WO2016039124A1

WO2016039124A1 - Detection device and detection method

Info

Publication number: WO2016039124A1
Application number: PCT/JP2015/073670
Authority: WO
Inventors: 恵美福永; 藤岡　一志; 伸佳石野; 義裕假家
Original assignee: シャープ株式会社
Priority date: 2014-09-11
Filing date: 2015-08-24
Publication date: 2016-03-17
Also published as: JPWO2016039124A1

Abstract

In the present invention, a detection device collects particles within a liquid (S1) and measures the fluorescence intensity of the collected particles (S2). Then, the detection device heats the collected particles (S3) and measures the fluorescence intensity of the collected particles after such particles have been heated (S4). The detection device calculates the amount of organism-derived particles within the liquid on the basis of the amount of change, before and after heating, in the fluorescence intensity of the collected particles (S5).

Description

Detection apparatus and detection method

This disclosure relates to a detection apparatus and a detection method, and more particularly, to a detection apparatus and a detection method for detecting biological particles in a liquid.

When detecting particles derived from organisms such as microorganisms in the liquid, particles in the liquid are collected as the first step, and particles derived from the organism are detected from the collected material as the second step. A method of evaporating a liquid portion by mist-forming a specimen in the first stage and performing measurement by staining with a fluorescent labeling reagent in the second stage is disclosed in International Publication No. 2013/132630 (Patent Document 1).

As another method for collecting particles in a liquid, there is a method for filtering a specimen as disclosed in JP 2013-15512 A (Patent Document 2).

International Publication No. 2013/132630 JP 2013-15512 A International Publication No. 2011/104770

In the detection process in the second stage of Patent Document 1, the method of performing the measurement by staining with a fluorescent labeling reagent may involve a complicated reagent spraying process, an expensive reagent, and a complicated apparatus structure. It is a problem. In order to solve the problem, a method disclosed in International Publication No. 2011/104770 (Patent Document 3) can be employed. In other words, the collected particles are heated, and the difference between the fluorescence intensities from the preceding and succeeding particles is applied to the relationship between the difference in fluorescence intensity stored in advance and the amount of biological particles. The amount of living organism-derived particles can be calculated. In the following description, this detection method is also referred to as “heated fluorescence detection method”. Since this method does not involve the use or spraying of reagents, the above-described problems can be solved.

However, if the collection method of the first stage of Patent Document 1 is adopted for the heating fluorescence detection method, biological particles in the liquid may be released out of the liquid during spraying, or biological particles in the air. May get mixed in the liquid. As a result, the detection accuracy may be reduced.

Further, since the method disclosed in Patent Document 2 is not a collection method for the heating fluorescence detection method, there is a problem that it is not suitable for heating after collection and measurement of fluorescence intensity.

This indication is made in view of such a problem, and one of the purposes is detection which can detect particles derived from living organisms with high accuracy by collecting particles from liquid with high accuracy. An apparatus and detection method are provided.

According to one embodiment, the detection device is a detection device for detecting biological particles in a liquid, and collects particles contained in the liquid, a light emitting element, a light receiving element for receiving fluorescence. And a computing device for measuring the fluorescence intensity before and after heating of the particles collected by the collection mechanism and calculating the amount of biologically derived particles in the liquid based on the amount of change in fluorescence intensity before and after heating With.

Preferably, the collection mechanism includes separation means for separating particles from the liquid.
Preferably, the separating means includes a heater that can be heated at a temperature at which the liquid collected by the collecting mechanism can be heated and evaporated.

More preferably, the separation means performs heating and evaporation at the first temperature and performs heating for calculating the amount of particles at the second temperature, and the first temperature is lower than the second temperature.

More preferably, the first temperature is 100 ° C. or lower and the second temperature is higher than 100 ° C.

Preferably, the separation means includes a filter capable of separating particles from the liquid by filtration.
According to another embodiment, the detection method is a method for detecting biologically-derived particles in a liquid, the step of collecting particles, the step of measuring the fluorescence intensity of the collected particles, and the collected particles The step of measuring the fluorescence intensity of the collected particles after heating, and the step of calculating the amount of biologically derived particles in the liquid based on the amount of change in the fluorescence intensity of the particles before and after heating. With.

According to a certain aspect, particles in a liquid can be collected with high accuracy, and biological particles among the particles can be detected with high accuracy.

It is the schematic for demonstrating the structure of the detection apparatus concerning embodiment. It is a graph which shows the fluorescence intensity of the particle | grains of biological origin before a heating and after a heating. It is a graph which shows the fluorescence intensity of the dust before a heating and after a heating. It is a schematic diagram for demonstrating a collection process. It is a schematic diagram for demonstrating a collection process. It is a schematic diagram for demonstrating a collection process. It is a schematic diagram for demonstrating a fluorescence measurement process. It is a schematic diagram for demonstrating a heating process. It is a graph which shows the correlation with the increase amount of the fluorescence amount by heating, and the density | concentration of the particle | grains of biological origin. It is a block diagram which shows the specific example of a function structure of a detection apparatus. It is a flowchart showing the flow of the detection operation in a detection apparatus.

Hereinafter, embodiments of the present invention will be described with reference to the drawings. In the following description, the same parts and components are denoted by the same reference numerals. Their names and functions are also the same. Therefore, these descriptions will not be repeated.

[First Embodiment]
<Device configuration>
FIG. 1 is a schematic diagram for explaining a configuration of a detection apparatus 100 according to the present embodiment. The detection device 100 includes a light receiving element 9, a signal processing unit 30, a heater 91, and a measurement unit 40. The light receiving element 9 receives light emitted from the surface of the collecting plate 12 which is a part of the collecting mechanism. The collection plate 12 can collect particles in the liquid. The signal processing unit 30 is electrically connected to the light receiving element 9 and processes a sensor signal from the light receiving element 9. The heater 91 heats the particles collected on the surface of the collection plate 12. The measurement unit 40 is electrically connected to the heater 91 and the signal processing unit 30 and measures the amount of biological particles by controlling the entire detection device 100.

The surface of the collecting plate 12 is irradiated with light from the light emitting element 6. The light emitting element 6 is preferably a semiconductor laser or an LED (Light Emitting Diode) element. The wavelength of the light emitted from the light emitting element 6 may be any wavelength as long as biological particles can be excited to emit fluorescence, and may be in the ultraviolet or visible region. The wavelength of light emitted from the light emitting element 6 is preferably 300 nm to 450 nm. The light receiving element 9 receives the fluorescence from the surface of the collecting plate 12 by irradiating the surface of the collecting plate 12 with light having such a wavelength. Preferably, the light emitting element 6 is also connected to the measurement unit 40, and the light emission is controlled by the measurement unit 40.

Preferably, a lens and an aperture are arranged in front of the light emitting element 6 in the irradiation direction. Thereby, the width of the light from the light emitting element 6 is uniform or the light has a predetermined size. Preferably, in front of the light receiving element 9 in the light receiving direction, a condensing lens for condensing the fluorescence from the surface of the collecting plate 12 onto the light receiving element 9 and the irradiation light entering the light receiving element 9. A filter for preventing is arranged.

The particles collected on the surface of the collecting plate 12 are heated by the heater 91. Heating of the heater 91 is controlled by the measurement unit 40. As the heater 91, a ceramic heater, a far infrared heater, a far infrared lamp, or the like is preferably used.

Preferably, the heater 91 is disposed on the surface (back surface) opposite to the surface on which the particles are collected with respect to the collection plate 12, and heats the collection plate 12 from the back surface side. Thereby, the influence of the heat with respect to apparatuses, such as the light emitting element 6 and the light receiving element 9 which are arrange | positioned at the surface side of the collection board 12, can be suppressed.

<Detection principle>
The detection principle of the biological particle in the detection apparatus 100 will be described with reference to FIGS. The biological particles refer to particles derived from microorganisms, fungi such as mold, and organisms including pollen and mites.

In the liquid, biological particles and minerals are mixed. Hereinafter, the particles in the liquid are also referred to as mixed particles. When the mixed particles are irradiated with ultraviolet light or blue light, the biological particles emit fluorescence. However, the mixed particles include chemical fiber dust (hereinafter also referred to as dust) that emits fluorescence, in addition to biological particles. Therefore, the amount of biologically derived particles cannot be measured only by detecting the fluorescence emitted from the mixed particles.

Therefore, in the detection apparatus 100, the detection method previously developed by the applicant of the present application and disclosed in Patent Document 3 is adopted. This method uses the fact that the fluorescence intensity (fluorescence amount) changes when biological particles are heated, but the fluorescence intensity does not change even when dust such as chemical fibers is heated.

FIG. 2 is a graph showing the fluorescence intensity of biological particles before and after heating. FIG. 3 is a graph showing the fluorescence intensity of the dust before and after heating. 2 and 3 show the fluorescence intensity on the vertical axis and the wavelength of the fluorescent light on the horizontal axis.

As shown in FIG. 2, in the case of biologically derived particles, the amount of fluorescence after heating is remarkably increased in comparison with the amount of fluorescence before heating in a wide wavelength range. On the other hand, as shown in FIG. 3, in the case of dust, the amount of fluorescence after heating and the amount of fluorescence before heating are substantially the same.

Therefore, the amount of biological particles contained in the mixed particles can be calculated by measuring the amount of fluorescence before and after heating the mixed particles and obtaining the difference in the amount of fluorescence before and after heating.

Hereinafter, each step of the detection operation in the detection apparatus 100 will be described. The detection operation of the detection apparatus 100 includes a mixed particle collecting step, a mixed particle fluorescence measurement step before heating, a mixed particle heating step, a mixed particle fluorescence measurement step after heating, and the amount of biological particles. Including the step of calculating.

FIG. 4, FIG. 5 and FIG. 6 are schematic diagrams for explaining the collection process. FIG. 7 is a schematic diagram for explaining the fluorescence measurement process before heating. FIG. 8 is a schematic diagram for explaining the heating step. FIG. 9 is a graph showing the correlation between the amount of increase in fluorescence due to heating and the concentration of biological particles. In FIG. 9, the vertical axis indicates the amount of increase in fluorescence due to heating, and the horizontal axis indicates the concentration of biological particles.

The detection device 100 detects biological particles contained in the liquid. Therefore, as shown in FIG. 4, in the collecting step, as an example, a liquid specimen is dropped onto the surface of the collecting plate 12. The specimen includes mixed particles 600 including biological particles 600A and dust 600B such as chemical fiber dust.

As shown in FIG. 5, when the collection plate 12 is heated, the liquid evaporates and the mixed particles 600 are separated from the liquid. Thereby, the mixed particles 600 remain on the collecting plate 12 and are collected. The heater 91 may be used for heating the liquid on the surface of the collection plate 12, or another heater may be used, and the collection plate 12 after heating may be set in the detection device 100.

Preferably, the liquid to be dropped is a concentrated liquid that is a specimen. The concentration method may be based on general heating, or may be filtered once using a filter, and the mixed particles 600 may be detached from the filtered filter in a liquid containing a surfactant.

As shown in FIG. 7, in the fluorescence measurement step before heating, excitation light is irradiated toward the mixed particles 600 collected on the collection plate 12 from the light emitting element 6 such as a semiconductor laser. The lens condenses the fluorescence emitted from the mixed particles 600 irradiated with the excitation light, and the light receiving element 9 receives the fluorescence.

As shown in FIG. 8, in the heating process, the detection apparatus 100 heats the collection plate 12 with a heater 91 attached to the back surface of the collection plate 12, thereby collecting the mixed particles 600 collected on the collection plate 12. Heat. After heating, the collection plate 12 is air-cooled.

In the fluorescence measurement step after heating, the excitation light is irradiated from the light emitting element 6 toward the mixed particles 600 collected on the collecting plate 12 as in the case of FIG. The fluorescence emitted from the mixed particles 600 irradiated with the excitation light is collected by the lens and received by the light receiving element 9.

The detection apparatus 100 is based on the difference ΔF1 obtained by subtracting the fluorescence amount before heating from the fluorescence amount after heating based on the relationship between the increase amount ΔF of the fluorescence amount illustrated in FIG. 9 and the biological particle concentration N. The concentration (particles / m ³ ) of biological particles is calculated. Note that the correlation between the increase ΔF and the biological particle concentration N is obtained by conducting an experiment in advance.

<Functional configuration>
FIG. 10 is a block diagram illustrating an example of a functional configuration of the detection apparatus 100 for performing the above-described detection operation. FIG. 10 shows a configuration example in which the function of the signal processing unit 30 is realized mainly by an electric circuit. However, although some of these functions are not illustrated, a configuration realized by a CPU (Central Processing Unit) included in the signal processing unit 30 executing a predetermined program may be used. In addition, an example in which the measurement unit 40 is realized by software is shown. However, some of these functions may be realized by a hardware configuration such as an electric circuit.

10, the signal processing unit 30 includes a current-voltage conversion circuit 34 connected to the light receiving element 9 and an amplification circuit 35 connected to the current-voltage conversion circuit 34.

The measurement unit 40 includes a control unit 41, a storage unit 42, a clock generation unit 43, and a drive unit 48.

The light receiving element 9 inputs the amount of received light to the signal processing unit 30. The amount of received light is a current signal (sensor signal) corresponding to the fluorescence intensity on the surface of the collection plate 12. The current signal is input to the current-voltage conversion circuit 34.

The current-voltage conversion circuit 34 detects the peak value of the current signal input from the light receiving element 9, that is, the peak current value H, and converts it into the voltage value Eh. The voltage value Eh is amplified to a preset amplification factor by the amplifier circuit 35, and the amplified voltage value Eh is output to the measurement unit 40. The control unit 41 of the measurement unit 40 receives an input of the voltage value Eh from the signal processing unit 30 and sequentially stores the voltage value Eh representing the fluorescence intensity in the storage unit 42.

The clock generation unit 43 generates a clock signal and outputs it to the control unit 41. The control unit 41 outputs a control signal for controlling ON / OFF of heating of the heater 91 to the driving unit 48 at timing based on the clock signal, and controls ON / OFF of heating of the heater 91. . Preferably, the control unit 41 is electrically connected to the light emitting element 6 as shown in FIG. 10, and also controls ON / OFF of the light emitting element 6 at a timing based on the clock signal.

Furthermore, the control unit 41 also controls the heating temperature of the heater 91. As the first heating, the control unit 41 performs heating at the first temperature T1 in order to vaporize the liquid on the collecting plate 12 in the collecting step. Next, the control unit 41 performs heating at the second temperature T2 in the above heating process as the second heating. The temperature T1 and the temperature T2 have a relationship of T1 <T2. That is, the control unit 41 performs the first heating at a temperature lower than that of the second heating. Preferably, the temperature T1 is a temperature at which the liquid dropped on the collection plate 12 can be evaporated and is 100 ° C. or lower (T1 ≦ 100 ° C.). On the other hand, the temperature T2 is a temperature higher than 100 ° C. (T2> 100 ° C.). More preferably, the temperature T1 is 80 ° C. to 100 ° C. (80 ° C. ≦ T1 ≦ 100 ° C.), and the temperature T2 is 100 ° C. to 250 ° C. (100 ° C. ≦ T2 ≦ 250 ° C.). By setting the temperature T1 in such a range, the liquid on the collection plate 12 can be vaporized and the particles can be separated from the liquid while suppressing an increase in the fluorescence intensity from the biological particles. On the other hand, the temperature T2 is a temperature range in which the effect expected in the heating step, that is, the fluorescence intensity from the biological particles can be expected to increase.

The control unit 41 includes a calculation unit 411. The calculation unit 411 calculates the amount of biological particles in the liquid using the voltage value Eh stored in the storage unit 42. That is, the calculation unit 411 calculates the difference between the fluorescence intensity F1 before the second heating and the fluorescence intensity F2 after the second heating as the increase amount ΔF. As described above, the amount of increase ΔF is related to the amount of biological particles (number of particles, particle concentration, etc.). The calculation unit 411 stores a correlation between the increase amount ΔF illustrated in FIG. 9 and the biological particle concentration N in advance. Then, the calculation unit 411 calculates the amount of biologically derived particles in the collected particles by substituting the calculated increase amount ΔF into the correlation.

In the above example, the increase amount ΔF uses the difference in fluorescence intensity before and after the heat treatment for a predetermined heating amount (product of a predetermined heating temperature and a heating time Δt). Instead of ΔF, the ratio of the fluorescence intensity before and after the heat treatment may be used.

<Detection operation>
FIG. 11 is a flowchart showing the flow of detection operation in the detection apparatus 100. When the start of the detection operation is instructed, first, the detection apparatus 100 executes an operation of separating particles from the specimen that is a liquid (step S1). In the present embodiment, it is assumed that an operation of separating particles by vaporizing a liquid is performed. In step S1, the detection apparatus 100 heats the liquid dropped on the surface of the collection plate 12 at the first temperature T1. The temperature T1 is lower than the second temperature T2, and is preferably 100 ° C. or lower (T1 ≦ 100 ° C.). More preferably, the first temperature T1 is 80 ° C. to 100 ° C. (80 ° C. ≦ T1 ≦ 100 ° C.).

In addition, preferably, the detection apparatus 100 performs the operation | movement which cools the collection board 12 after the 1st heating of the said step S1. The cooling operation may also be performed after the second heating which is step S3 described later.

Next, the detection apparatus 100 performs an operation of measuring the fluorescence intensity F1 before the second heating from the surface of the collection plate 12 (step S2). That is, the signal processing unit 30 converts the current signal from the light receiving element 9 into the voltage value Eh and inputs it to the measurement unit 40. Then, the measurement unit 40 stores the voltage value Eh representing the fluorescence intensity F1 in the storage unit 42. When the control unit 41 of the measurement unit 40 can control the light emitting element 6, in step S <b> 2, the control unit 41 further controls the light emitting element 6 to emit light.

Next, the detection apparatus 100 executes a second heating operation for heating the particles collected on the surface of the collection plate 12 (step S3). The second temperature T2 is higher than the first temperature T1, and is preferably higher than 100 ° C. (T2> 100 ° C.). More preferably, the second temperature T2 is 100 ° C. to 250 ° C. (100 ° C. ≦ T2 ≦ 250 ° C.).

Next, the detection apparatus 100 performs an operation of measuring the fluorescence intensity F2 after the second heating from the surface of the collection plate 12 (step S4). The operation in step S4 is the same as that in step S2.

Next, the detection apparatus 100 calculates the amount of biological particles collected on the collection plate 12 (step S5). More specifically, first, the control unit 41 of the measurement unit 40 calculates the difference (increase ΔF) between the fluorescence intensities F1 and F2 before and after the second heating from the surface of the collection plate 12 by the calculation unit 411. Subsequently, the control unit 41 stores in advance a correlation between the increase amount ΔF illustrated in FIG. 9 and the biological particle concentration N, and the increase amount ΔF calculated by the calculation unit 411 is stored in the correlation. By substituting into the relationship, the amount of biological particles in the collected particles is calculated.

<Effect of Embodiment>
By using the detection apparatus 100 according to the present embodiment, the difference in properties due to the heat treatment between the fluorescence from the biological particles and the fluorescence from the dust is utilized, and the biological particles are derived from the mixed particles contained in the liquid specimen. Particles can be detected. At this time, the detection apparatus 100 separates particles from the liquid by heating and vaporizing the liquid dropped on the surface of the collection plate 12. The method of collecting separately by this method can prevent the surrounding particles from mixing in and dissipate the particles and can collect the particles more accurately than the method of collecting by misting and spraying on the surface of the collecting plate. . Furthermore, at this time, in order to separate and collect the particles from the liquid, the detection apparatus 100 heats at a temperature lower than the heating for detecting the biological particles. Thus, by using the detection apparatus 100, it is possible to detect biologically-derived particles contained in the specimen that is a liquid with high accuracy.

[Second Embodiment]
The detection principle of the first embodiment describes a case where a method of heating and evaporating a liquid is employed in the separation step of the mixed particle collecting step. However, the separation step is not limited to the above method.

The method employed in the separation step of the present embodiment is a method of filtering using a filter as shown in FIG. In this case, in the separation step, the liquid is filtered using the filter 21 that can be set on the collection plate 12. The case where the filter 21 after filtration is set on the surface of the collection plate 12 is assumed to be collection on the collection plate 12. The filter has fineness that can separate particles from the liquid.

In the detection operation, in step S1, the detection apparatus 100 receives a set of filters that can be set on the collection plate 12 after separating the particles from the liquid that is the specimen.

[Third Embodiment]
The method employed in the separation step of the present embodiment includes a step of filtering using a filter and a step of vaporizing the liquid by heating. By including the step of filtering using a filter in the separation step, the particles are collected in a state of being completely separated from the liquid. Or it collects in the state by which the liquid component to which particle adheres was reduced significantly. Then, by passing through the process of heating and vaporizing a liquid, it can prevent that the heating efficiency in 2nd heating falls with the liquid adhering to particle | grains. Therefore, the detection accuracy in the detection apparatus 100 can be further improved by including both steps in the separation step.

As described above, the separation method employed in the separation step is not limited to a specific method such as a method of heating and vaporizing a liquid, a method of filtering using a filter, and a method of combining them.

The embodiment disclosed this time should be considered as illustrative in all points and not restrictive. The scope of the present invention is defined by the terms of the claims, rather than the description above, and is intended to include any modifications within the scope and meaning equivalent to the terms of the claims.

6 light emitting element, 7 lens, 9 light receiving element, 12 collecting plate, 21 filter, 30 signal processing unit, 34 current-voltage conversion circuit, 35 amplification circuit, 40 measurement unit, 41 control unit, 42 storage unit, 43 clock generation Part, 48 drive part, 91 heater, 100 detection device, 411 calculation part, 600 mixed particles, 600A biological particles, 600B dust.

Claims

A detection device for detecting biological particles in a liquid,
A light emitting element;
A light receiving element for receiving fluorescence;
A collection mechanism for collecting particles contained in the liquid;
Measure the fluorescence intensity before and after heating the particles collected in the collection mechanism, and based on the amount of change in fluorescence intensity before and after the heating, comprising an arithmetic unit for calculating the amount of biological particles in the liquid, Detection device.
The detection device according to claim 1, wherein the collection mechanism includes a separation unit for separating the particles from the liquid.
The detection device according to claim 2, wherein the separation unit includes a heater capable of being heated at a temperature at which the liquid collected by the collecting mechanism can be heated and evaporated.
The detection device according to claim 2, wherein the separation means includes a filter capable of separating the particles from the liquid by filtration.
The separation means performs the heating and evaporation at a first temperature, performs heating for calculating the particle amount at a second temperature, and the first temperature is lower than the second temperature. Item 4. The detection device according to Item 3.
The detection apparatus according to claim 5, wherein the first temperature is 100 ° C or lower and the second temperature is higher than 100 ° C.
A method for detecting biological particles in a liquid,
Collecting the particles;
Measuring the fluorescence intensity of the collected particles;
Heating the collected particles;
Measuring the fluorescence intensity of the collected particles after the heating;
Calculating the amount of biologically derived particles in the liquid based on the amount of change in fluorescence intensity of the particles before and after the heating.