JP2012005381A - Collection mechanism, detection apparatus, and method for collecting and detecting - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a detection apparatus for effectively and accurately collecting particles derived from living organisms in a fluid in a short time.SOLUTION: The detection apparatus takes air in by driving a pump 6 from an opening 9 to a chamber 16 in which a liquid for dielectrophoresis is stored inside. The liquid in the chamber 16 is circulated with contacting an electrode 14 by driving a pump 7 by opening a bulb 22a. In the state, microorganisms taken in the liquid by dielectrophoresis adhere to the electrode 14 by applying a predetermined voltage to the electrode 14. In the state, by closing the bulb 22a and discharging water by opening a bulb 22c, the application of pressure to the electrode 14 is stopped. As the result, concentration of the microorganisms is concentrated. Then, the liquid is transferred to a measurement cell 3 by opening the bulb 22b. An amount of the microorganisms is determined based on fluorescence amount or the like.

Description

  The present invention relates to a collection mechanism, a detection device, a collection method, and a detection method, and more particularly, to a collection mechanism, a detection device, a collection method, and a detection method for detecting organism-derived particles in a fluid.

  Conventionally, in the detection of microorganisms in the air, after collecting microorganisms in the air by methods such as the falling bacteria method, collision method, slit method, perforated plate method, centrifugal collision method, impinger method, and filter method, culture However, a culture method for counting the number of appearing colonies has been proposed. However, this method requires 2-3 days for culturing and is difficult to detect in real time.

  Therefore, in recent years, as disclosed in Japanese Patent Laid-Open No. 2003-00224 (Patent Document 1), a pair of electrodes for dielectrophoresis, a dielectrophoresis power supply unit that applies an AC voltage connected between the electrodes, There has been proposed a microbial activity measuring apparatus having a configuration including a measuring unit that measures the impedance of the microbial activity. In this apparatus, a solution containing microorganisms is supplied into a measurement chamber provided with an electrode for dielectrophoresis. By applying an alternating voltage having a predetermined frequency to a predetermined microorganism between both electrodes, the corresponding microorganism is concentrated between the electrodes. As a result, the impedance between the electrodes changes. From this impedance value, the amount of microorganisms is calculated using calibration curve data of the impedance and the amount of microorganisms obtained in advance.

  Japanese Patent Application Laid-Open No. 11-299477 (Patent Document 2) proposes a method of calculating the number of microorganisms by irradiating the microorganisms gathered in the vicinity of the electrode with light and measuring the scattering intensity from the microorganisms. According to the above-mentioned dielectrophoresis method, it is possible to separate live bacteria from partially damaged bacteria and dead bacteria by selecting the frequency to be applied, so it has been attracting attention as a detection method that replaces the conventional culture method. .

Japanese Patent Laid-Open No. 2003-224 Japanese Patent Laid-Open No. 11-299477

  By the way, when using the apparatus proposed in Patent Document 1, it is necessary to first concentrate the microorganisms in the solution by drawing them to the electrode using the dielectrophoresis phenomenon. However, the range in which the attractive force to the microorganisms by dielectrophoresis works is about several tens of microns to one hundred microns, and the microorganisms existing in the range beyond that can only be waited until the microorganisms enter the range by diffusion. Therefore, when the concentration of microorganisms in the solution is low, there is a problem that it takes time until the impedance changes.

On the other hand, when the amount of microorganisms in the air is measured using the apparatus proposed in Patent Document 1, microorganisms in the air are taken into a filter or a solution by a filter method or an impinger method, and the solution is put into the apparatus to form a dielectric. Concentrate by electrophoresis and measure impedance to calculate the amount of microorganisms. For example, consider the case where microorganisms are collected in air in a 10 milliliter solution at a collection speed of 100 liters per minute. The microbial concentration in the air in a quiet state in a normal room is set to the order of 10 4 per m ³ . As described above, when considering that only microorganisms near the electrode can be detected in a short time, the concentration of microorganisms in the solution to obtain a detectable impedance requires an order of 10 5 per milliliter, The collection time of microorganisms is 1,000 minutes, which takes about 17 hours. In practice, a large amount of solution for collection is required, and the collection speed is small, so the time is long. As described above, the conventional method can be used for the detection of high-concentration microorganisms, but has a problem that it is difficult to grasp a normal indoor situation.

  There is also a method of concentrating by collecting microorganisms in the air in the solution and then evaporating the solution or precipitating the microorganisms by using a centrifugal method and taking the supernatant liquid. And there is a problem that handling becomes complicated.

  In addition, the detection method proposed in Patent Document 2 measures the scattering intensity of microorganisms concentrated between electrodes, and the scattering intensity depends on the particle concentration on the low concentration side, but the particles overlap locally on the high concentration side. The scattering intensity depends on the particle size rather than on the particle concentration, which causes a problem that accurate measurement cannot be performed. Therefore, there arises a problem that the measured density range is limited to the low density side.

  The present invention has been made in view of such a problem, and is a collection mechanism, a detection apparatus, and the like, capable of efficiently collecting and detecting biologically-derived particles in a fluid in a short time. It aims at providing the collection method and the detection method.

  In order to achieve the above object, according to one aspect of the present invention, the collection mechanism is a mechanism for collecting biological particles, and is provided in a chamber for holding a liquid, and in the chamber. An electrode for collecting biological particles in a liquid by dielectrophoresis when a voltage of a predetermined frequency is applied, and a discharge mechanism for discharging the liquid in the chamber, the biological particles in the electrode By discharging a predetermined amount of the liquid in the chamber in a state where is collected by dielectrophoresis, the concentration of biological particles is concentrated and collected.

  Preferably, the collection mechanism further includes a circulation mechanism for circulating the liquid in the chamber.

  Preferably, a substrate having a through-hole for allowing liquid to pass therethrough is provided in the chamber.

Preferably, a guide is provided in the chamber for guiding the liquid to the electrode.
Preferably, the collection mechanism further includes an intake mechanism for taking in the biological particles in the fluid into the liquid by taking the fluid containing the biological particles into the chamber.

  According to another aspect of the present invention, a detection device includes the above-described collection mechanism and a measurement unit for measuring the amount of biological particles collected by the collection mechanism, and is collected by the collection mechanism. A moving mechanism is further provided for moving the collected biological particles to a cell for measurement by the measuring means.

  According to still another aspect of the present invention, the collection method is a method for collecting biological particles, the step of circulating a liquid containing biological particles in the chamber, and a method provided in the chamber. Applying a voltage of a predetermined frequency to the electrode, collecting biological particles in the liquid on the electrode by dielectrophoresis, discharging a predetermined amount of liquid from the chamber, and stopping the application to the electrode. Separating biological particles collected from the electrodes and dispersing them in the liquid remaining in the chamber.

  According to still another aspect of the present invention, the collection method is a method for collecting biological particles, the step of circulating a liquid containing biological particles in the chamber, and a method provided in the chamber. Applying a voltage of a predetermined frequency to the electrode, collecting biological particles in the liquid on the electrode by dielectrophoresis, and supplying a predetermined amount of a new solution to the chamber after discharging the liquid from the chamber And dissociating the biological particles collected from the electrodes by stopping application to the electrodes and dispersing them in a new liquid supplied to the chamber.

  Preferably, the collection method further includes the step of taking in the biological particles in the fluid into the liquid by taking the fluid containing the biological particles in the chamber, and the step of taking in the biological particles in the liquid This is performed before the step of circulating the liquid in the chamber.

  Preferably, the step of circulating the liquid in the chamber includes the step of taking the biological particles in the fluid into the liquid by taking the fluid containing the biological particles into the chamber.

  According to still another aspect of the present invention, the detection method is a method for detecting biological particles collected by the above-described collection method, and is based on imaging of an electrode from which biological particles are collected. Detecting a particle of origin.

  According to still another aspect of the present invention, the detection method is a method for detecting biological particles collected by the above-described collection method, from a liquid in which biological particles collected by an electrode are dispersed. Detecting a biological particle based on the intensity of the scattered light.

  According to still another aspect of the present invention, the detection method is a method for detecting biological particles collected by the above-described collection method, wherein the biological particles collected by the electrode are dispersed in a liquid. Detecting a biological particle based on the fluorescence intensity under irradiation of excitation light.

  According to the present invention, it is possible to efficiently collect biologically-derived particles in a fluid in a short time and detect them with high accuracy. Furthermore, even when the concentration of biological particles is low in the fluid, the biological particles can be collected and detected with high accuracy.

It is a figure which shows the specific example of a structure of the detection apparatus concerning 1st Embodiment. It is a figure which shows one specific example of the flow path provided in the discharge hole side of the chamber of the board | substrate provided in the chamber contained in a detection apparatus. It is a figure which shows one specific example of a structure of the board | substrate provided in the chamber contained in a detection apparatus. It is a figure which shows the modification of a structure of a detection apparatus. It is a figure which shows the example formed with a several pairs comb-shaped electrode as another example of a structure of the electrode for dielectrophoresis. It is a figure explaining the flow of the liquid in the electrode vicinity when the electrode for dielectrophoresis is formed with a plurality of pairs of comb electrodes. It is a figure for demonstrating the guide provided in a chamber when an electrode is formed with a several pairs comb-shaped electrode. It is a figure for demonstrating the guide provided in a chamber when an electrode is formed with a several pairs comb-shaped electrode. It is a figure explaining the image of the electrode vicinity taken in by CCD. It is a figure which shows the other specific example of the structure for moving the microorganisms in a liquid to the surface of the electrode for dielectrophoresis by circulation. It is a figure which shows the specific example of a structure of the detection apparatus concerning the modification of this Embodiment. It is a figure which shows the specific example of a structure of the collection apparatus carrying a collection chamber. It is a figure which shows the specific example of a structure of the collection chamber filling part filled with one collection chamber contained in a detection apparatus. It is a figure which shows the specific example of a structure of the concentration part and measurement part of a detection apparatus. It is a figure explaining the specific example of the usage method of a detection apparatus.

  Embodiments of the present invention will be described below 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.

  FIG. 1 is a diagram illustrating a specific example of the configuration of the detection apparatus 100 according to the embodiment. Referring to FIG. 1, a detection device 100 includes, as basic components, a collection and concentration unit 11, a measurement system 1, a circulation mechanism 2, a flow path 5, and an optical system 13, which are an integrated collection unit and concentration unit. including.

  The collection and concentration unit 11 is a mechanism for taking in microorganisms floating in the external air into the chamber 16, and the collection unit includes the collection opening 9, the collection pump 6, the collection pump. A collection filter 10 and a chamber 16 are included. The opening 9 is a suction flow path 18 and the pump 6 is an exhaust flow path 19 connected to one side of the chamber 16. The filter 10 is provided in the flow path 18. By operating the pump 6, an external fluid (here, air) passes through the filter 10 from the opening 9 and is supplied into the chamber 16 through the flow path 18.

  The chamber 16 has a structure in which a liquid suitable for dielectrophoresis can be stored. Inside the substrate, a substrate 15 having a plurality of through holes 20 drilled is provided. An electrode 14 for dielectrophoresis is provided on the surface of the substrate 15.

  A discharge hole 17 is provided on a surface (lower surface in the drawing) of the chamber 16 opposite to the surface to which the opening 9 and the pump 6 are connected. A space may be provided between the substrate 15 and the discharge hole 17, and the substrate 15 may be supported by the wall 31 with respect to the inner surface of the chamber 16 on the side where the discharge hole 17 is provided.

  FIG. 2 is a diagram illustrating one specific example of the flow path provided on the discharge hole 17 side of the substrate 15. Referring to FIG. 2, the wall 31 may be connected to the through hole 20 provided in the substrate 15, and may have a spiral channel 21 formed in a spiral shape from the through hole 20 toward the discharge hole 17. .

  FIG. 3 is a diagram showing one specific example of the configuration of the substrate 15. Referring to FIG. 3, a pair of comb-shaped members having a plurality of circular through holes 20 and electrode terminals 35 and 36 are formed on the surface of substrate 15 on the side of opening 9 and pump 6 (upper surface in the figure). Electrode 14 is formed. The shape of the through hole 20 is not limited to a circular shape, and may be another shape as long as the liquid can pass through the substrate 15. Moreover, the number may be either one or more. Further, as will be described later, the position and arrangement of the through-holes 20 flow on the surface of the electrode 14 when the liquid on the substrate 15 in the chamber 16 flows through the discharge hole 17 or the surface of the electrode 14. If it forms so that it may collide with and flow, it will not be limited to a specific position and arrangement | positioning.

  The circulation mechanism 2 is a mechanism for circulating the liquid in the chamber 16, and includes a circulation channel 4, a circulation pump 7, and a circulation system filter 12. The flow path 4 connects a surface (a side surface in the figure) different from the surface where the discharge hole 17 of the chamber 16 is provided and the discharge hole 17 via the valve 22a, and the pump 7 and the filter 12 are connected to the chamber 16 along the way. They are provided in that order in the direction toward the discharge hole 17.

  The measurement system 1 includes a measurement cell 3, a pump 8 for filling the measurement cell 3 with a solution, a light source 32, and a light receiving element 34. The measurement cell 3 has a shape extending along the long axis in one direction, and has an injection hole 23 on one end side of the axis and a discharge hole 24 on the other end side. The injection hole 23 is connected to the discharge hole 17 of the chamber 16 via the valve 22 b, and the discharge hole 24 is connected to the pump 8.

  The discharge hole 17 is further connected to the drainage reservoir 25 through the discharge channel 5 via the valve 22c. The drainage reservoir 25 is provided with a pump 26 for discharging.

  The optical system 13 includes a lens group 27 and a CCD (Charge Coupled Device Image Sensor) 28 for focusing at least near the electrode 14.

  The material of the chamber 16, each flow path for passing a liquid, and the valves 22 a, 22 b, and 22 c is not limited to a specific material. For example, a metal material, a plastic material, glass or the like may be used. Preferably, a material that does not cause corrosion or the like because the liquid held in the chamber 16 is an aqueous solution is used. Specifically, a metal material is used as the material of the electrode 14 for dielectrophoresis, and a material that is unlikely to be oxidized such as gold, platinum, silver, and titanium is preferable, and an alloy may be used. Plastic or glass can be used as the material of the measurement cell 3, and quartz that transmits ultraviolet light is preferable when measuring fluorescence from a liquid containing microorganisms. Conventional pumps and optical systems can be used as they are.

  The size of the chamber 16 is determined according to the amount of liquid to be processed and the processing time. As an example, the planar size may be 10 mm to 500 mm in width, 10 mm to 500 mm in depth, and 0.1 mm to 500 mm in height. The size of the flow path is also determined according to the amount of liquid to be processed and the processing time. Each flow path may be different. As an example, the cross-sectional size of the flow path may be 0.1 mm to 10 mm in diameter with a circular cross section. The cross section of the flow path is not limited to a circle but may be an ellipse or a rectangle. Of course, these sizes are not limited to the above sizes.

  A comb-shaped electrode is generally proposed as the electrode 14 for dielectrophoresis, but a conventionally proposed electrode can be used as it is. 3 shows an example in which the electrodes 14 are formed of a pair of comb-shaped electrodes, a plurality of pairs may be formed on the surface of the base 15. As will be described later, the formation of a plurality of pairs is advantageous because more microorganisms in the liquid can be collected in the vicinity of the electrode in a short time.

  In the detection apparatus 100 according to the present embodiment, the concentration step and the measurement step are separated and the amount of biological particles floating in the air is detected. That is, first, the concentration of the microorganisms in the collected air in the liquid is concentrated (concentration step), and then the microorganisms are measured from the concentrated liquid (measurement step). Therefore, the detection apparatus 100 is provided with the chamber 16 for concentrating microorganisms and the measurement cell 3 separately.

  In the following description, “biologically-derived particles” are represented by microorganisms (including dead bodies) such as bacteria and viruses, but those that carry out life activities regardless of life or death. This refers to a size that floats in the air, including non-microorganisms. Specifically, in addition to microorganisms (including dead bodies) such as bacteria and viruses, mites (including dead bodies) and the like may be included. In the following description, it is assumed that “microorganism” represents this “biological particle”.

  In the concentration step, the detection apparatus 100 attaches microorganisms taken in the liquid to the electrode by using the principle of dielectrophoresis. Dielectrophoresis used here uses the difference in electrical properties such as dielectric constant between biological particles and a medium (here, water) to attach or separate biological particles from electrodes. It refers to the phenomenon. Specifically, by generating an alternating electric field in the liquid in which the microorganism is taken in, the inside of the microorganism is polarized to plus and minus. By using a specific frequency band, only normal microbial particles (live bacteria) whose cell membrane is not damaged are attracted to the high electric field (positive dielectrophoresis), and damaged microbial particles (dead or damaged bacteria) are live bacteria. And the dielectric constant is different, the phenomenon of repulsion (negative dielectrophoresis) occurs. In the detection apparatus 100, by utilizing this phenomenon, microorganisms in the liquid are attached to the electrode 14 to which a predetermined voltage is applied, the liquid is discharged, and the microorganism concentration in the liquid is concentrated. As described above, when a frequency capable of separating live bacteria and dead or damaged bacteria is used, only one of them can be concentrated. Moreover, when the frequency attracted to the electrode is used for both live and dead or damaged bacteria, both bacteria can be concentrated.

  In the measurement process, in the detection apparatus 100, for example, a linear light source 32 is arranged along the long axis of the measurement cell 3 to irradiate the measurement cell 3 with excitation light, and receive fluorescence from microorganisms in the liquid. The amount of microorganisms is measured by measuring with the element 34. Here, by irradiating the entire cell with excitation light using the linear light source 32, fluorescence from all the microorganisms existing in the liquid can be measured, and there is an advantage that the measurement sensitivity can be increased.

  As the line-shaped light source 32, for example, blue or ultraviolet semiconductor LEDs (Light Emitting Diodes) or semiconductor lasers arranged in an array may be used, or a conventionally proposed line light source may be used. Also good.

  Further, an optical filter 33 may be provided between the measurement cell 3 and the light receiving element 34 in order to prevent the excitation light from entering the light receiving element 34. This has the advantage that even when the amount of microorganisms in the liquid is small, measurement can be performed with high sensitivity.

  The measurement method is not limited to a specific method. As an example, there is a method of measuring scattered light from microorganisms and measuring the amount of microorganisms based on the intensity. In addition, a conventional method may be applied. In any method, the detection apparatus 100 does not directly measure the microorganisms collected on the electrode for concentration dielectrophoresis, but once transferred to the measurement cell 3 having a shape extending along the long axis. Detect microorganisms from With this configuration, even when the concentration of microorganisms in the solution in the chamber 16 is high, it is possible to avoid local overlapping of microorganism particles, and to measure microorganisms in the solution. As a result, there is an advantage that the measurement concentration range is wider than that of the conventional measurement method.

Hereinafter, a method for detecting microorganisms using the detection apparatus 100 will be described in detail.
First, the liquid suitable for dielectrophoresis with the valves 22 a, 22 b, and 22 c connected to the discharge hole 17 of the chamber 16 is closed until the liquid level is above the position where the flow path 4 is connected. Supply in. As the liquid, a liquid having characteristics such as a dielectric constant according to the microorganism to be detected is selected. For example, pure water, a buffer solution, an aqueous solution containing a compound such as mannitol at a predetermined concentration, and the like can be cited as candidates.

  Thereafter, with the valves 22b and 22c closed, only the valve 22a is opened and the pump 7 is driven. As a result, the liquid in the chamber 16 passes through the through hole 20 provided in the substrate 15 on which the electrode 14 is formed, passes through the spiral channel 21 provided under the substrate 15, and is discharged from the discharge hole 17. It circulates in the direction of arrow A in FIG. 1 while filling the chamber 16, the discharge hole 17, and the flow path 4 by a route of returning to the chamber 16 through the flow path 4.

  Next, the pump 6 is driven. Thereby, external air is taken into the liquid in the chamber 16 from the opening 9. At that time, the pump 7 may be driven to circulate the liquid. Or you may repeat circulating or stopping for a fixed time, taking in external air for a fixed time.

  During the circulation, a voltage having a predetermined frequency is applied to the electrode 14 (electrode terminals 35 and 36 in the example of FIG. 3). Thereby, dielectrophoresis is performed. As the liquid circulates, microorganisms in the liquid sequentially approach the vicinity of the electrode 14, so that microorganisms in the liquid sequentially adhere to the surface of the electrode 14 due to the dielectrophoretic force formed on the surface of the electrode 14. In addition, the flow which cannot approach the electrode surface as shown by the arrow B also arises. However, by increasing the number of circulations, most microorganisms in the liquid adhere to the surface of the electrode 14 during the circulation. As a result, the microorganism concentration in the liquid is efficiently concentrated in the vicinity of the electrode 14 in a shorter time than the conventional method. Note that dust and dust mixed in the liquid are captured by the filter 12 during circulation.

  FIG. 4 is a diagram illustrating a modification of the configuration of the detection apparatus 100. As shown in FIG. 4, a guide 30 having an opening at a position corresponding to the electrode 14 for guiding the liquid in the vicinity of the electrode 14 of the substrate 15 may be provided. The opening of the guide 30 may be formed in a mortar shape so that the liquid above the substrate 15 can easily flow into the surface of the electrode 14. By adopting such a configuration, as shown by an arrow C, the liquid is more effectively guided to the surface of the electrode 14 as compared with the case where the guide 30 is not provided. As a result, microorganisms in the liquid can be concentrated on the surface of the electrode 14 in a shorter time with a smaller number of circulations.

  FIG. 5 is a diagram showing an example in which a plurality of pairs of comb electrodes are formed as another example of the configuration of the electrode 14 for dielectrophoresis. Moreover, FIG. 6 is a figure explaining the flow of the liquid in the vicinity of the electrode 14 in this case.

  When the electrode 14 is formed of a plurality of pairs of comb-shaped electrodes as shown in FIG. 5, a flow from the upper part of the electrode pair to the electrode pair as shown by the arrow E in FIG. Thereby, microorganisms in the liquid efficiently move to the surface of the electrode 14 and are captured on the surface of the electrode 14 by the dielectrophoretic force formed on the surface of the electrode 14.

  Even in this case, a flow that does not pass through the surface of the electrode 14 as shown by an arrow B in FIG. 1 (arrow D in FIG. 6) also occurs. However, by increasing the number of circulations, the flow is shorter than conventional. The microorganisms in the liquid can be concentrated on the electrode surface over time.

  By forming the electrode 14 with a plurality of pairs of comb-shaped electrodes as shown in FIG. 5, concentration occurs simultaneously on the plurality of electrode pairs. As a result, it is possible to capture and concentrate microorganisms in the liquid on the electrode surface in a shorter time than when concentration is caused by a pair of electrodes.

  In this configuration, the same guide 30 as that described with reference to FIG. 4 may be provided. 7 and 8 are diagrams for explaining the guide 30 provided when the electrode 14 is formed by a plurality of pairs of comb-shaped electrodes. Referring to FIGS. 7 and 8, in this case, guide 30 has an opening for guiding liquid to each of a plurality of pairs of comb-shaped electrodes. By adopting such a configuration, the liquid is more effectively guided to the respective electrode pairs as compared with the case where the guide 30 is not provided. As a result, microorganisms in the liquid can be concentrated on the surface of the electrode 14 in a shorter time with a smaller number of circulations.

  Here, the flow rate and circulation speed for taking in the external air are set so that the microorganisms are efficiently concentrated according to the microorganisms to be detected. Therefore, it differs depending on the size of the flow path 4 and the size of the chamber 16. As an example, the flow rate for taking in external air is 0.01 liter / min to 100 liter / min, and the circulation rate is 1 ml / hour to 100 ml / min.

  After taking in the external air for a predetermined time, the driving of the pump 6 is stopped. As a result, the intake of the external air is stopped and only the circulation of the liquid is continued, and the microorganisms in the liquid are concentrated near the electrode 14.

  Thereafter, the driving of the pump 7 is stopped and the valve 22a is closed. Thereby, the circulation of the liquid is stopped. Then, the valve 22c is opened to drive the pump 26. Accordingly, the liquid in the chamber 16 moves to the drainage reservoir 25 while the microorganisms in the liquid are concentrated near the electrode 14.

  When the liquid level is close to the surface of the electrode 14, the valve 22 c is closed to stop the driving of the pump 26 and the application of the AC voltage to the electrode 14 is stopped. As a result, the dielectrophoresis is completed, and the microorganisms concentrated in the vicinity of the electrode 14 are dispersed in the liquid remaining in the chamber 16. This increases the concentration of microorganisms in the liquid.

  Thereafter, the valve 22b is opened and the pump 8 is driven. Thereby, the liquid remaining in the chamber 16 in which the microorganisms are dispersed moves to the measurement cell 3. The measurement cell 3 to which the liquid has moved is irradiated with light having a wavelength of ultraviolet to blue from a linear light source 32, and fluorescence emission from the end face is measured. Thereby, the quantity of the microorganisms in the measurement cell 3 can be measured.

  In addition, there is a possibility that some dust emits fluorescence in the dust that cannot be removed by circulating filtering, and there is a concern that the amount of microorganisms calculated from the amount of fluorescence becomes inaccurate. There are few such concerns in an environment with a high concentration of microorganisms, but there is a concern that the amount of microorganisms may be inaccurate when the concentration is low. Therefore, preferably, when the liquid in the chamber 16 is moved to the drainage reservoir 25, all the liquid in the chamber 16 is moved, and after washing the portion below the electrode 14 with another liquid, the new liquid is changed to the chamber. 16 and the application of the AC voltage to the electrode 14 is stopped. Thereby, the microorganisms concentrated in the vicinity of the electrode 14 are dispersed in the new liquid after washing. Therefore, the amount of dust or the like in the liquid in which microorganisms are dispersed can be reduced, and the amount of fluorescence measured can be correlated with the amount of microorganisms.

  As described above, the detection apparatus 100 according to the present embodiment has a configuration in which dielectrophoresis is performed as a mechanism for concentrating the concentration of the microorganism in the liquid, and further includes a measurement mechanism as a mechanism different from the mechanism for concentration. It has a configuration provided separately. With this configuration, it is possible to measure at a higher concentration than in the prior art, and the measurement concentration range is expanded.

  In the above example, external air intake and circulation are performed simultaneously, but the timing of external air intake and circulation is not limited to a specific timing. For example, outside air may be taken in for a predetermined time and then circulated.

  In the above example, quantitative measurement using fluorescence or light scattering is performed, but microorganisms concentrated in the vicinity of the electrode 14 may be captured by the CCD using the optical system 13. FIG. 9 is a diagram illustrating an image captured by the CCD. Referring to FIG. 9, when an image near the electrode 14 is captured by the CCD using the optical system 13, an image of the microorganism 37 gathered between the pair of electrodes 14 is obtained. From this image, the amount of microorganisms may be observed qualitatively. Of course, the number of microorganisms may be calculated by image processing by combining conventional techniques.

  Further, FIG. 10 is a diagram showing another specific example of the configuration for moving microorganisms in the liquid by circulation to the surface of the electrode 14 for dielectrophoresis. FIG. 10 shows only the vicinity of the chamber 16 for ease of explanation. Referring to FIG. 10, as another configuration, substrate 15 may not be drilled, and instead, a configuration in which discharge hole 17 of chamber 16 is provided along the upper surface of substrate 15 may be employed. A guide 30 having an opening may be provided at a position corresponding to the electrode 14 in the chamber 16. With this configuration, the liquid supplied to the chamber 16 passes through the opening along the guide 30, and is discharged along the upper surface of the substrate 15 from the upper part of the electrode pair through the surface of the electrode pair as indicated by an arrow G. It flows toward the hole 17. As a result, like the configuration shown in FIG. 8, microorganisms in the liquid can be captured and concentrated on the surface of the electrode pair in a shorter time than the conventional measurement method.

[Modification]
FIG. 11 is a diagram illustrating a specific example of the configuration of the detection apparatus 200 according to the modification. The detection device 200 has a configuration separated into a collection chamber filling unit, a concentration device, and a measurement device. That is, referring to FIG. 11, detection device 200 includes collection chamber filling unit 201, concentration unit 202, and measurement unit 203. One or more collection chamber filling units 201 and concentration units 202 are provided in the detection device 200. The valve may be provided as necessary, and in the following description, only the main ones are not shown.

  The collection chamber filling unit 201 has a configuration capable of filling the collection chamber 116. A liquid in which microorganisms in the air are dispersed is supplied to the collection chamber 116. Similar to the concentration mechanism of the detection apparatus 100, the concentration unit 202 concentrates the concentration of microorganisms using a dielectrophoresis method.

  The collection chamber 116 is mounted on the collection device 130 (FIG. 12), and is used for collecting microorganisms in the external air. As the collection device 130, a collection device used in a conventional impinger method can be used. FIG. 12 is a diagram illustrating a specific example of the configuration of the collection device 130 on which the collection chamber 116 is mounted. In FIG. 12, the collection device 130 is shown as a collection device used in the conventional impinger method.

  Referring to FIG. 12, the collection device 130 includes a collection opening 109 for taking outside air into the collection chamber 116, a filter 110 for removing large dust, dust, pollen, and the like, and air A flow path 119 for guiding the air to the collection chamber 116, a pump 106 for taking in external air, and a flow path 119 for connecting the pump 106 and the collection chamber 116.

  The collection chamber 116 can be separated from the flow path 118 and the flow path 119. The opening 109 is a flow path 118, and the pump 106 is an exhaust flow path 119, which are connected to one side (upper part in the drawing) of the collection chamber 116. Further, a discharge hole 117 having an openable / closable joint 150 is provided on a surface (bottom portion in the drawing) different from a surface to which the flow paths 118 and 119 of the collection chamber 116 are connected.

  The collection chamber 116 is supplied with a liquid for collecting and dispersing the collected microorganisms. As the liquid, a liquid having characteristics such as a dielectric constant is selected depending on the microorganism to be detected as described above. For example, pure water, a buffer solution, an aqueous solution containing a compound such as mannitol at a predetermined concentration, and the like can be cited as candidates.

  The size of the collection chamber 116 is determined according to the amount of liquid to be processed, the processing time, and the collection speed. As an example, the plane size is 10 mm to 500 mm in width, 10 mm to 500 mm in depth, and 0.1 mm to 500 mm in height. The channel size is also determined according to the amount of liquid to be processed and the processing time. Each flow path may be different. As an example, the cross-sectional size of the flow path may be 0.1 mm to 10 mm in diameter with a circular cross section. The cross section of the flow path is not limited to a circle but may be an ellipse or a rectangle. Further, these sizes are not limited to the above sizes.

  The flow rate and circulation speed for taking in external air are set so that the microorganisms are efficiently concentrated according to the microorganisms to be detected. As an example, the flow rate for taking in external air is 0.01 liter / min to 100 liter / min, and the circulation rate is 1 ml / hour to 100 ml / min.

  The collection chamber filling unit 201 according to the modification has a configuration capable of filling one or more collection chambers 116. FIG. 13 is a diagram illustrating a specific example of the configuration of the collection chamber filling unit 201 that fills one collection chamber 116. Referring to FIG. 13, the collection chamber filling unit 201 includes flow paths 102 and 204 having joints 207 and 206, respectively. The joints 207 and 206 are connected to the joints 150 and 151 of the collection chamber 116, respectively. By connecting these joints, the flow paths 102 and 204 of the collection chamber filling unit 201 are connected to the collection chamber 116.

  The channel 102 is further connected to the concentration unit 202. As a result, the discharge hole 117 of the collection chamber 116 is connected to the concentrating unit 202 through the flow path 102 via the joints 150 and 207.

  The flow path 204 is further connected to a liquid reservoir 205. Thereby, the collection chamber 116 is connected to the liquid reservoir 205 through the flow path 204 via the joints 151 and 206. The liquid reservoir 205 is further connected to the flow path 4. The liquid reservoir 205 holds the same liquid as that supplied to the collection chamber 116 in advance.

  As will be described later, the concentrating unit 202 is provided with an air vent mechanism for filling the collecting chamber 116 and filling the collecting chamber 116 and the concentrating unit 202 with a liquid at an appropriate position.

  FIG. 14 is a diagram illustrating a specific example of the configuration of the concentration unit 202 and the measurement unit 203 in the detection apparatus 200. The configuration of the concentration unit 202 shown in FIG. 14 includes an opening 9 for taking in external air included in the concentration mechanism shown in the detection device 100 shown in FIG. 1, a filter 10, flow paths 18 and 19, and Instead of the pump 6, a liquid injection flow channel 102 connected to the collection chamber filling unit 201 is provided. Further, the configuration represented as the measurement system 1 in FIG. In addition, the thing with the same function as the detection apparatus 100 shown by FIG. 1 attaches | subjects the same code | symbol.

  Specifically, referring to FIG. 14, the measurement unit 203 includes a measurement cell 3, a filling pump 8, the same as the configuration of the measurement system 1 downstream from the valve 22 b of the detection device 100 shown in FIG. 1. A flow path connecting the measurement cell 3 and the pump 8, a linear light source 32, an optical filter 33, and a light receiving element 34 are included. The chamber 16 is connected to the collection chamber filling unit 201 through the flow path 102. As shown in FIG. 13, when the collection chamber filling unit 201 is filled with the collection chamber 116, the chamber 16 is connected to the collection chamber 116 through the discharge hole 117 and the joints 150 and 207.

Hereinafter, a method for detecting microorganisms using the detection apparatus 200 will be described.
First, the collection chamber 116 in which the liquid is supplied is mounted in the collection device 130, and the pump 106 is driven for a predetermined time. As a result, external air is taken into the liquid in the collection chamber 116 through the opening 109 and the filter 110. The taken-in air is bubbled in the liquid, so that microorganisms in the air are taken into the liquid. Large dust, pollen and the like are removed by the filter 110.

  After the collection operation for a predetermined time, the collection chamber 116 is separated from the flow path 118 and the flow path 119. At this time, it is possible to prevent the liquid in the collection chamber 116 from leaking by closing the joints 151 and 152. The joints 151 and 152 may be configured to be automatically closed when the collection chamber 116 is separated from the flow path 118 and the flow path 119.

  Next, the joints 151 and 150 of the collection chamber 116 holding the liquid in which the microorganisms in the air are taken in are connected to the joints 206 and 207 of the collection chamber filling unit 201, respectively. Thereby, the collection chamber 116 is filled in the collection chamber filling unit 201.

  In this state, the valve 22a is opened, and the pump 7 of the concentration unit 202 is driven. As a result, the liquid in the liquid reservoir 205 is injected into the collection chamber 116, and the collection chamber 116 and the concentration unit 202 are filled with the liquid.

  Thereafter, a voltage having a predetermined frequency is applied to the electrode 14 for dielectrophoresis while performing the above-described circulation operation for a predetermined time. Thereby, the microorganisms in the liquid in the chamber 16 are concentrated near the electrode 14 by the dielectrophoresis method. As described above, the chamber 16 of the concentrating unit 202 has a structure in which the circulating fluid collides with the electrode surface or flows in contact therewith. Therefore, it is possible to concentrate microorganisms near the electrode more efficiently than the conventional measuring apparatus. As a result, there is an advantage that the concentration time can be shortened and the measurement time can be shortened.

  After the circulation is completed, the valve 22c is opened for a predetermined time while a voltage having a predetermined frequency is applied to the electrode 14. As a result, a predetermined amount of the liquid in the chamber 16 is discharged, and the liquid level is close to the surface of the electrode 14.

  Thereafter, the valve 22c is closed to stop the driving of the pump 26, and the application of the AC voltage to the electrode 14 is stopped. Thereby, microorganisms disperse in the liquid remaining in the chamber 16. This increases the concentration of microorganisms in the liquid.

  Thereafter, the valve 22b is opened and the pump 8 is driven. Thereby, the liquid remaining in the chamber 16 in which the microorganisms are dispersed moves to the measurement cell 3. The measurement cell 3 to which the liquid has moved is irradiated with light having a wavelength of ultraviolet to blue from a linear light source 32, and fluorescence emission from the end face is measured. Thereby, the quantity of the microorganisms in the measurement cell 3 can be measured. This operation is the same as the measurement using the detection apparatus 100.

  FIG. 15 is a diagram illustrating a specific example of how the detection apparatus 200 is used. That is, as shown in FIG. 15, a large number of collection devices 130 having a simple configuration are arranged in a space (room) to be detected, and microorganisms in the air are collected and detected by the detection device 200. By the above method, there is an advantage that the amount of microorganisms in each part of the room can be measured, and in the second and subsequent times, collection, concentration and measurement can be performed in parallel, and the time change of the amount of microorganisms in each part of the room There is an advantage that can be obtained efficiently in almost real time.

  In such a case, the collection chamber filling unit 201 of the detection apparatus 200 is filled with a plurality of collection chambers 116 used for collecting microorganisms in each part of the room. The liquid in each collection chamber 116 circulates independently, and microorganisms are concentrated in the vicinity of the electrode of each concentration unit 202. Next, if the measurement is sequentially performed on the liquid in each concentration unit 202, the amount of microorganisms in each part in the room can be determined, and the distribution of microorganisms in the room can be determined. Moreover, the total amount of microorganisms of the whole room can also be obtained by mixing and measuring the liquid of each concentration part 202. FIG. In this method, even if the amount of microorganisms per part is small, the microorganisms collected by the plurality of collection chambers 116 are added together, so that the total amount of microorganisms is increased and the collection time can be shortened. Arise. In addition, by repeating the above method, it is possible to obtain information on temporal changes in the distribution of microorganisms in the room and temporal changes in the total amount of microorganisms in the entire room.

  In the above description, microorganisms in the air are detected using the detection devices 100 and 200. However, the present invention is not limited to air, and microorganisms floating in a fluid such as a liquid can be similarly detected.

  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.

  1 measurement system, 2 circulation mechanism, 3 measurement cell, 4, 5, 18, 19, 21, 102, 118, 119, 204 flow path, 6, 7, 8, 26, 106 pump, 9,109 opening, 10 , 12, 110 Filter, 11 Collection and concentration unit, 13 Optical system, 14 Electrode, 15 Base, 16 Chamber, 17, 24 Discharge hole, 20 Through hole, 22a, 22b, 22c Valve, 23 Injection hole, 25 Drainage reservoir , 27 lens group, 30 guide, 31 wall, 32 light source, 33 optical filter, 34 light receiving element, 35, 36 electrode terminal, 37 microorganism, 100, 200 detector, 116 collection chamber, 117 discharge hole, 130 collection Apparatus, 150, 151, 152, 206, 207 joint, 201 collection chamber filling unit, 202 concentrating unit, 203 measuring unit, 205 liquid reservoir.

Claims (13)

A mechanism for collecting biological particles,
A chamber for holding liquid;
An electrode provided in the chamber for collecting the biological particles in the liquid by dielectrophoresis by applying a voltage of a predetermined frequency;
A discharge mechanism for discharging the liquid in the chamber;
A collection mechanism for collecting and concentrating the concentration of the biological particles by discharging a predetermined amount of the liquid in the chamber in a state where the biological particles are collected by dielectrophoresis on the electrode.   The collection mechanism according to claim 1, further comprising a circulation mechanism for circulating the liquid in the chamber.   The collection mechanism according to claim 1 or 2, wherein a substrate having a through-hole for allowing the liquid to pass therethrough is provided in the chamber.   The collection mechanism according to claim 1, wherein a guide for guiding the liquid to the electrode is provided in the chamber.   5. The apparatus according to claim 1, further comprising an uptake mechanism for taking up the biogenic particles in the fluid into the liquid by taking the fluid containing the biogenic particles into the chamber. The collection mechanism described. The collection mechanism according to any one of claims 1 to 5,
Measuring means for measuring the amount of the particles derived from the organism collected by the collection mechanism,
A detection apparatus further comprising a moving mechanism for moving the biological particles collected by the collecting mechanism to a cell for measurement by the measuring means. A method of collecting biological particles,
Circulating a liquid containing biological particles in the chamber;
Applying a voltage of a predetermined frequency to an electrode provided in the chamber, and collecting the biological particles in the liquid on the electrode by dielectrophoresis;
Discharging a predetermined amount of the liquid from the chamber;
A step of separating the collected biological particles from the electrode by stopping application to the electrode and dispersing the particles in the liquid remaining in the chamber. A method of collecting biological particles,
Circulating a liquid containing biological particles in the chamber;
Applying a voltage of a predetermined frequency to an electrode provided in the chamber, and collecting the biological particles in the liquid on the electrode by dielectrophoresis;
Supplying a predetermined amount of new solution to the chamber after draining the liquid from the chamber;
A step of separating the collected biological particles from the electrode by stopping application to the electrode and dispersing the particles in the new liquid supplied to the chamber. Incorporating the biological particles in the fluid into the liquid by incorporating fluid containing the biological particles into the chamber;
The collection method according to claim 7 or 8, wherein the step of taking the biological particles into the liquid is performed before the step of circulating the liquid in the chamber.   The step of circulating the liquid in the chamber includes the step of taking the biological particles in the fluid into the liquid by taking the fluid containing the biological particles into the chamber. Or the collection method of 8. A method for detecting the organism-derived particles collected by the collection method according to claim 7,
A detection method comprising detecting the biological particle based on imaging of the electrode from which the biological particle is collected. A method for detecting the organism-derived particles collected by the collection method according to claim 7,
A detection method comprising detecting the biological particles based on the intensity of scattered light from a liquid in which the biological particles collected by the electrode are dispersed. A method for detecting the organism-derived particles collected by the collection method according to claim 7,
A detection method comprising the step of detecting the biological particle based on fluorescence intensity of the liquid in which the biological particle collected by the electrode is dispersed under excitation light irradiation.

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