JP4679197B2 - Microbial separator - Google Patents

Description

  The present invention simplifies specific microorganisms from sample water that may contain microorganisms in raw water quality management, purified water quality management, other water treatment water quality management, environmental water quality management, food sanitation management, aquaculture facility management, etc. The present invention relates to a microorganism separation apparatus for selectively capturing, separating and recovering using a simple apparatus.

  Microorganisms are detected in the inspection of purified water quality in waterworks, eutrophication of environmental water, food and product inspection, process control of food processing facilities, and water quality inspection of aquaculture and fishery facilities. In particular, the spread of pathogenic microorganisms through treated water and products can have a serious impact on human health and can be used as an indicator of the detection of pathogenic microorganisms or the occurrence of pathogenic microorganisms. There is a need for detection of microorganisms. In order to prevent the occurrence and spread of obstacles, it is necessary to detect microorganisms as quickly as possible.

  However, the conventional method for detecting microorganisms is mainly a method for observing colonies formed by culture. Although this method has the advantage of being easy to perform, it takes time until results are obtained, and further, it is difficult to operate. There was a problem of requiring skill.

  In addition, in the case of protozoa such as Cryptosporidium and Giardia, which are problematic in water purification plants, detection by culture as described above is impossible, and there are many cases from the pretreatment to detection by the current detection method. The process was complicated, and the operation required skill. Furthermore, in the case of protozoa, there is a possibility that even a small amount may cause a problem. Therefore, it is required to detect only the microorganism from a large amount of diluted sample water.

  In recent years, methods for applying molecular biological techniques such as genes, DNA, and immunity to detection and identification of these microorganisms have been proposed. In these molecular biological techniques, it is possible to selectively identify a base sequence and an antigen unique to the target microorganism, and to specifically detect the target microorganism.

  However, DNA fragments and antibodies used in these methods may adsorb nonspecifically to microorganisms other than the target microorganisms, or contaminants such as turbidity of minerals, which may cause misrecognition. Become. In practice, microorganisms do not exist in a pure state, but coexist with many other microorganisms and suspended solids, and it is difficult to completely eliminate misrecognition.

  In general, when detecting organism DNA, the cells are disrupted and DNA or RNA is removed. In some cases, these are amplified by PCR or RT-PCR, and then the target DNA and the complementary probe are bound. By detecting this, it is confirmed that the target organism exists. However, there is a possibility of false positives in this case as well, but the structure of the microorganism has already been destroyed, and it is impossible to verify it from information such as the shape factor and internal structure.

In view of these circumstances, a separation and recovery system using a hollow fiber membrane (see, for example, Patent Document 1), a double label detection method using an antibody and DNA on an immobilized plate (for example, see Patent Document 2). A method for detecting underwater and its oocysts is disclosed (for example, see Patent Document 3). By the technology described in these patent documents, the target microorganism is continuously and efficiently produced in a large amount by attaching a multi-stage separation process using a hollow fiber membrane filter and the like and a process for improving the recovery rate. In addition, the microorganisms immobilized on the plate are double-stained with antibody and DNA, and the shape factor of the cells is added to the discriminating factor to improve accuracy. Can be selectively detected.
JP 10-314552 A Japanese Patent No. 3127244 Japanese Patent Laid-Open No. 11-193

  Conventionally, as an immobilization plate used for detecting microorganisms, a disk-like filter such as a nitrocellulose membrane filter has been assumed, and since the area of the detection unit is relatively large, image processing is performed for detection. In application, a stage scanning mechanism for moving the microscope field of view or a scanning mechanism for the excitation laser light is required, which increases the size of the apparatus, increases the cost, and delays in response due to the time required for scanning. There was such a problem.

  An object of the present invention was made to solve the above-described problems, and provides a microorganism separation apparatus that can selectively capture, separate, and recover microorganisms to be detected in a micro area. There is.

In order to achieve the above object, the invention according to claim 1 is a microorganism separation apparatus that captures, separates and recovers specific microorganisms contained in sample water, wherein a flow path of sample water is formed on a substrate, A microchip having a capturing part in which a plurality of microorganism capturing holes for capturing separation target microorganisms are disposed in the middle of the path, and sample water supply means for flowing sample water containing the separation target microorganisms into the microchip And a sample water circulation means for collecting the waste water that has passed through the microchip and returning it to the sample water supply means, and when the microorganisms to be separated contained in the sample water come into contact with the plurality of microorganism capture holes, The sample water circulation operation by the sample water supply means and the sample water circulation means is repeated a plurality of times so that the number of microorganisms to be separated captured by the sample water increases .

The invention according to claim 2 is the microorganism separation apparatus according to claim 1, characterized in that a physical cleaning means is attached to the microchip.

According to a third aspect of the present invention, in the microorganism separation apparatus according to the first or second aspect of the present invention, a cleaning liquid supply means for pouring the cleaning liquid into the microchip is provided.

According to a fourth aspect of the present invention, in the microorganism separation apparatus according to any one of the first to third aspects, the microchip includes a shielding plate that covers the surface of the substrate, the substrate is made of a conductive material, and the microorganism A flat plate portion other than the catching hole is electrically insulated by the insulating layer, and an electrode is embedded through the insulating layer in a portion facing the catching portion of the shielding plate, and a voltage is applied between the electrode and the substrate. Thus, a voltage application device for generating an electric field in the flow path of the sample water is provided.

The invention according to claim 5 is the microorganism separation apparatus according to any one of claims 1 to 3, wherein the microchip includes a shielding plate that covers the surface of the substrate, the substrate is made of a non-conductive material, and captures microorganisms. A first electrode is provided inside the hole, and a second electrode is embedded via an insulating layer in a portion facing the capturing portion of the shielding plate, and a voltage is applied between the first and second electrodes. And a voltage applying device for generating an electric field in the flow path of the sample water.

The invention according to claim 6 is the microorganism separation device according to any one of claims 1 to 5, comprising an antibody that is immobilized in the microorganism capturing hole and recognizes the target microorganism as an antigen. .

The invention according to claim 7 is the microorganism separation device according to any one of claims 1 to 3 , wherein the projection is provided on the flow path surface of the shielding plate so as to create a flow of the sample water in a direction toward the microorganism capturing hole. It is provided.

The invention according to claim 8 is the biological material labeled with a fluorescent dye or a luminescent dye capable of selectively identifying and adsorbing the target microorganism in the microorganism separating apparatus according to any one of claims 1 to 7. It is characterized by comprising a biological material supply means for staining for supplying the microchip to the microchip.

The invention according to claim 9 is the microorganism separation apparatus according to any one of claims 1 to 8 , characterized in that the substrate of the microchip is made of a transparent material.

  By configuring as described above, the present invention can provide a microorganism separation apparatus that selectively captures, separates, and collects microorganisms to be detected in a minute region.

  Hereinafter, the present invention will be described in detail based on preferred embodiments shown in the drawings.

  FIGS. 1A and 1B are a plan view and a longitudinal sectional view of a microchip 1A constituting a first embodiment of a microorganism separation apparatus according to the present invention. The microchip 1A includes a substrate 1a and a shielding plate 1b that covers the surface of the substrate 1a. On the surface of the substrate 1a, a flow path 2 of sample water and a capturing part 3 for capturing the separation target microorganisms are provided in the middle. And the sample water supply port 5 which penetrates the shielding board 1b is formed in the one end part of the flow channel 2, ie, the left side part of drawing, and the other end part of the flow channel 2, ie, the right side part of drawing Is formed with a sample water drain port 6 penetrating in the bottom direction.

  The capturing part 3 spreads in the shape of a turtle shell in the lateral direction in the longitudinal direction intermediate part of the flow channel 2, and a plurality of microorganism capturing holes capable of capturing the microorganisms to be separated by their shape characteristics in the expanded part. 4 is arranged. The sample water is introduced into the flow channel 2 from the sample water supply port 5 by an external sample water supply means described later. As the sample water supply means for this purpose, a micro pump capable of controlling a minute flow rate such as a tube pump or a plunger pump is suitable. In particular, since the sample water containing microorganisms contains particulate matter, the above pump suitable for feeding turbidity is desirable.

  As the material of the microchip 1A, silicon, metal, plastic, glass, ceramic, or the like can be used. For processing the flow channel 2 and the microorganism capturing hole 4, a method combining photolithography and chemical etching, laser processing, micromachining, or the like is used.

  2 (a) and 2 (b) are perspective views showing the microchip 1A designed for the purpose of selectively capturing Cryptosporidium partially cut out and the shape of the microorganism capture hole 4 shown in FIG. It is a perspective view shown. Cryptosporidium has a spherical shape with a diameter of 5 μm, and in order to capture this, cylindrical microorganism capturing holes 4 having an opening diameter of 10 μm and a depth of 10 μm are arranged at intervals of 50 μm. The material is a silicon substrate.

20 ml of a suspension containing oocysts of Cryptsporidium parvum (1.0 × 10 6 cells / m) on the substrate 1a through the sample water supply port 5 of the microchip 1A.
l) was dropped and washed, followed by staining with anti-crypt antibody and staining with FISH method. The results are shown in FIGS. 3 (a) and 3 (b).

  For staining with an antibody, a FITC-labeled antibody was dropped on the substrate 1a and incubated at 37 ° C. for 1 hour. For staining by FISH method, use Cy3-labeled Cryptosporidium-specific probe in Hybridization buffer (0.9 mol / l NaCl, 20 mmol / l Tris-HCl, 0.05% SDS, 2 pmol / μl oligonucleotide probe, pH 7.2) Hybridization was performed at 48 ° C. for 1 hour. Since the light emission of both dyes is confirmed at the positions where regularly arranged holes are present, it can be seen that microorganisms are trapped in the holes. Regarding the antibody, light emission due to nonspecific adsorption to particles other than Cryptosporidium is confirmed. On the other hand, luminescence other than Cryptosporidium cannot be confirmed by the FISH method. As described above, it is effective to apply a detection method using biological information such as antibody staining or FISH method as a means for confirming whether the captured particles are target microorganisms. Furthermore, comparing the results of the two staining methods is an effective means for preventing misrecognition.

  4 (a) to 4 (d) show modified examples of the microorganism capturing hole 4 disposed in the capturing unit 3 of the microchip 1A. When the opening is a circle, in addition to a cylindrical shape, a conical microorganism capturing hole 4a shown in (a), a mortar-shaped microorganism capturing hole 4b shown in (b), and the like can be applied. In the case where the opening is a square, shapes such as a rectangular parallelepiped microorganism capturing hole 4c shown in (c) and a square pyramid microorganism capturing hole 4d shown in (d) can be applied.

  FIG. 5 is a system diagram of a microorganism separation apparatus that simplifies and shows the end of the microchip 1A shown in FIGS. 1 and 2 and incorporates sample water supply means and its circulation mechanism. Here, the sample water is introduced into the reservoir tank 9 through the sample water supply line 7. After introducing a predetermined amount of sample water into the reservoir tank 9, the sample water supply valve 8 is closed, and the sample water is supplied to the sample water supply port 5 of the microchip 1A by the sample water supply pump 10. The sample water passes through the capturing unit 3 and is then discharged from the sample water drain 6. The discharged liquid is returned again to the reservoir tank 9 by the circulation pump 12 and supplied to the microchip 1A. In this circulation process, the circulation valve 13 is opened and the drain valve 14 is closed. After repeating the circulation for a predetermined time, the circulation valve 13 is closed, the drain valve 14 is opened, and the liquid is discharged. In this circulation process, the particulate matter containing microorganisms passes through the capturing part 3 of the microchip 1A a plurality of times, and the capturing rate is improved.

  After the liquid is discharged from the sample water drain port 6, an antibody or DNA probe stained with a fluorescent dye or a luminescent dye is injected into the capture unit 3 from the stained antibody supply means 15 or the stained DNA probe supply means 16. As the antibody used at this time, an antibody prepared using the target microorganism as an antigen can be applied, but it is desirable that the antibody be a monoclonal antibody if possible. A DNA probe designed to pair with a base sequence specific to the target microorganism is used.

  After reacting for a certain period of time, washing treatment is performed to remove the nonspecifically adsorbed antibody or DNA probe, and then the microchip 1A is observed. At this time, the presence or absence of the target microorganism can be detected by confirming the fluorescence image or the emission image.

  FIG. 6 is a system diagram showing a schematic configuration of a second embodiment of the microorganism separation device according to the present invention, in which the same elements as those in FIG. 5 showing the first embodiment are denoted by the same reference numerals. The description is omitted. Here, a cleaning liquid storage container 17 is newly connected to a connection path between the reservoir tank 9 and the sample water supply pump 10 via a cleaning liquid supply valve 18, and an outer bottom portion of the substrate 1a on which the capturing unit 3 is formed. 5 differs from FIG. 5 in that a microchip 1B equipped with an ultrasonic transducer 19 driven by an ultrasonic oscillator (not shown) is used.

  In this embodiment, similar to the first embodiment, the steps of introducing, circulating and discharging the sample water are executed. Next, the cleaning liquid is supplied from the cleaning liquid storage container 17 to the microchip 1 </ b> B by the sample water supply pump 10. Here, a surfactant, alcohol, organic solvent, acid, alkali or the like is used as the cleaning liquid. As an effect of the surfactant, the cleaning liquid penetrates into the interface between the surface of the microchip 1B and the particles, and the potential energy necessary for separating the particles from the surface is reduced by separating the distance between the surface and the particles. Sometimes. As a result, the target microorganism has a large interaction with the inner surface of the hole, whereas the other particles have a small interaction, so that the target microorganism is easily detached and only the target microorganism is captured in the microorganism capturing hole.

  When the particulate material is a metal oxide, cleaning with an acid is effective. These oxides may cause non-specific adsorption with antibodies and DNA probes, and it is desirable to remove them in order to prevent erroneous recognition. In this case, it is effective to use hydrochloric acid to remove an acid capable of dissolving the target oxide, for example, iron oxide. In addition, alkali cleaning can be applied to amphoteric oxides such as zinc and aluminum. However, needless to say, it is necessary to select a microchip 1B and a target microorganism that are not damaged by the cleaning liquid.

  At this time, in order to promote the detachment of the particulate matter other than the target microorganism from the microorganism capturing hole 4, it is effective to provide a physical cleaning means. In the example of FIG. 6, the ultrasonic vibrator 19 is mounted on the bottom of the microchip 1b, and energy necessary for desorption is supplied by applying mechanical energy by vibration to the particulate matter. Similarly, desorption can be promoted by supplying energy by heating. However, the target microorganism may also be detached due to excessive vibration or overheating. In order to prevent this and effectively remove other particulate matter, it is necessary to set optimum cleaning conditions.

  The cleaning with the cleaning liquid and the physical cleaning means may be used alone or in combination with each other.

  The use of antibodies is the most effective way to improve selectivity for organisms. By immobilizing the antibody on the inner surface of the hole, the target microorganism that has reached the hole can be firmly captured. Immobilization of the anti-cryptospodium antibody on the silicon substrate can be realized by the process exemplified below.

That is, a 10% KOH-ethanol solution was dropped into the hole of the silicon substrate on which the microorganism capturing hole 4 was produced, left to wash for 30 minutes, dried with argon (Ar) gas, and dried with 2% -MEPTES (3-mercaptopropyl Immerse in triethoxysilane) toluene solution for 60 minutes. Then, after washing and argon gas drying, it is immersed in 1 mM-GMBS (N- (γ-maleimidobutyryloxy) succinimide ester) ethanol solution for 30 minutes. Further, after washing, 0.5 mg / ml-Strptavidin is added dropwise and incubated overnight. Biotin-labeled anti-cryptospodium mixed in Deposition buffer (10 mM-PBS + 10 mM-NaCl + 10 mM-Sucrose, 0.1% BSA) after soaking in Blocking buffer (1% -BAS, 0.01% -NaN ₃ ) for 20 minutes or more By spotting the antibody in the hole, an anti-cryptospodium antibody is introduced into the hole.

  Since the binding between the antibody and the organism is strong, it is desirable to limit the fixing site of the antibody within the microorganism capturing hole 4 as much as possible. However, even when an antibody is fixed to the peripheral surface of the microorganism capture hole 4, the energy required for desorption differs from that in the microorganism capture hole 4, so that if an appropriate cleaning condition is set, the microorganism capture hole The target microorganisms can be preferentially collected within 4.

  FIG. 7 is a schematic configuration diagram of a third embodiment of the microorganism separation device according to the present invention. In the figure, a microchip 1C is provided with a plate-like electrode 20 on the surface portion of the shielding plate 1b facing the capturing portion 3 of the substrate 1a on the flow channel 2 side, and this electrode 20 prevents entry of sample water. In addition, it is covered with an insulating layer 21 for electrical insulation. In this case, a conductive material such as a conductive silicon substrate or a metal plate is used as the substrate 1a. And the lead wires of the positive electrode and the negative electrode of the voltage application device 23 are connected between the substrate 1a and the electrode 20, whereby a microorganism collecting mechanism for effectively capturing microorganisms by applying an electric field to the sample water. It is configured.

By this microorganism collecting mechanism , charged particles can be moved to the microorganism capturing hole 4. In general, microorganisms are negatively charged. By applying a voltage so that the substrate 1a side is positive and the electrode 20 side is negative, an electric field is generated from the substrate 1a toward the shielding plate 1b. It is possible to capture microorganisms.

  At this time, by covering the part other than the opening of the microorganism capturing hole 4 with the insulating layer, the electric field can be concentrated in the microorganism capturing hole 4 and the introduction of the microorganism into the microorganism capturing hole 4 can be promoted. As a method for forming the insulating layer, there are a method of oxidizing or nitriding the substrate 1a while covering the organic or inorganic material or masking the microorganism capturing holes 4. The latter is desirable for carrying out finer processing with high accuracy.

  8A and 8B are a cross-sectional view of a microchip constituting a fourth embodiment of the microorganism separation apparatus according to the present invention and a perspective view showing a partial configuration thereof. In the figure, the same elements as those in FIG. 7 showing the third embodiment are designated by the same reference numerals, and the description thereof is omitted. The microchip 1D shown here collects microorganisms by an electric field, as in the third embodiment. For the substrate 1a, for example, glass, plastic, ceramic, etc. can be used. By using a material having high properties, there is an advantage that optical microscope observation of a phase difference image, a differential interference image or the like becomes easy.

  These materials are non-conductive, and an auxiliary electrode for generating an electric field is required when collecting microorganisms by an electric field. Therefore, in this embodiment, the ring-shaped auxiliary electrode 24a is formed on the peripheral end portion of the bottom surface of the microorganism capturing hole 4, or the auxiliary electrode 24b is formed on the inner peripheral surface of the microorganism capturing hole 4. . By applying a voltage between the auxiliary electrode 24a or 24b and the electrode 20, it is possible to accumulate charged particles by an electric field as in the third embodiment shown in FIG. With this configuration, collection by an electric field and observation with an optical microscope can be realized without sacrificing the transparency of the bottom surface. In particular, when observation with an optical microscope is not necessary, needless to say, the entire bottom surface of the microorganism capturing hole 4 can be used as an auxiliary electrode.

  FIGS. 9A and 9B are a cross-sectional view of a microchip constituting a fifth embodiment of the microorganism separation apparatus according to the present invention and a perspective view showing an essential part thereof. In the microchip 1E according to this embodiment, a plurality of microorganism capturing holes 4 are provided on a substrate 1a to form a capturing part 3. A plurality of protrusions 25 project from the surface of the shielding plate 1b on the flow channel side so as to correspond to the microorganism capturing holes 4 respectively. These protrusions 25 are formed so as to be positioned on the upstream side where the sample water flows from the microorganism capturing hole 4 when the shielding plate 1b is attached to the substrate 1a. It is possible to act to direct the water flow. As a result, the probability that the particulate matter contained in the sample water reaches the microorganism capturing hole 4 is increased, and the capturing rate is improved.

  The protrusion 25 can have a columnar shape, a rod shape, a conical shape, or the like. As for the shape and size of the protrusion 25, it is better to increase the shape in order to improve the flow direction switching action. However, if the flow path becomes too narrow, the resistance increases, and the flow velocity is lowered and the particulate matter may be blocked. There is. It is necessary to select an optimum size in consideration of these situations.

The top view and longitudinal cross-sectional view of the microchip which comprise 1st Example of the microorganisms separator which concerns on this invention. The perspective view which partially cut and showed the capture part of the microchip designed in order to capture | acquire cryptosporidium selectively, and the perspective view which shows the shape of a microorganisms capture | acquisition hole. The figure which showed the example of dyeing | staining of Cryptosporidium by antibody dyeing and FISH method. The figure which showed the modification of the microorganisms capture hole arrange | positioned in the capture part of a microchip. The system diagram which took in the sample water supply means and its circulation mechanism in 1st Example. The systematic diagram which showed schematic structure of 2nd Example of the microorganisms separator which concerns on this invention. The schematic block diagram of 3rd Example of the microorganisms separator which concerns on this invention. Sectional drawing of the microchip which comprises 4th Example of the microorganisms separator which concerns on this invention, and a perspective view which shows the partial structure. Sectional drawing of the microchip which comprises 5th Example of the microorganisms separator which concerns on this invention, and a perspective view which shows the principal part.

Explanation of symbols

1A to 1E Microchip 1a Substrate 1b Shield plate 2 Flow channel 3 Capture unit 4 Microorganism capture hole 5 Sample water supply port 6 Sample water drain port 7 Sample water supply line 8 Sample water supply valve 9 Reservoir tank 10 Sample water supply pump 12 Circulating pump 13 Circulating valve 14 Drain valve 15 Stained antibody supply means 16 Stained DNA probe supply means 17 Washing liquid storage container 18 Washing liquid supply valve 19 Ultrasonic vibrator 20 Electrode 21 Insulating layer 23 Voltage application devices 24a and 24b Auxiliary electrode 25 Protrusion

Claims (9)

In a microorganism separation device that captures, separates and recovers specific microorganisms contained in sample water,
A microchip having a capturing portion in which a flow path of sample water is formed on a substrate, and a plurality of microorganism capturing holes for capturing microorganisms to be separated are disposed in the middle of the flow path;
Sample water supply means for pouring sample water containing microorganisms to be separated into the microchip;
Sample water circulation means for collecting waste water that has passed through the microchip and returning it to the sample water supply means,
And when the separation target microorganisms contained in the sample water come into contact with the plurality of microorganism capture holes, the sample water supply means and the separation target microorganisms captured in the microorganism capture holes are increased. The sample water circulation operation by the sample water circulation means was repeated a plurality of times.
Microorganism separation device, characterized in that. 2. The microorganism separation apparatus according to claim 1 , wherein a physical cleaning means is attached to the microchip. The microorganism separation apparatus according to claim 1, further comprising a cleaning liquid supply unit for pouring a cleaning liquid into the microchip. The microchip includes a shielding plate that covers the surface of the substrate, the substrate is made of a conductive material, and a flat plate portion other than the microorganism capturing hole is electrically insulated by an insulating layer, and the capturing of the shielding plate An electrode was embedded in a part facing the part through an insulating layer, and a voltage applying device for generating an electric field in the flow path of the sample water by applying a voltage between the electrode and the substrate was provided. The microorganism separation device according to any one of claims 1 to 3 , wherein The microchip includes a shielding plate that covers the surface of the substrate, the substrate is made of a non-conductive material, a first electrode is provided inside the microorganism capturing hole, and a portion facing the capturing portion of the shielding plate And a voltage applying device for generating an electric field in the flow path of the sample water by applying a voltage between the first and second electrodes through the insulating layer. The microorganism separation device according to any one of claims 1 to 3 , wherein the device is a microorganism separation device. The microorganism separation apparatus according to any one of claims 1 to 5 , further comprising an antibody that is immobilized in the microorganism capturing hole and recognizes a target microorganism as an antigen. The microorganism separation apparatus according to any one of claims 1 to 3 , wherein a projection is provided on a flow path surface of the shielding plate so as to create a flow of sample water in a direction toward the microorganism capturing hole. Claim, characterized in biological material labeled with a fluorescent dye or luminescent dye capable of absorbing the target microorganism selectively identify and to be supplied to the microchip, further comprising a dyeing biological material supply means The microorganism separation device according to any one of 1 to 7 . The microorganism separating apparatus according to any one of claims 1 to 8 , wherein the substrate of the microchip is made of a transparent material.

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