JP2008096157A - Fine particle detector - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a fine particle detector for rapidly separating and detecting various kinds of fine particles, at high sensitivity. <P>SOLUTION: The fine particle detector is constituted so as to subject a sample, containing a plurality of kinds of fine particles to capillary electrophoresis, by using a migration liquid to detect the fine particles and equipped with an exciting light emitter for irradiating the fine particles, which are subjected to fluorescence dyeing so as to be selectively bonded, corresponding to the kind of the fine particles and a fluorescence detector for detecting fluorescence, having a plurality of wavelengths that are different, according to the kind of the fine particles and emitted from the fine particles subjected to fluorescence dyeing by using an optical system. A plurality of kinds of filters, permitting only a wavelength region to be detected to transmit, are installed, and a plurality of fluorescences are passed through the respective filters, by replacing the respective filters to be detected with respect to time, by the fluorescence detector. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a microparticle detection apparatus for detecting various microparticles such as cells and the like present in a specimen sample using microtubule electrophoresis. The present invention relates to a fine particle detection apparatus capable of separating and detecting with sensitivity.

In general, in order to investigate whether or not desired microparticles are present in a sample, or to examine the type and amount of microparticles such as microorganisms contained in a sample, or the distinction between live and dead microbes, these are temporarily used. It is necessary to grow microparticles that are target microorganisms by inoculating the sample in a suitable medium and culturing.
For this reason, conventionally, detection of microorganisms requires a long time of at least several days for culturing, and there has been a problem that results cannot be obtained quickly.
In addition, since microorganism culture is basically performed aseptically, there is a problem that special culture techniques and facilities are required.
On the other hand, there is a method for identifying the type of microorganism from genetic information, but there is a problem that the number of microorganisms and the distinction between live and dead bacteria cannot be identified.

For these reasons, there is a demand for a method that can quickly and easily detect the types and properties of harmful microorganisms that can be contained in a specimen sample without the need for culturing microorganisms and advanced techniques and devices.
For the detection of microorganisms classified into many types, conventionally, after culturing in selective separation culture, it is necessary to catch suspicious colonies and determine with confirmation medium, identification kit or antiserum. It took a lot of experience and skill to identify. Moreover, when time for culture | cultivation etc. was match | combined, the determination result required the long term of 3 to 5 days or more.
For this reason, in the environment / food and medical fields, establishment of a quick and safe method for specifically detecting microorganisms, particularly harmful microorganisms, for each species and strain is required.

By the way, in recent years, as a rapid method for separating and detecting microorganisms, an electrophoresis technique has been proposed in which microorganisms that have been pre-fluorescently stained in a microtubule are electrophoresed and irradiated with excitation light to detect the fluorescence generated at that time ( For example, see Patent Document 1).
Further, although there is a paper on the fine tube isoelectric focusing technique, details of specific detection means are not described (for example, see Non-Patent Document 1).
On the other hand, when migrating and separating microorganisms in a microtubule, the interaction between the microbial cells and the inner wall of the tubule or between the cells and the cells is large. It is effective to contain an alginate (see, for example, Patent Document 2).
In this method, alginate as an additive that improves the separation efficiency of microorganisms does not function in the same manner for all microorganisms, and more types of separation efficiency improvers that can handle a wide variety of microorganism species. The introduction is expected.

By the way, in order to quickly take appropriate measures against environmental water, food contamination and infectious diseases, and to quickly identify and deal with the contamination sources and infection sources, it is important There is a need for rapid and specific detection methods.
In addition, it is important to confirm whether or not all harmful microorganisms have been killed when such microorganisms are subjected to a treatment such as chlorine sterilization.
In addition, when it is difficult to kill all harmful bacteria, it is necessary to know the status of chlorine sterilization and the sterilization efficiency by checking the number of live and dead bacteria after sterilization.
As a method for detecting these multiple types of microorganisms, an example has been proposed in which only light of a desired wavelength is separated and analyzed using a plurality of reflection mirrors. Since a mirror is used, there is a problem that the amount of light to be detected is attenuated (see Patent Document 3).
JP 2002-345451 A JP 2002-181781 A US Pat. No. 5,982,497 Microbial electrophoresis separation and detection method (Monthly Food Chemical 2005-2)

  In view of the problems of the conventional fine particle detection apparatus, the present invention is capable of separating and detecting a plurality of types of microorganisms, the number of living microorganisms of the target microorganism, the number of dead bacteria, and the like quickly and with high sensitivity. An object is to provide a detection device.

  In order to achieve the above object, the microparticle detection apparatus of the present invention treats a sample with fluorescent staining that selectively binds depending on the type of microparticle, and performs microtube electrophoresis of the sample using an electrophoresis solution. In a fine particle detection apparatus for detecting fine particles, an excitation light emitter that irradiates fluorescence-stained fine particles with excitation light, a fluorescence detector that detects fluorescence emitted by the excitation light irradiation, and a plurality of light that transmits only a specific wavelength region It is characterized by comprising a filter of a type and a filter switching device that sequentially switches and arranges each filter in front of the fluorescence detector, and the fluorescence of the fine particles that have passed through each filter is sequentially detected by the fluorescence detector. .

  In this case, the apparatus includes a particulate detection control device that detects the fluorescence signal obtained from the fluorescence detector in synchronization with the switching timing of each filter, and a signal recording device that records the detection signal of the particulate detection control device. Can do.

  Further, the fluorescence detector can be an image detection sensor.

  Further, the particle detection signal can be the luminance of the image, the area of the image, or the number of particles.

  According to the fine particle detection apparatus of the present invention, an excitation light emitter that irradiates fluorescent light-stained fine particles with excitation light, a fluorescence detector that detects fluorescence emitted by the excitation light irradiation, and a specific wavelength region are transmitted. It is equipped with a plurality of types of filters and a filter switching device that sequentially switches and arranges each filter in front of the fluorescence detector, and the fluorescence detector sequentially detects the fluorescence of the particles passing through each filter. Depending on the signal from the fluorescence detector, when the fluorescence of different wavelengths is emitted or when the same kind of fine particles emit fluorescence of different wavelengths by distinguishing between live and dead bacteria, Fine particles according to the detection wavelength range can be detected, which allows the type and characteristics of various fine particles contained in the sample to be detected without the need for special techniques. In addition to separation with high reproducibility and reproducibility, the target microparticles are fluorescently stained in advance so that even minute amounts of microparticles can be detected with high sensitivity without culturing or other growth treatment, and with higher resolution. Desired fine particles can be detected with high accuracy, and furthermore, the fine particles mixed in the sample can be detected not only simply, but also can be quantitatively detected separately for each type of fine particles and for each characteristic.

  In this case, by including a particulate detection control device that detects the fluorescence signal obtained from the fluorescence detector in synchronization with the switching timing of each filter, and a signal recording device that records the detection signal of the particulate detection control device. The plurality of types of fluorescence can be detected almost simultaneously with the plurality of types of filters, and the processing results can be displayed at the same time or stored and recorded in a memory such as a personal computer.

  Further, by using the fluorescence detector as an image sensor, the signal recording apparatus can record as an image signal, and the particle detection control apparatus can use an image processing technique. In addition, when a large amount of image signals are used, a large amount of images can be recorded and reproduced by configuring the signal recording device with a DVD or the like.

  Further, by using the fine particle detection signal as the luminance of the image, the area of the image, or the number of fine particles, the detailed data of the detection result of the fine particles becomes clear.

  Hereinafter, embodiments of the particulate detection device of the present invention will be described with reference to the drawings.

The microparticle detection apparatus of the present invention separates a plurality of types of microparticles such as microorganisms included in a sample with high resolution and high reproducibility by utilizing microtubule electrophoresis using an electrophoresis solution.
In addition, in this microtubule electrophoresis, by using a fluorescence detector as a detection means, the target microparticles are preliminarily fluorescently stained, so that even a minute amount of microparticles can be highly sensitive without being subjected to growth treatment or the like. Therefore, the desired fine particles can be detected with high accuracy with an even higher resolution.
Furthermore, the fine particles mixed in the sample can be detected not only simply but also quantitatively.
The detection in the present invention includes the concentration of desired fine particles contained in a sample, separation from others, qualitative detection, quantitative detection, screening, life / death discrimination, and the like.

Specifically, for example, it is a fine particle detection device having the following part or all as constituent elements.
(1) A sample containing fine particles is subjected to microtubule electrophoresis using an electrophoresis solution.
(2) A sample containing fine particles is fluorescently stained so as to selectively bind to the fine particles, and this is subjected to microtubule electrophoresis to detect fluorescence of the fluorescently stained fine particles.
(3) A device for detecting microparticles using microtubule electrophoresis, a water tank containing an electrophoretic solution, a microtubule for concentrating and separating microparticles injected into the electrophoretic solution, and a detection for detecting the concentrated microparticles Means.
(4) An apparatus for detecting microparticles using microtubule electrophoresis, wherein the microparticles are concentrated by a pH gradient provided in the microtube.
(5) A fluorescence detector is provided as a detection means. The fluorescent dyed fine particles are irradiated with excitation light, and fluorescence emitted from the fluorescently stained fine particles is detected using an optical system. Is detected.
(6) A plurality of types of filters that transmit only a specific wavelength region, and a filter switching device that sequentially switches and arranges each filter in front of the fluorescence detector, and by switching each filter in time, A plurality of fluorescences are detected by a fluorescence detector after passing through each filter.
(7) Further, a signal recording device for recording a detection signal detected by fluorescence with an optical system and a particle detection control device for recording a particle detection signal for detecting particles from the detection signal are provided.
(8) Furthermore, an image sensor is used as a fluorescence detector, and a fluorescence image is detected as a particulate detection signal. The number of fine particles detected by characterization.

FIG. 1 shows a basic configuration of a microtubule isoelectric focusing system of the present invention as an embodiment.
In FIG. 1, 1 is an excitation light emitter such as a diode, 2 is a microtube, 3 is its detection window, 4a and 4b are electrodes, 5a and 5b are solutions, 6 is fine particles contained in the solution, and 7 is excitation light. Excitation light emitted from a light emitter, 8 is an objective lens, 9 is fluorescence emitted from fine particles, and 10 is a dichroic filter.
Also, 11a and 11b are two types of filters that allow only specific wavelengths to pass, 12 is a fluorescence detector such as a CCD camera, 13 is a particulate detection control device, and 14 is a filter 11a and 11b in front of the fluorescence detector 12. A filter switching device that is sequentially switched, 15 is a display device, and 16 is a signal recording device.

Microorganisms such as harmful bacteria that are detection objects, for example, microparticles such as microorganisms harmful to the human body such as Escherichia coli and Legionella, are previously fluorescently stained so as to selectively bind only to the microorganisms, for example, capillary Is injected from the solution 5a or the solution 5b installed at both ends thereof.
The solution 5a or the solution 5b is changed according to the detection stage, and a high voltage power source is applied to both ends of the microtube 2 by the electrodes 4a and 4b.
The high voltage is supplied from a power source (not shown), and the magnitude and application time of the voltage are controlled by the particulate detection control device 13 such as a personal computer, for example.

The excitation light 7 emitted from the excitation light emitter 1 is reflected by the dichroic filter 10 and is irradiated from the objective lens 8 to the fluorescently stained fine particles from the detection window 3 of the fine tube.
The fluorescently stained fine particles emit fluorescent light 9, enter the apparatus main body through the objective lens 8, and pass through the dichroic filter 10.
At this time, the fluorescence 9 emitted from the fluorescently stained fine particles has various wavelengths based on the characteristics of the fluorescent staining combined according to the type of the fine particles. For example, when a microbe fluorescently stained with a LIVE / DEAD reagent (manufactured by Invitrogen) is irradiated with blue excitation light, green fluorescence is emitted in the case of live bacteria, and red fluorescence is emitted in the case of dead bacteria. .
After passing through the filter 11a or the filter 11b, the fluorescence 9 is imaged on a fluorescence detector 12 composed of, for example, a CCD camera, and the image signal is sent to the particle detection control device 13 for detection processing. The
This detected image signal is recorded in the signal recording device 16.
The fluorescently stained fine particles are concentrated by a method described later and passed through the detection window 3. The particulate detection operation can be performed during the passage.
The fluorescence detector 12 may be a photoelectric tube, and the fluorescence signal that has passed through the filters 11a and 11b may be converted into an electrical signal and sent to the particle detection control device 13 for detection processing.

FIG. 2 is a diagram for explaining the switching procedure between the first filter 11a and the second filter 11b. The filter switching device 14 arranges the filters 11a and 11b at, for example, substantially right angles, and uses a motor. A reciprocating rotation (oscillation) 17 is performed.
At this time, the fluorescence 9 passes through the filter 11a, the filter 11b, or both the filters 11a and 11b, and forms an image on the fluorescence detector 12. Therefore, if each filter 11a, 11b is configured to have a combination of characteristics that allows only the fluorescence having a desired wavelength to pass therethrough, only the fluorescence having the desired characteristic can be selectively acquired.
Since the image signal imaged on the fluorescence detector 12 passes through one or more filters, image correction may be performed when there is image distortion due to light refraction or the like.

In the particulate detection control device 13, an image signal is detected in synchronization with the rotation signal of the motor of the filter switching device 14.
That is, image processing can be performed in synchronization with the timing using only the filter 11a, the timing using both the filter 11a and the filter 11b, and the timing using only the filter 11b, respectively. Details of the processing result will be described later.

FIG. 3 shows a migration state of microorganisms that are harmful bacteria in the microtubule 2 as an example of fine particles, and the following detection process is performed.
First, a test solution, which is a sample containing microorganisms as detection targets, is prepared as, for example, a solution 5a and sucked from the solution 5b side to be injected as a target test solution 20 into the microtube 2.
Next, an acidic solution such as phosphoric acid is used as the solution 5a, and an alkaline (basic) solution such as caustic soda is used as the solution 5b. The electrodes 4a and 4b are respectively connected to a high voltage power source (not shown). By the particulate detection control device 13, the electrode 4a (acidic solution 5a side) is an anode, and the electrode 4b (alkaline solution 5b side) is a cathode. Connect to apply voltage.
Here, both ends of the microtube 2 are immersed in the above acidic or alkaline solution (referred to as an electrophoretic solution), respectively, and then when a high-voltage power supply is operated, a potential gradient is generated in the sample solution 20 in the microtube 2. As a result, the entire liquid in the migration region (in the microtube) in the microtube generates a pH gradient between the cathode direction and the anode direction due to the isoelectric point phenomenon.

At this time, since the microorganisms 21 that may exist in the test solution 20 are diffused to different pH regions, they migrate toward the region that does not have the charge, that is, the isoelectric point position 24 or 25. .
That is, the microorganism 21 moves to a region (the isoelectric point position 24 or the isoelectric point position 25) having the same pH condition as the isoelectric point it has.
For example, assuming that the pH range of a microorganism having an isoelectric point pH of pH (A) is the isoelectric point position 24, the microorganism is moved from the isoelectric point position 24 to the electrode (cathode) 4b side by the formed pH gradient. When it exists, it moves in the moving direction 22, and when it exists on the electrode (anode) 4 a side from the isoelectric point position 24, it moves in the moving direction 23 and is concentrated at the isoelectric point position 24.
Similarly, if the pH range of a microorganism having an isoelectric point pH of pH (B) is the isoelectric point position 25, the microorganism is moved from the isoelectric point position 25 to the electrode (cathode) 4b side by the formed pH gradient. Is present in the moving direction 22, and when present on the electrode (anode) 4 a side from the isoelectric point position 25, it moves in the moving direction 23 and is concentrated at the isoelectric point position 25.
That is, the microorganisms are accumulated at the isoelectric point position 24 or the isoelectric point position 25 in the microtube 2 by the isoelectric point that the microorganisms have, thereby forming a concentrated microbial mass 24a or microbial mass 25a. The isoelectric point position changes depending on the type of the target microorganism, the live bacteria, and the dead bacteria. In general, since the surface charge component of the microorganism has a specific isoelectric point, the microorganism is concentrated at the specific electric points 24, 25, etc., which are the specific positions, by the pH gradient in the microtube.
In this state, the electrophoretic current between the electrodes 4a and 4b is in a steady state and hardly flows.

Next, as shown in FIG. 4, when the solution 5a is an acidic solution as it is and a voltage of an anode is applied to the electrode 4a and a cathode is applied to the electrode 4b using a salt (mobilizer), for example, The microbial mass 24a or the microbial mass 25a concentrated at the isoelectric point position 24 or the isoelectric point position 25 in FIG. 3 is directed toward the electrode (cathode) 4b, for example, in the form of flowing in a mobilizer due to the potential gradient generated again. Move in the moving direction 26.
At this time, the microbial masses 24 a and 25 a pass through the detection window 3. This microbial mass is fluorescently stained, and as shown in FIG. 1, when excitation light is irradiated from the objective lens 8, it emits a fluorescent color having a wavelength corresponding to the characteristic of the fluorescent staining.

The method of injecting the specimen sample into the microtube 2 is not particularly limited, and any of the conventionally used gravity method, pressurization method, and decompression method can be used.
Although the injection amount is not particularly limited, it depends on the size of the microtubule in order to fill the sample in the entire microtubule to be used, and it is 0.1 to 100 μL, preferably 0.5 to 100 μL, more preferably 0.5 to 10 μL. Can be illustrated.

In addition, the pair of electrodes 4a and 4b and the high-voltage power source exemplified above, the potential gradient of the intensity necessary for the microbe injected into the microtube to migrate in the electrophoretic solution in the microtube, It is a means for applying with respect to a liquid.
Any means (other means for applying a potential gradient) can be used without being limited to these (power supply and a pair of electrodes) as long as the object can be achieved.

The voltage applied between the electrodes is generally about 1 kV / m to about 500 kV / m, preferably about 2 kV / m to about 100 kV / m, more preferably about 5 kV / m to about 20 kV / m, relative to the length of the microtube. Can be mentioned.
Examples of the fine tube include a hollow tube having an inner diameter of 1 to 150 μm and a length of 0.1 to 100 cm, preferably an inner diameter of 20 to 100 μm and a length of 1 to 50 cm. The material is not particularly limited, and glass (fused silica) or the like can be arbitrarily selected.

FIG. 5 shows, for example, an enlarged view of the microorganism 21 to be detected here, and shows an antibody 30 that selectively binds to the outer wall 21a, a fluorescent dye 31 that is fluorescently labeled with the antibody 30, and the like.
Although not shown, in addition to the antibody and fluorescent dye 31 bound to the microorganism 21 to be detected, fluorescence is also emitted from the antibody and fluorescent dye floating in the solution without being bound to the microorganism. Since the position is different from the microorganism to be detected, it is possible to separate them by observing the passage of time.

FIG. 6 is a flowchart showing the detection process of the fine particles such as the target microorganism described above.
First, a sample solution is injected into a microtube (step 32), and a fine particle is subjected to isoelectric focusing by providing a pH gradient to form a fine particle mass (concentration) (step 33).
Next, the fine particle lump is migrated in a fine tube to search for the fine particle lump (step 34), and the fine particle is detected by a CCD camera or the like through a filter (step 35).
In the above process, the target fine particles are detected.

FIG. 7 is a diagram showing a fine particle image processing display example in the fine particle detection control device 13 such as a personal computer.
Main keys include a start key 40, a stop key 41, an end key 42, and the like.
When the particle detection operation is started by clicking the start key 40, the status display 43 is being detected, the elapsed time is displayed in the comment column 44, and whether or not there is an abnormality (normal here) is displayed in the comment column 45. .
Click the stop key 41 to stop the detection operation in the middle of detection, and click the end key 42 to end the detection operation.

When the fluorescence 9 has passed through the filter 11a, an image obtained in synchronization therewith is shown as a region 46 on the left side of the screen, and when the fluorescence 9 has passed through both the filter 11a and the filter 11b, it has been obtained in synchronization therewith. The image is shown as a region 47 in the center of the screen, and when the fluorescence 9 passes through the filter 11b, the image obtained in synchronism with it is shown as a region 48 on the right side of the screen.
Here, 51a and 51b indicate the wall of the fine tube, and when the fluorescently stained fine particles are detected, the fine particles 52, 53 and 54 are displayed on the image.
Image tracking is performed from the brightness, shape, area, spatial position, direction of movement, and the like of these detected images, and a unique number is assigned to the detected fine particles, and the number is counted.
In this embodiment, three fine particles 52 are detected in the region 46, two fine particles 53 are detected in the region 47, and four fine particles 54 are detected in the region 48.
From the detection position and detection time of these fine particles, it is possible to determine which fine particles emit what kind of fluorescence.

The image brightness, the area, and the like of the detected fine particles vary depending on the intensity of the excitation light 7 and fluorescence 9, the type of target fine particles, the filter characteristics, and the like.
Therefore, the level adjustment of the image luminance value detected by the image processing is changed by clicking on the luminance value key 60 (▽ is black in the figure. The same applies hereinafter).
Further, the adjustment of the image area to be detected is changed by clicking ▽ of the area key 61.
Such fine particle detection technology and image tracking technology by image processing are existing technologies and can be easily realized.

Furthermore, in this embodiment, the total luminance value 62 detected in the region 46 (passed through the first filter 11a), the total area 63 of the detected fine particles, the instantaneous value 64 of the number of detected fine particles, and detection after the detection operation is started. The sum total 65 of the detected fine particles, the total brightness 66 detected in the region 48 (passed through the second filter 11b), the total sum 67 of the detected fine particles, the instantaneous value 68 of the number of detected fine particles, and the detection operation The sum total 69 of the fine particles detected after the start is displayed.
Thereby, it can be confirmed that the image processing is operating normally. The obtained data is stored in a waveform control device such as a memory built in the particle detection control device 13.
Here, although the detailed data of only the areas 46 and 48 is displayed, the area 47 can be displayed together, or any area can be configured not to be displayed.

FIG. 8 shows another embodiment of the filter switching device and the filter.
In the present embodiment, a third filter 11c and a fourth filter 11d are further added to the first filter 11a and the second filter 11b.
The rotation signal of the motor of the filter switching device 14 is connected to the particulate detection control device 13 as in FIG. 1, and image processing is performed in synchronization with the image.
If comprised in this way, it will have a fluorescence detection function equivalent to eight types of filter configurations including the case where two filters are used simultaneously by four types of filters 11a-11d.

FIG. 9 shows another embodiment of the particle detection apparatus, and parts having the same functions as those in FIG. 1 are denoted by the same reference numerals.
Filters 80 a and 80 b arranged so as to be substantially orthogonal to the fluorescence 9 are switched by rotating the filters 80 a and 80 b by the motor of the filter switching device 81. Other operations are the same as those in FIG.

FIG. 10 is a plan view of the filter in FIG.
The filters 80a and 80b are arranged in a disc shape, each being a semicircle. In this case, there is no function to use both filters 80a and 80b at the same time.

FIG. 11 shows another embodiment of the filter switching device and the filter.
The filter unit configured by the filters 84a and 84b is reciprocated in the direction of the arrow 86 or the direction of the arrow 87 by the motor of the filter switching device 85 to switch the filter.
The other operations are the same as those in FIG. 1 or FIG. In this case, there is no function of using both filters 84a and 84b simultaneously.

Usually, since the time required for detection of fine particles ranges from several minutes to several tens of minutes, when using an image sensor for the fluorescence detector 12 and performing fluorescence image detection, the signal recording device 16 outputs many image signals. It is desirable to use a recordable DVD deck or the like.
Further, the fine particle detection control device 13 can sufficiently function on a normal personal computer, but since it is difficult to record images during the entire detection period, only the images before and after detecting the target fine particles are recorded, or image recording is a signal. Dedicated to the recording device 16, the fine particle detection control device 13 has only the image processing results as shown in the present embodiment, that is, only the image luminance, the number of fine particles, the size thereof, the electrophoresis parameters, etc. for the target fine particles. You may limit to.

Further, instead of the method of concentrating the fine particles by providing a pH gradient in the microtube, a potential gradient is generated in the microtube in the migration region, and an electroosmotic flow that is the flow of the entire liquid in the microtube is used by this potential gradient. Electrophoresis can be used. This electroosmotic flow is a flow from the anode toward the cathode.
The fine particles injected into the microtube move in the direction of the electrode having a polarity opposite to the surface charge of the fine particles in response to the generated potential gradient.
As a result, the migration direction (velocity from the cathode to the anode) and velocity of the fine particles in the microtube are determined by the electroosmotic flow velocity and the mobility of the fine particles, and are concentrated near the opposite end of the microtube (near the anode). The method can be changed as appropriate, such as a method for detecting fine particles by the method.

In addition, as a means for detecting fine particles concentrated and separated in a microtube, generally, any of spectroscopic, electrochemical, and weight detection methods can be used, but preferably UV-visible detection, fluorescence Examples thereof include optical detection methods such as detection, luminescence detection, and light scattering detection.
Among these, a fluorescence detection method excellent in detection specificity and sensitivity is preferable, and a laser-induced fluorescence detection method capable of detecting with higher sensitivity is desirable when the light quantity of the LED is insufficient. These fluorescence-based detections can be performed by combining reactions such as immune reactions (antigen-antibody reactions), intracellular enzyme reactions, and nucleic acid complementary chain formation reactions as appropriate according to the target microparticles. Can be detected.
Among these methods, the immune reaction is one of the preferred methods because it does not require special pretreatment of the microparticle sample and is excellent in specificity. As such an immune reaction, specifically, a method of labeling target fine particles with fluorescence, an enzyme, or the like using a fluorescent dye-labeled antibody (also simply referred to as a fluorescent antibody), an enzyme-labeled antibody (also simply referred to as an enzyme antibody), or the like. Can be mentioned.

Thus, the particle detector of the present embodiment includes an excitation light emitter 1 that irradiates fluorescence-stained particles with excitation light, a fluorescence detector 12 that detects fluorescence emitted by the excitation light irradiation, and a specific wavelength region. And a filter switching device 14 that sequentially switches and arranges each filter 11 in front of the fluorescence detector, and the fluorescence detector 12 sequentially detects the fluorescence of the fine particles that have passed through each filter 11. Therefore, fine particles (for example, microorganisms) in the specimen sample can be separated, detected, and quantified with high accuracy in a short time. That is, it becomes possible to isolate the microorganism species and strains individually. In addition, since it is fluorescently stained so as to selectively bind to the target microorganism, it can be detected easily and accurately in a short time without requiring a special technique and without a culture operation. Accurate microbial testing and monitoring system can be established.
Further, by detecting fine particles using an image processing technique, the image processing result and the fine particle detection image can be stored and recorded in a memory such as a personal computer. Therefore, by recalling the stored data, it is possible to reconfirm the detection result and report the detailed result to the sample solution supplier. In addition, it is possible to contribute to quick measures against microorganisms, measures against sterilization, and the like.
In addition, by using an inexpensive LED or laser-induced fluorescence detector or the like as the excitation light emitting means, it is possible to detect the desired fine particles with high sensitivity and specificity, so that a proliferation treatment such as culturing the sample is performed. Therefore, a minute amount of fine particles can be separated and detected with high accuracy.
In addition, since the fine particle detection apparatus of this example has quantitative properties, if used for microorganism detection, it is possible to determine and measure (quantitative detection) the number of microorganisms such as microorganisms present in a sample, It is possible not only to detect microorganisms in a sample, but also to identify bacterial species by using specific detection reagents (for example, antibodies) specific to various microorganisms.
As described above, since the particulate detection device of this embodiment is suitable for detection of harmful microorganisms (qualitative detection, quantitative detection), environmental water and food contaminated with pathogenic microorganisms can be eliminated at an early stage, and the occurrence of diseases. It is useful for prevention. In addition, since early diagnosis of patients with pathogenic microorganisms becomes possible, it helps prevent the spread of damage caused by harmful microorganisms and helps early treatment. Furthermore, the quality control period of the food can be shortened, and the shelf life period can be extended due to the strictness of hygiene management, which has the effect of greatly contributing to the economics of food distribution.

  In this case, a particle detection control device 13 that detects the fluorescence signal obtained from the fluorescence detector 12 in synchronization with the switching timing of each filter 11, and a signal recording device 16 that records the detection signal of the particle detection control device 13. , The plurality of types of fluorescence can be detected almost simultaneously by the plurality of types of filters 11, and the processing results can be simultaneously displayed or stored and recorded in a memory such as a personal computer.

  Further, by using the fluorescence detector 12 as an image sensor, the signal recording device 16 can record as an image signal, and the particle detection control device 13 can use an image processing technique.

  Further, by using the fine particle detection signal as the luminance of the image, the area of the image, or the number of fine particles, detailed data of the detection result of the fine particles can be clarified.

  As mentioned above, although the particulate detection device of the present invention has been described based on the embodiments thereof, the present invention is not limited to the configurations described in the embodiments, and the configuration is appropriately changed without departing from the gist thereof. can do.

  The fine particle detection device of the present invention has the property of separating and detecting various fine particles quickly and with high sensitivity. For example, environmental water such as bath water, pools, cooling water for cooling towers, food and medical fields For example, it can be suitably used for the purpose of accurately detecting harmful microorganisms mixed in a sample sample collected in the above.

It is a schematic block diagram which shows one Example of the microparticle detection apparatus of this invention. It is explanatory drawing which shows operation | movement of a filter. It is explanatory drawing which shows the detection process of the microparticles | fine-particles by microtube electrophoresis. It is explanatory drawing of the next step of the detection process of microparticles | fine-particles by microtube electrophoresis. It is an enlarged view showing details of fine particles. It is a flowchart which shows the detection operation of microparticles | fine-particles. It is a figure which shows the screen which displayed the image processing result in a microparticle detection control apparatus. It is explanatory drawing which shows the other Example of a filter and a filter switching apparatus. It is a schematic block diagram which shows the other Example of a microparticle detection apparatus. It is a top view of the filter of the Example. It is explanatory drawing which shows the further another Example of a filter and a filter switching apparatus.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Excitation light emitter 2 Fine tube 3 Detection window 4a Electrode (anode)
4b Electrode (cathode)
5a Solution 5b Solution 6 Fine Particle 7 Excitation Light 8 Objective Lens 9 Fluorescence 10 Dichroic Filter 11a Filter 11b Filter 11c Filter 11d Filter 12 Fluorescence Detector 13 Fine Particle Detection Control Device 14 Filter Switching Device 15 Display Device 16 Signal Recording Device 20 Analytical Solution 21 Fine Particle 24 isoelectric point position 25 isoelectric point position 30 antibody 31 fluorescent dye 40 start key 41 stop key 42 end key 43 status display 44 elapsed time 45 comment field 46 left image area 47 center image area 48 right image area 51a microtubule tube Wall 51b Tube wall of fine tube 52 Left region fine particle image 53 Center region fine particle image 54 Right region fine particle image 60 Luminance value adjustment key 61 Area adjustment key 62 Left region luminance total value 63 Left region fine particle area total 64 Left region fine particle number Instantaneous value 65 Sum of left area fine particles 66 Right area luminance total value 67 Area total area of right area fine particles 68 Instantaneous value of right area fine particle number 69 Sum of right area fine particles 80a Filter 80b Filter 81 Filter switching device 84a Filter 84b Filter 85 Filter switching device

Claims (4)

  The sample is treated with fluorescent staining that selectively binds depending on the type of fine particles, and the sample is subjected to microtubule electrophoresis using an electrophoresis solution. An excitation light emitter that emits excitation light, a fluorescence detector that detects fluorescence emitted by the excitation light irradiation, a plurality of types of filters that transmit only a specific wavelength region, and each filter sequentially in front of the fluorescence detector A particulate matter detection device comprising: a filter switching device arranged to be switched, wherein the fluorescence of the particulate matter that has passed through each filter is sequentially detected by a fluorescence detector.   A particulate detection control device that detects a fluorescence signal obtained from the fluorescence detector in synchronization with the switching timing of each filter, and a signal recording device that records a detection signal of the particulate detection control device. The fine particle detection apparatus according to claim 1.   The fine particle detection apparatus according to claim 1 or 2, wherein the fluorescence detector is an image detection sensor.   4. The particle detection apparatus according to claim 1, wherein the particle detection signal is an image brightness, an image area, or the number of particles.

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