KR102288766B1 - Filter coted Luminescent substance and Microbial measurement apparatus having the same - Google Patents

Abstract

The present invention relates to a method for manufacturing a collection plate and an apparatus for measuring suspended microorganisms. A method for manufacturing a collection plate according to one aspect includes: preparing a filter for collecting or filtering microorganisms; preparing a solvent for the coating part for coating the filter; mixing a surfactant with the solvent; preparing a coating by diluting the bioluminescent material in the solvent; and coating the prepared coating part on the filter.

Description

Collection plate manufacturing method and airborne microorganism measurement apparatus {Filter coated Luminescent substance and Microbial measurement apparatus having the same}

The present invention relates to a method for manufacturing a collection plate and an apparatus for measuring suspended microorganisms.

Recently, as avian influenza and new influenza have become an issue, the problem of air infection is emerging. Accordingly, airborne microbial measurement is more important, and the biosensor market is growing significantly accordingly.

In the existing method for measuring airborne microorganisms, the biological particles suspended in the sample gas are collected on a solid or liquid surface suitable for proliferation, cultured in an appropriate temperature and humidity environment for a certain period of time, and then the number of collected microorganisms from the number of colonies that appeared on the surface. There are a culture method for obtaining

In recent years, ATP bioluminescence method using the principle that ATP (adenosine triphosphate) and luciferin/luciferase react to emit light, a series of steps that take up to the removal of ATP, extraction of ATP, and measurement of luminescence The process was shortened to about 30 minutes, enabling rapid work.

However, in order to apply the ATP bioluminescence method, an ATP extractant is basically required, but when it is used in a gaseous airborne microorganism measurement system, it may have adverse effects such as toxicity to the human body. It should be continuously supplied, but there is a problem in that the cost burden is large to continuously supply the ATP extractant that is currently commercially available.

In addition, since a series of manual operations are required, such as continuously injecting a luminescent material for luminescence of airborne microorganisms into the captured microorganisms, there is a limitation in that an automatic measurement system for airborne microorganisms in the gas phase cannot be developed using these methods.

US Patent Publication 2007/207514 (published on Sep. 06, 2007)

The present invention has been proposed to improve the above problems, and it is an object of the present invention to provide a method for manufacturing a collection plate and an apparatus for measuring suspended microorganisms for quickly measuring suspended microorganisms without having to go through a series of manual operations for light emission of suspended microorganisms do it with

According to one aspect of the present invention to achieve the above or other object, the step of preparing a filter for collecting or filtering microorganisms; preparing a solvent for the coating part for coating the filter; mixing a surfactant with the solvent; preparing a coating by diluting the bioluminescent material in the solvent; and coating the prepared coating part on the filter.

According to the proposed invention, there is an advantage in that a luminescent material can be applied to suspended microorganisms without a separate manual operation, thereby simplifying the process of measuring suspended microorganisms.

In addition, by adding a surfactant to the coating portion, there is an advantage that the light emitting material is uniformly distributed on the filter.

In addition, by adding the antifoaming agent to the coating portion, there is an advantage in that the formation of a film on the filter by the surfactant is suppressed.

In addition, by adding an enzyme stabilizer to the coating portion, the luminescence duration of the suspended microorganisms is maintained for a long time, and the luminescence value of the suspended microorganisms is increased.

1 is a conceptual diagram illustrating an apparatus for measuring airborne microorganisms according to an embodiment of the present invention.
2 is a conceptual diagram illustrating various embodiments of a particle classification unit.
3 is a perspective view of a collecting plate according to an embodiment of the present invention;
4 is a cross-sectional view of a collecting plate according to an embodiment of the present invention.
5 is a flowchart of a method for manufacturing a collection plate according to an embodiment of the present invention.
6 is a graph showing the degree of bioluminescence according to the concentration of the enzyme stabilizer according to an embodiment of the present invention.
7 is an enlarged cross-sectional view of the filter when only the bioluminescent material is coated on the filter.
8 is an enlarged cross-sectional view of the filter when the coating according to the present embodiment is formed on the filter according to the embodiment of the present invention.

Hereinafter, the embodiments disclosed in the present specification will be described in detail with reference to the accompanying drawings, but the same or similar components are assigned the same reference numerals regardless of reference numerals, and overlapping descriptions thereof will be omitted. The suffixes "module" and "part" for the components used in the following description are given or mixed in consideration of only the ease of writing the specification, and do not have a meaning or role distinct from each other by themselves. In addition, in describing the embodiments disclosed in the present specification, if it is determined that detailed descriptions of related known technologies may obscure the gist of the embodiments disclosed in the present specification, the detailed description thereof will be omitted. In addition, the accompanying drawings are only for easy understanding of the embodiments disclosed in the present specification, and the technical spirit disclosed herein is not limited by the accompanying drawings, and all changes included in the spirit and scope of the present invention , should be understood to include equivalents or substitutes.

In the present specification, for convenience of explanation, an apparatus for measuring suspended microorganisms using ATP emission will be described as an example. However, the present invention is not limited thereto, and the collection plate according to an embodiment of the present invention may be applied to various measuring devices for emitting light.

1 is a conceptual diagram illustrating an apparatus for measuring airborne microorganisms according to an embodiment of the present invention, and FIG. 2 is a conceptual diagram illustrating various embodiments of a particle classification unit.

1 and 2 , the airborne microorganism measuring apparatus 1 according to an embodiment of the present invention includes a particle classification unit 10 for trapping and emitting microorganisms and collecting the particles in the particle classification unit 10 . The microorganism dissolving unit 20 for dissolving the microorganism and extracting ATP (adenosine triphosphate), DNA, RNA, etc. in the microorganism, and installed on one side of the particle classification unit 10 to emit light from the particle classification unit 10 A light receiving element 30 that detects the light of the microorganism and extracts the concentration or contamination level of the microorganism, a display 40 that displays the data detected by the light receiving element 30, and the particle classification unit 10 It is installed on the other side of the flow generating unit 50 for flowing air.

In FIG. 1 , the particle classification unit 10 is shown in a flat plate shape, but this is to mainly express the action between the particle classification unit 10 and the microorganism dissolving unit 20 and the light receiving element 30 . As a result, only the components corresponding to the collection plate 11 are conceptually illustrated, and the shape and structure of the particle classification unit 10 are not specifically limited, and the particle classification unit 10 is described below. It can be applied to various embodiments.

The particle classification unit 10 is, such as an electrostatic precipitator (electrostatic pricipitator), inertial impactor (inertial impactor), a cyclone (cyclone), a centrifuge (centrifuge), etc., particles in the air to the solid collecting method, or the liquid collecting method. A dust collector or filter system having a collecting plate or a collecting space that can be collected by

In the electrostatic precipitator, when a negative (-) voltage (or (+) voltage) is applied to the discharge electrode by a DC high voltage, corona discharge occurs. At this time, the generated negative (-) ions (or positive (+) ions) are It is a dust collector using the electrostatic principle of being charged and collected by electric force to a dust collecting electrode (collecting plate) to which a (+) voltage (or a (-) voltage) is applied.

FIG. 2(a) shows an example of a wire to plate type that is most widely applied among various structures of an electrostatic precipitator. An electric field is formed between a charging wire and a collecting plate, and charging The charged particles passing between the line and the collecting plate are collected by the collecting plate.

The inertial impact device has a structure in which an impact plate or a receiving tube (hereinafter referred to as a 'collection plate') is installed under an acceleration nozzle (impaction nozzle).

Figure 2 (b) shows an example of such an inertial collision device, and the air passing through the acceleration nozzle or jet changes its flow direction by 90° by the collecting plate, and among the particles contained in the air, Particles with a mass greater than a certain amount collide and collect on the collecting plate without completely changing the flow direction due to inertia.

A cyclone is one of the separation devices using centrifugal force that is widely used to separate solid particles in a fluid or to separate liquid droplets from gas, and has various types and specifications. will show

After air containing particles is introduced tangentially inside the circular cyclone, it rotates along the inner wall of the cylinder to form a swirling flow. It is separated from the flow by pushing it toward the inner wall. The flow (air) from which particles are removed rises from the lower end of the cone to the upper part and is discharged through the outlet. Hereinafter, collectively referred to as a 'collection plate').

A centrifugal separator is a device that applies a continuous centrifugal force generated when continuously rotating at a high speed. A cyclone is also a separation device using centrifugal force, but it uses a rotating vessel that rotates at high speed compared to a cyclone to separate the particles contained in the air to the outer wall side of the rotating vessel. can do it

The electrostatic precipitator has low pressure loss, so it is suitable for large-capacity or high-flow agents, and has high dust collection efficiency even for nano-sized (100 nm or less) fine particles. On the other hand, the inertial collision device, the cyclone, etc. have a simple structure, and thus have the advantage of low cost and maintenance cost.

The solid collection method is a method of collecting a substance to be measured on a solid by adsorption, reaction, etc. by sucking sample air through a particle layer of a solid. It can be applied in the process of collecting on a collecting plate or collecting space.

The liquid collection method is a method of collecting the material to be measured in the liquid by dissolution, reaction, precipitation, suspension, etc. by passing sample air through the liquid or contacting the surface of the liquid. will lose

The liquid may be applied or accommodated on the collecting plate or the collecting space of the particle classification unit 10, and airborne microorganisms may be collected by applying the liquid collecting method.

In addition, using the particle sorting unit 10, the sample air is passed through the filter medium to collect the material to be measured on the filter medium, and the material to be measured is collected after condensing the sample air by contacting it with a cooled tube. After collecting using the principle of molecular diffusion, the collection method is a cooling condensation collection method that collects the sample air directly without dissolving, reacting, or adsorbing the sample air. It is also possible to apply the diffusion capture method for analysis.

Microorganisms suspended in the atmosphere are collected by the particle classification unit 10 while passing through the particle classification unit 10, and the ATP reaction luminescent agent required for ATP bioluminescence is collected on the particle classification unit 10 where the microorganisms are collected. A coated collecting plate 11 is provided.

Therefore, by configuring the collecting plate, the filter medium, and the filter system described above as the collecting plate 11, when microorganisms pass through or are collected by the collecting plate 11, ATP bioluminescence is achieved.

The microorganism dissolving unit 20 uses ions, electromagnetic force of electrons, antibacterial substances, thermal energy, catalyst, etc. to dissolve microorganisms collected in the particle classification unit 10 or flowing toward the particle classification unit 10 side. It is a generic term for device components that extract ATP (adenosine triphosphate), DNA, RNA, etc. in microorganisms. Here, dissolving microorganisms does not melt microorganisms into a liquid state, but decomposes one microorganism into multiple elements. or extracting multiple elements.

When the microorganism dissolving unit 20 is configured as an ion generator, the larger the diameter of the discharge tip provided in the ion generator, the greater the power consumption. Therefore, it is preferable to apply an ozone-free ion generator using a carbon brush having a diameter of 10 μm or less of the discharge tip.

According to the ozone-free ion generator using a carbon brush with a discharge tip diameter of 10㎛ or less, it has a low power consumption of 4W or less, and ozone is generated less than 0.01 ppm　. It can stably satisfy the ozone management standard of 0.06 ppm or less of Article 27 Paragraph 1 of the Industrial Safety and Health Act.

When the microorganism dissolving unit 20 is configured as an ion generator, the cell wall of the microorganism is damaged by the repulsive force between charged ions attached to the microorganism and ATP is extracted. , it damages the cell wall of microorganisms by collision of electrons and extracts ATP.

ATP extracted by the microorganism dissolving unit 20 is exposed to the outside of the cell of the microorganism and at the same time reacts with the ATP-reactive light-emitting agent in the particle sorting unit 10 to generate light, converting light into electricity The light receiving device 30 such as a photodiode (PD) or an avalanche photodiode (APD) detects light generated by ATP bioluminescence to extract the concentration or contamination level of microorganisms.

All living things store the energy generated from the oxidation of organic matter in a compound called ATP, and then hydrolyze it as needed to exercise and maintain body temperature using the energy released at that time. It can also cause bioluminescence.

The light-receiving element measures the photon flux or light power by converting the energy of the photon absorbed by the element into a measurable form. It is widely used as a device for detecting optical signals in optical fiber communication systems operating in the near-infrared region (0.8~1.6 μm).

In particular, in photoelectric detectors among light receiving elements, carriers such as electrons and holes are generated in the material constituting the element by the photons absorbed by the element, and by the flow of the carrier, A device in which a measurable current is generated, that is, based on the photoeffect, is suitable for application to the present invention.

Among the electromagnetic waves, the wavelength that the human eye feels bright as light is in the range of about 380 nm to 780 nm, and as monochromatic light, the wavelength ranges from short wavelengths to 400 to 500 nm for blue, 450 to 500 nm for blue, 500 to 570 nm for green, 570 to 590 nm for yellow, and 590 nm for orange. At 610 nm and red 610 to 700 nm, the light receiving element 30 has a sensitivity capable of receiving a wavelength band of 400 nm or more to 700 nm or less.

In collecting airborne microorganisms in the air in the particle classification unit 10, a flow generating unit 50 such as a blower or a pump is used to forcibly flow the air from one side to the other side based on the particle classification unit 10. A pneumatic difference is generated, and the microorganism dissolving unit 20 and the light receiving element 30 are located on the path through which the atmosphere flows to the particle classification unit 10 , that is, one side of the particle classification unit 10 . is installed, and the flow generating unit 50 is installed on the other side of the particle classifying unit 10 .

As the concentration of microorganisms increases, the amount of ATP extracted increases and the degree of luminosity also increases, and the light receiving element 30 converts the received light into electrical signals such as voltage, current, and frequency and outputs it The microbial concentration calculator (not shown) dataizes or formulates the relationship between the electrical signal output from the light receiving element 30 and the bioluminescence value according to the microbial concentration, or compares the data converted or modified values.

The light detected by the light-receiving element 30 is subjected to a signal processing process that is formalized or data-formed through the microorganism concentration calculator, etc., and then displays the concentration or contamination level of microorganisms in real time through the display unit 40 .

The particle classification unit 10 includes a collecting plate 11 for collecting microbial particles. Hereinafter, the configuration of the collecting plate 11 will be described. ,

3 is a perspective view of a collection plate according to an embodiment of the present invention, and FIG. 4 is a cross-sectional view of the collection plate according to an embodiment of the present invention.

3 and 4 , in an embodiment of the present invention, the collecting plate 11 includes a filter 12 for collecting airborne microorganisms, and a coating unit coated on the filter 12 to emit airborne microorganisms. (14) includes.

The coating part 14 is formed by coating the luminescent solution on the filter 12 .

In this specification, the filter 12 is a concept that collectively refers to a filter paper for filtering and collecting airborne microorganisms, and a collecting substrate. Accordingly, the filter 12 may be configured in various forms, such as a porous filter through which air can flow, or a plate form for collecting airborne microorganisms.

The filter 12 is formed in a substantially plate shape. The filter 12 is a component included in the particle classification unit in the airborne microorganism measuring device to be described later, and the airborne microorganisms contained in the air are collected by the filter 12 . The filter input to the airborne microorganism measuring device is a plate having an approximate diameter of 15 mm, but is not limited thereto and may be formed in various sizes and shapes.

A coating portion 14 for emitting suspended microorganisms is provided on one or both surfaces of the filter 12 . Accordingly, the coating unit 14 emits light from the airborne microorganisms captured by the filter 12 to measure the concentration of the airborne microorganisms.

Hereinafter, a process of coating the coating portion 14 on the filter 12 will be described.

5 is a flowchart of a method for manufacturing a collection plate according to an embodiment of the present invention.

First, the disc usable for the filter 12 is cut to a size usable for the floating microorganism measuring device (S100), and the cut filter 12 is sterilized to remove the remaining fine dust and bacteria ( S110). In addition, by sterilizing the filter 12, it is possible to reduce errors in the measurement of airborne microorganisms.

As the sterilization method, wet heat sterilization using steam or autoclave method using pressurized steam can be used. Moist heat sterilization is a heating method using steam, and the autoclave method is a steam sterilization method using a pressure-resistant vessel or high heat used to react at high temperature and high pressure.

However, the present invention is not limited thereto, and various sterilization methods may be used to remove fine dust and bacteria remaining in the filter.

After the filter 12 is prepared, a solvent for preparing the coating portion 14 is prepared (S200). The coating portion 14 uses a solvent as a solvent. The solubilizing agent includes an extractant, a degradable buffer (Lysis-Buffer), or distilled water. The solvent also undergoes a sterilization process like the filter 12 .

After the sterilization process is completed, a surfactant is added to the dissolving agent (S300). When only the bioluminescent material is added to the solubilizing agent without the addition of the surfactant, the bioluminescent material is not uniformly mixed with the solubilizing agent, so that there is a difficulty in bioluminescence. Therefore, in this embodiment, the bioluminescent material can be uniformly mixed in the solubilizing agent by adding a surfactant to the solubilizing agent. For example, the surfactant may use TritonX-100.

The surfactant is mixed so that the final concentration in the solubilizing agent is approximately 0.1%.

After mixing the surfactant with the solubilizer, the bioluminescent material is diluted (S400). For example, the bioluminescent material may be luciferin. Bioluminescence is a kind of photochemical reaction in which the energy released when an organic compound is oxidized by the action of an enzyme is released out of the body in the form of light energy. create two molecules. Here, luciferin is a reduced form, so it is denoted as LH2. (LH2+ATP → LH2-AMP+2H3PO4)

Since LH2-AMP generated in the above reaction reacts with oxygen and is oxidized and is in an unstable energy state, the oxidation product in this unstable state is soon decomposed to generate oxidized luciferin and AMP while generating light (hv). Here, L is oxidized luciferin, and L-AMP* refers to the luciferin-AMP complex in an unstable energy state (LH2-AMP+1/2 O2 → L-AMP*+H2O) (L-AMP* → L+AMP+hv) (light energy)).

Bioluminescent materials include luciferase as an enzyme that performs a catalytic action.

Since LH2-AMP reacts with oxygen (1/2 O2) and is oxidized by the catalytic action of an enzyme called luciferase, bioluminescence occurs in the presence of luciferin ATP luciferase and oxygen, and one molecule of luciferin It is calculated that one photon is emitted from the oxidation of

Therefore, if the bioluminescent material is composed of luciferin, airborne microorganisms can be quickly measured within 5 minutes by the same process as above, and the maximum luminous intensity is measured within 3 minutes (180 sec), from this It can be seen that the measurement time is less than 3 minutes.

The bioluminescent material is diluted in the solubilizer to a concentration of 400 mg/ml.

After diluting the bioluminescent material in the solubilizer, a foam inhibitor is added (S500). The antifoaming agent may be added in a sterile state at a concentration of approximately 0.02%. Since a surfactant is added to the solubilizer to uniformly mix the bioluminescent material, a film may be formed on the surface of the solubilizer due to the chemical properties of the surfactant. Therefore, when a film is formed on the solubilizer, since the coating portion 14 may be non-uniformly distributed on the filter, it is possible to prevent a film from being formed on the surface of the solubilizer by adding the antifoaming agent.

An enzyme stabilizer may be added to the coating portion 14 (S510).

6 is a graph showing the degree of bioluminescence according to the concentration of the enzyme stabilizer according to an embodiment of the present invention.

In the graph of FIG. 6 , the horizontal axis represents the duration of light emission, and the vertical axis represents the degree of light emission.

Referring to FIG. 6 , when an enzyme stabilizer is added to the coating part 14 , it can be confirmed that the bioluminescence reaction continues for a longer time. Therefore, the emitted suspended microorganisms can be measured for a longer period of time. In addition, it can be confirmed that the bioluminescence value increases due to an appropriate amount of the enzyme stabilizer.

As the enzyme stabilizer, sucrose or fructose may be used, and it is added at an approximately 0.1-0,5 molar concentration (M) in the coating portion 14 .

When the preparation of the filter 12 and the luminescent solution is completed, the prepared luminescent solution is dropped onto the filter 12 prepared to form the coating portion 14 (S600). The coating portion 14 is distributed to the filter 12 in an amount of 0.01 to 0.1 ml per ^{1 cm 2 area.} After the luminescent solution is dripped, the collecting plate 11 coated with the luminescent solution is dried in an environment where the humidity is 10% or less, and the luminescent solution is deposited on the filter 12 (S700). Therefore, by coating the filter 12 with the luminescent solution, the storage period of the luminescent material at room temperature can be maintained for a long time.

7 is an enlarged cross-sectional view of the filter when only the bioluminescent material is coated on the filter, and FIG. 8 is an enlarged view of the filter when the coating according to the present embodiment is formed on the filter according to an embodiment of the present invention. This is a cross-sectional view.

Referring to FIG. 7 , when only the bioluminescent material (eg, luciferin) is coated on the filter, the pressure loss increases because a film is formed on the filter by the bioluminescent material. Therefore, the luminous efficiency of the luminescent material with respect to airborne microorganisms is reduced.

Referring to FIG. 8 , when the coating part 14 according to this embodiment is coated on the filter, it can be confirmed that porosity is secured by the surfactant and the foam inhibitor. Accordingly, the pressure loss is also reduced and the luminous efficiency of the luminescent material for airborne microorganisms is increased.

According to the proposed invention, there is an advantage in that a luminescent material can be applied to suspended microorganisms without a separate manual operation, thereby simplifying the process of measuring suspended microorganisms.

In addition, by adding a surfactant to the coating portion, there is an advantage that the light emitting material is uniformly distributed on the filter.

In addition, by adding the antifoaming agent to the coating portion, there is an advantage in that the formation of a film on the filter by the surfactant is suppressed.

In addition, by adding the enzyme stabilizer to the coating portion, there is an advantage in that the luminescence duration of the suspended microorganisms is maintained for a long time, and the luminescence value of the suspended microorganisms is increased.

Claims (17)

preparing a filter for collecting or filtering microorganisms;
preparing a solvent of a luminescent solution for forming a coating portion on at least one surface of the filter;
mixing a surfactant with the solvent;
diluting the bioluminescent material in the solvent to prepare a luminescent solution;
adding an antifoaming agent to the luminescent solution diluted with the bioluminescent material;
mixing an enzyme stabilizer with the luminescent solution to which the antifoam is added;
forming the coating part by coating the prepared luminescent solution on the filter; and
After coating the luminescent solution on the filter, comprising the step of drying the coating in an environment of 10% or less humidity,
The method of manufacturing a collecting plate in which the coating unit is coated in an amount of 0.01ml to 0.1ml per 1cm2 area of the filter.
delete The method of claim 1,
Foam inhibitor is sterilized, the method for producing a collection plate, characterized in that it consists of a concentration of 0.02%.
delete The method of claim 1,
The enzyme stabilizer, a method for producing a collection plate, characterized in that it contains sucrose or fructose.
The method of claim 1,
Enzyme stabilizer method for manufacturing a collection plate, characterized in that it consists of a concentration of 0.1 ~ 0.5M
delete The method of claim 1,
The method of manufacturing a collection plate further comprising the step of sterilizing the filter before coating the luminescent solution on the filter.
The method of claim 1,
The method for producing a collection plate, characterized in that the solvent is distilled water.
The method of claim 1,
The method for producing a collection plate, characterized in that the solvent is a dissolving agent.
delete 9. The method of claim 8,
The sterilization is a method of manufacturing a collection plate, characterized in that the moist heat sterilization or autoclave (Autoclave).
The method of claim 1,
The method of manufacturing a collection plate, characterized in that the concentration of the surfactant in the luminescent solution is 0.1%.
Particle classification unit comprising a filter through which airborne microorganisms are collected or filtered;
a microorganism dissolving unit for dissolving suspended microorganisms collected or filtered in the particle classification unit to extract adenosine triphosphate (ATP);
a light receiving element installed on one side of the particle classification unit and configured to detect the light of the suspended microorganisms emitted from the particle classification unit and extract the concentration or contamination level of the suspended microorganisms;
a display unit for displaying the data detected by the light receiving element; and
and a flow generating unit installed on the other side of the particle classification unit to flow air,
In the filter,
A coating portion formed by dripping a luminescent solution on at least one surface and drying in an environment of 10% or less humidity is included,
The coating portion is coated in 0.01ml to 0.1ml per 1cm2 area on the filter,
The luminescent solution is
Airborne microorganism measuring device comprising a solvent, a bioluminescent material, a foam inhibitor and an enzyme stabilizer.
15. The method of claim 14,
Suspended microorganism measuring device, characterized in that the surfactant is composed of Triton X-100 at a concentration of 0.1%.
15. The method of claim 14,
The bioluminescent material includes a floating microorganism measuring device including luciferin and luciferase. 15. The method of claim 14,
In the enzyme stabilizer, sucrose or fructose is a floating microorganism measuring device, characterized in that it is composed of a concentration of 0.1 ~ 0.5M.

Images