into the reaction chamber to wash the inside of the reaction chamber and washing step; and further comprising a pre-treatment and metering method for testing bacteria in drinking water. A filter for filtering and collecting bacterial particles;
Injecting the measurement sample and the reaction sample through the filter,
A reaction chamber for obtaining light energy generated by the reaction between the collected bacterial particles and the injected cell lysate;
A photodiode measuring the amount of light generated from the reaction chamber and outputting a voltage corresponding to the magnitude of the amount of light;
A main controller for receiving the voltage output from the photodiode and outputting data quantitatively calculating the number of bacteria through a predetermined calculation process; And
And a LCD display unit for receiving the data output from the main controller and outputting the image signal as an image signal.

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