KR101815751B1 - Apparatus and method for microorganism measurement - Google Patents

Apparatus and method for microorganism measurement Download PDF

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KR101815751B1
KR101815751B1 KR1020160019531A KR20160019531A KR101815751B1 KR 101815751 B1 KR101815751 B1 KR 101815751B1 KR 1020160019531 A KR1020160019531 A KR 1020160019531A KR 20160019531 A KR20160019531 A KR 20160019531A KR 101815751 B1 KR101815751 B1 KR 101815751B1
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collecting
light
unit
microorganisms
measuring
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KR20170097887A (en
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황정호
박지운
김형래
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연세대학교 산학협력단
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C3/00—Separating dispersed particles from gases or vapour, e.g. air, by electrostatic effect
    • B03C3/34—Constructional details or accessories or operation thereof
    • B03C3/40—Electrode constructions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/24—Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/66—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving luciferase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2304/00—Chemical means of detecting microorganisms
    • C12Q2304/60—Chemiluminescent detection using ATP-luciferin-luciferase system

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
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  • Wood Science & Technology (AREA)
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  • General Health & Medical Sciences (AREA)
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  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Toxicology (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

The apparatus for measuring microorganisms according to the present invention comprises a collecting and dissolving unit for collecting and dissolving microorganisms in the air by a collecting solution containing a solubilizing agent and a light emitting agent and a collecting and dissolving unit for measuring the concentration of microorganisms by the light generated from the collected microorganisms And a light measuring unit. According to this configuration, the microbial concentration measurement can be performed at the same time without being divided at each step, so that the real-time microbial concentration measurement can be performed and the accuracy can be improved.

Description

[0001] APPARATUS AND METHOD FOR MICROORGANISM MEASUREMENT [0002]

The present invention relates to an apparatus and a method for measuring microorganisms, and more particularly, to an apparatus and a method for measuring microorganisms that can accurately measure the concentration of microorganisms in real time.

Recently, a variety of pathogens such as avian influenza, swine influenza, and corn virus have been spreading worldwide and causing many casualties. These pathogens are present in extremely small amounts in the air, and studies on high sensitivity measurement techniques for microorganisms floating in the air such as pathogens have been continuously carried out.

Recently, a technique for measuring airborne floating microorganisms using ATP (adenosine triphosphate) bioluminescence has been widely used. This ATP microorganism concentration measurement technique includes a series of steps such as a collection step of floating microorganisms, a liquefaction step of captured microorganisms, a step of reacting a liquefied sample of a liquefied microorganism with a bioluminescent reagent, and a step of measuring the generated light I have.

However, a series of steps for measuring airborne microorganisms in the air are independently performed separately and manually performed by an operator. As a result, the waiting time for the measurement value of the desired suspended microorganism takes a long time, and the measurement accuracy depends on the skill of the operator. Therefore, in recent years, studies on the method of measuring floating microorganisms capable of rapid measurement while increasing the measurement accuracy have been continuously carried out.

-. Korean Registered Patent No. 10-1163641 (Registered on July 2, 2012)

SUMMARY OF THE INVENTION It is an object of the present invention to provide an apparatus and method for measuring microorganisms capable of continuously measuring microorganisms in real time as well as continuous automatic measurement.

In order to achieve the above object, a microorganism measuring apparatus according to the present invention comprises a collecting and dissolving unit for collecting and dissolving microorganisms in the air by a collecting solution containing a solubilizing agent and a light emitting agent, And a light measuring unit for measuring the concentration of the microorganism.

According to one aspect of the present invention, the collecting and dissolving unit includes a collecting unit for collecting the charged microorganisms into the collecting liquid, a charge collecting unit collecting the charged microorganisms by the corona discharge to charge the microorganisms in the air, And a supply part for supplying a collecting solution containing a light emitting agent.

According to one aspect, the light measurement unit includes a light measurement unit that measures light emitted from the microorganisms captured by the collection unit, and an operation unit that calculates light measured by the light measurement unit.

According to one aspect, the supply section includes a waste section for discarding the collection liquid discharged from the collection section.

According to one aspect of the present invention, the charging unit and the collecting unit are provided so as to face each other on a body portion provided on the path of the air so that the air is introduced and discharged, and the charging unit includes at least one electrode body .

According to one aspect, the at least one electrode body has a shape of at least one of an electrode line, an electrode, an electrode wire, an electrode pin, a nanofiber electrode, and a sawtooth electrode.

According to one aspect, the light measuring unit includes an optical sensor for measuring light intensity and a reflector for collecting the light.

According to one aspect, the solubilizer includes a lysis buffer for dissolving cells to extract adenosine triphosphate (ATP), wherein the luminescent agent is luciferin, luciferase, and magnesium (Mg 2 + ).

A method for measuring a microorganism according to a preferred embodiment of the present invention comprises the steps of charging a microorganism in the air, collecting the charged microorganism with a trapping solution containing a solubilizer and a light emitting agent, dissolving and emitting the microorganism, Measuring light, and calculating the concentration of the microorganism with the measured light.

According to one aspect, in the charging step, a charge unit including at least one electrode body to which a positive electrode (+) or a negative electrode (-) is applied generates a corona discharge to charge the microorganisms in the air.

According to one aspect, in the collecting, dissolving, and luminescent steps, the microorganisms are trapped, dissolved, and emitted simultaneously.

According to one aspect, the light measuring step measures the light of the microorganism by an optical sensor for measuring the light intensity and a reflector for collecting the light.

According to one aspect, the calculating step includes visually or audibly notifying the concentration of the calculated microorganisms.

According to one aspect, the solubilizer includes a lysis buffer for dissolving cells to extract adenosine triphosphate (ATP), wherein the luminescent agent is luciferin, luciferase, and magnesium (Mg 2 + ).

According to the present invention having the above-described configuration, firstly, by collecting and simultaneously dissolving microorganisms in the air to emit light, the concentration of microorganisms can be measured in a short time.

Secondly, it is possible to measure the concentration of microorganisms in real time through the simultaneous capture, dissolution and luminescence of microorganisms in the air, thereby contributing to improvement of measurement accuracy due to improvement of real-time concentration measurement response speed.

Third, if the inflow of air is continuously performed, continuous and automatic microbial concentration measurement becomes possible.

Fourth, since the airborne microbial concentration measurement is not classified at each step, it is possible to improve the efficiency compared to the conventional manual measurement method.

1 is a block diagram schematically showing a microorganism measuring apparatus according to a preferred embodiment of the present invention,
FIG. 2 is a view schematically showing an example of a microorganism measuring apparatus according to the embodiment shown in FIG. 1;
FIG. 3 is a graph showing that the luminous intensity measured by the microorganism measuring apparatus shown in FIG. 1 changes with concentration when staphylococci are generated in the air and the concentration is adjusted with time,
FIG. 4 is a flowchart schematically showing a method for measuring microorganisms according to an embodiment using the apparatus for measuring microorganisms shown in FIG. 1. FIG.

Hereinafter, a preferred embodiment of the present invention will be described with reference to the accompanying drawings.

1, the apparatus 1 for measuring microorganisms according to a preferred embodiment of the present invention includes a collecting and dissolving unit 10 and a light measuring unit 50. [

The collecting and dissolving unit 10 collects and simultaneously dissolves the microorganisms (B) in the air (A) with the collecting liquid W containing the dissolving agent and the light emitting agent. To this end, the collecting and dissolving unit 10 includes a charging unit 20, a collecting unit 30, and a supplying unit 40. 2, the charge unit 20 and the collecting unit 30 are provided with an inlet 12 and an outlet 13 through which the air A is introduced and discharged, and the collecting and dissolving unit 10 And are arranged to face each other on the body portion 11 constituting the body.

The charge unit 20 generates a corona discharge to charge the floating microorganisms B in the air A. [ To this end, the charging unit 20 includes a plurality of electrode bodies 21 provided on one side of the body portion 11, as shown in Fig. (+) Or a negative (-) is applied to the charging unit 20 to generate a corona discharge, so that microorganisms B, which are particles of air A introduced into the inlet 12 of the body part 11, Charge.

For reference, in the present embodiment, the electrode bodies 21 of the charge unit 20 are a plurality of linear electrode lines patterned so as to be spaced apart from each other at equal intervals, but the present invention is not limited thereto. For example, the electrode unit 21 of the charging unit 20 may be a plurality of electrode lines provided with a curvature, not a straight line, or a plurality of concentric circles or concentric polygonal shapes. The electrode member 21 may be formed of any one of electrode members such as electrode bar, electrode wire, electrode pin, nanofiber electrode, sawtooth electrode, etc. instead of the electrode line patterned on one side of the body portion 11 . The shape and size of the charging unit 20 including the electrode unit 21 can be variously changed depending on the size of the microorganism measuring device 1 or the measurement environment.

The collecting unit 30 collects the charged microorganisms into the collecting liquid W. 2, the collecting part 30 is provided on the other side so as to face the charging part 20 patterned on one side of the body part 11. [ More specifically, the collecting portion 30 is disposed at the lower portion of the body portion 11 so as to face the charging portion 20 with respect to the gravity direction. The collecting section 30 includes a water tank 31 in which the collecting liquid W is stored and the water tank 31 is provided with an inlet port 32 and an outlet port 33 for introducing and discharging the collecting liquid W do.

On the other hand, the collecting liquid W supplied to and discharged from the water tank 31 of the collecting unit 30 includes a solubilizing agent and a light emitting agent based on water. The solubilizer includes a dissolution enzyme for dissolving cells. In this embodiment, the solubilizer includes a lysis buffer for extracting adenosine triphosphate (ATP) from the charged microorganism (B).

In addition, the luminescent agent includes a luminescent enzyme that reacts with adenosine triphosphate (ATP) extracted from cells dissolved by a solubilizer to generate light. In the present embodiment, will be described as the light-emitting agent comprises at least one of the luciferin (luciferin), luciferase (luciferase), and magnesium (Mg 2 +).

For reference, luciferin in the luminescent agent is activated by adenosine triphosphate (ATP) present in the cells of the dissolved microorganism (B) to be converted into active luciferin, and the active luciferin is oxidized by the action of luciferase, And converts the chemical energy into light energy to generate light.

As described above, since the charged microorganisms B are collected by the collecting liquid W of the collecting unit 30, the solubilizer contained in the collecting liquid W and the luminescent agent contained in the collecting liquid W collect And light emission can be simultaneously performed.

The collecting part 30 is disposed at the bottom position of the body part 11 so as to face the charging part 20 so that a grounding electrode (not shown) corresponding to the charging part 20 is provided on the collecting part 30 side, . When an anode (+) or a cathode (-) is applied to the charge unit 20, an electric field is formed between the charge unit 20 and the ground electrode (not shown). The microbial (B) collection operation by the charging of the charging unit 20 will be described later in more detail.

The supplying unit 40 supplies the collecting liquid W containing the dissolving agent and the light emitting agent to the collecting unit 30. [ The supply unit 40 includes a supply means such as a pump to supply the solution W containing the solubilizing agent and the light emitting agent to the inlet 32 of the water tank 31.

The supply unit 40 includes a waste unit 41 for discarding the collection liquid W discharged from the collection unit 30, that is, discharged through the discharge port 33 of the water tank 31. For reference, although not shown in detail, the waste liquid W discarded by the waste section 41 may be supplied through the supply section 40 again through a process such as filtering for reuse.

The light measuring unit 50 measures the concentration of the microorganism (B) by measuring light emitted from the microorganisms (B) collected and dissolved. For this purpose, the light measuring unit 50 includes a light measuring unit 60 and a calculating unit 70.

The light measuring unit 60 measures light emitted from the microorganisms B captured by the collecting unit 30. [ That is, the light measuring unit 60 measures the light of the microorganism B that is captured by the trapping unit 30 and dissolves and emits light. The light measuring unit 60 is provided at one side of the water tank 31 of the collecting unit 30 and measures the light of the microorganism B collected in the water tank 31. [

The light measurement efficiency by the light measuring unit 60 is shown in Fig. 3 is a graph showing that the luminous intensity measured by the microorganism measuring device 1 according to the present invention changes with concentration when staphylococcus is generated in the air and the concentration is adjusted with time. 3 (A) shows a case where the flow rate of the collecting solution W is 200 .mu.m, and FIG. 3 (B) shows a case where the flow rate of the collecting solution W is 600 .mu.m.

3 (A), there is a delay in the measurement signal due to the longer time taken to collect the microorganisms floating in the air (A) and move to the optical measuring unit 60. However, It can be seen that the light signal is strongly exhibited when the amount of microorganisms (B) In addition, in the case of FIG. 3B, it can be seen that as the flow rate of the collecting liquid W increases to 600 pm, the delay of the signal decreases but the light signal becomes relatively low.

That is, referring to the graph of FIG. 3, when the flow rate of the collecting liquid W is high, it is confirmed that the measurement sensitivity is lowered but the measurement reaction rate is faster. On the other hand, when the flow rate of the collection liquid W is low, it is confirmed that the measurement sensitivity is increased and the measurement reaction rate is slow. As described above, it can be confirmed that the concentration of the microorganisms (B) floating in the air (A) can be measured also by optical measurement of the microorganisms (B) captured in the collecting solution (W).

Although not shown in detail, the optical measuring unit 60 may include an optical sensor for measuring light intensity and a reflector for collecting light. Since the optical measuring technique of the optical measuring unit 60 including the optical sensor and the reflector can be understood from the known technology, detailed description and illustration are omitted.

The calculating unit 70 calculates the light measured by the light measuring unit 60. The calculation unit 70 calculates the concentration of the microorganism B with the measured light and the concentration value of the microorganism B calculated by the calculation unit 70 is supplied to the worker through at least one of visual or auditory Lt; / RTI >

A method of measuring the microbial concentration of the microorganism measuring apparatus 1 according to the present invention having the above-described structure will be described with reference to FIGS. 2 and 4. FIG.

First, as shown in Fig. 2 and Fig. 4, the microorganisms B in the floating air A are charged (100). When the positive electrode (+) or the negative electrode (-) is applied to the charge unit 20 provided at the upper part of the collecting and dissolving unit 10 at a high voltage, the collecting unit 20, which is the bottom position facing the charge unit 20, An electric field for corona discharge is formed due to the current flowing between the ground electrode (not shown) provided on the side of the discharge electrode 30 and the ground electrode.

At this time, when the air A flows through the inlet 12 provided in the body 11 of the collecting and dissolving unit 10, the electric field generated between the charging unit 20 and the earth electrode (not shown) Ions are generated from the air (A) around the electrode (20). The generated ions of the air A receive kinetic energy due to a strong electric field between the charge unit 20 and the earth electrode (not shown) and collide with the surrounding microbial particles (B) Energy is transferred to charge.

The particles of the charged microorganism B are separated from the air A and are discharged toward the ground electrode (not shown) by an electric field formed between the charging unit 20 and the unillustrated grounding electrode provided at the bottom position of the body unit 11 Accelerated. The particles of the charged microorganisms B are moved to the collecting part 30 provided at the bottom position of the body part 11 by changing the moving direction in the direction perpendicular to the inflow direction of the air A, do. Due to the acceleration of the charged microorganisms B, the charged microorganisms B are trapped in the water tank 31 provided in the collecting unit 30. [

The captured microorganism (B) is lysed by the dissolving agent contained in the collecting solution (W) and the luminescent agent to emit light (200). That is, the charged microorganisms (B) are collected in the collecting solution (W) and melted to emit light (200). The optical measuring unit 60 measures the light emission of the charged microorganism B (300), and the measured value is calculated by the arithmetic unit 70 and notified to the operator (400).

As described above, the concentration of the microorganism (B) in the air (A) is collected and dissolved simultaneously to emit light, so that the concentration of the microorganism (B) can be measured in real time in a short time. In addition, continuous charging and trapping can be performed in response to the inflow of the air (A), and the concentration of the microorganisms (B) can be automatically measured continuously.

Although the present invention has been described with reference to the preferred embodiments thereof, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit and scope of the invention as defined in the following claims. It can be understood that

1: Microorganism measuring device
10: Collecting and dissolving unit
20:
30: Collection section
40:
50: optical measuring unit
60: optical measuring unit
70:
A: air
B: Microorganisms

Claims (14)

A collecting and dissolving unit for collecting and dissolving microorganisms in the air by means of a collecting solution containing a solubilizing agent and a light emitting agent; And
An optical measuring unit for measuring the concentration of the microorganisms with light generated from the collected and dissolved microorganisms;
/ RTI >
In the collecting and dissolving unit,
A charge unit for generating a corona discharge to charge the microorganisms in the air;
A collecting unit collecting the charged microorganisms with the collecting liquid; And
A supplying unit for supplying the collecting unit with the solubilizing agent and the light emitting agent;
Lt; / RTI >
Wherein the charging unit and the collecting unit are disposed to face each other on a body part provided on the path of the air so that the air is introduced and discharged so that charging, collecting, and emitting light are simultaneously generated in one body part.
delete The method according to claim 1,
Wherein the light measuring unit comprises:
A light measuring unit for measuring light emitted from the microorganisms collected in the collecting unit; And
A calculator for calculating light measured by the light measuring unit;
And a microbial cell.
The method according to claim 1,
Wherein the supplying section includes a waste section for discarding the collecting liquid discharged from the collecting section.
The method according to claim 1,
Wherein the charge unit comprises at least one electrode body provided on the body portion.
6. The method of claim 5,
Wherein the at least one electrode body has a shape of at least one of an electrode line, an electrode, an electrode wire, an electrode pin, a nanofiber electrode, and a sawtooth electrode.
The method of claim 3,
Wherein the light measuring unit includes an optical sensor for measuring light intensity and a reflector for collecting the light.
8. The method according to any one of claims 1 to 7,
The dissolving agent includes a lysis buffer for dissolving cells to extract adenosine triphosphate (ATP)
Wherein the luminescent agent comprises at least one of luciferin, luciferase, and magnesium (Mg < 2 + >).
Charging the microorganisms in the air into the lower part;
Collecting the charged microorganisms as a collecting solution of a collecting part including a solubilizing agent and a light emitting agent to dissolve and emit light;
Measuring light emitted from the microorganism; And
Calculating the concentration of the microorganism with the measured light;
/ RTI >
Wherein the charging part and the collecting part are provided to face each other on a body part provided on the path of the air so that the air is introduced and discharged, so that charging, collecting, dissolving, and emitting light are simultaneously generated in one body part.
10. The method of claim 9,
Wherein the charging step comprises charging the microorganisms in the air by generating a corona discharge in the charge unit including at least one electrode body to which a positive electrode (+) or a negative electrode (-) is applied.
delete 10. The method of claim 9,
Wherein the light measuring step measures the light of the microorganism by an optical sensor for measuring the light intensity and a reflector for collecting the light.
10. The method of claim 9,
Wherein said calculating step comprises visually or audibly notifying the concentration of the calculated microorganisms.
The method according to any one of claims 9, 10, 12, and 13,
The dissolving agent includes a lysis buffer for dissolving cells to extract adenosine triphosphate (ATP)
Wherein the luminescent agent comprises at least one of luciferin, luciferase, and magnesium (Mg < 2 + >).
KR1020160019531A 2016-02-19 2016-02-19 Apparatus and method for microorganism measurement KR101815751B1 (en)

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