JP2007097444A - Chip for testing use - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a chip for testing use enabling a reagent to be put to reaction with a specimen while keeping the reagent's activity high enough. <P>SOLUTION: The chip 10 for testing use is characterized by that the main body 1 is furnished with a first well 2 for housing a specimen-containing specimen liquid, a second well 3 housed with the solid reagent 5 reactive with a specimen, a first opening 2a for making the first well 2 open to the air, a second opening 3a for making the second well 3 open to the air, and a flow channel 4 connecting the first well 2 and the second well 3 with each other. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to an inspection chip.

  In recent years, in order to detect microorganisms (bacteria, fungi, etc.) in food and drink, adenosine triphosphate (ATP) present in microorganisms has been detected. In addition, in order to analyze the DNA base sequence, pyrophosphate generated by the binding of deoxynucleotide triphosphate (dNTP) that constitutes DNA and dNTP complementary thereto is converted to ATP, and this ATP is detected. (For example, the following patent document 1).

Such detection of ATP has been conventionally performed by mixing a sample solution with a reagent solution prepared in advance using a test chip and causing luminescence based on a luciferin-luciferase reaction (for example, the following) Patent Document 1).
JP 2004-028589 A

  However, since the reagent used in the luciferin-luciferase reaction contains luciferase, which is a protein, and easily deteriorates in an aqueous solution state, in a conventional test chip that requires preparation of a reagent solution, There was a problem that the activity of the reagent was reduced and the amount of luminescence was reduced accordingly.

  Such a decrease in the activity of the reagent is not limited to luciferase as long as a conventional test chip is used, and generally a problem occurs when a test is performed using a reagent containing a protein.

  Accordingly, an object of the present invention is to provide a test chip that enables a reagent to be used for a reaction with a specimen while keeping the activity of the reagent sufficiently high.

  To achieve the above object, the present invention provides a first well for introducing a sample liquid containing a sample, a second well in which a solid reagent that reacts with the sample is stored, A first opening for opening the well to the atmosphere, a second opening for opening the second well to the atmosphere, and a flow path connecting the first well and the second well. An inspection chip is provided which is formed in a portion.

  In the test chip, after the sample liquid is introduced into the first well, the sample liquid is circulated through the flow path and stored in the second well. When the sample liquid is stored in the second well, the solid reagent stored in the second well is dissolved in the sample liquid and reacts with the sample in the second well. Distribution of the sample liquid introduced into the first well into the flow path and accommodation in the second well are performed by adjusting the pressure applied to the sample liquid through the first and second openings. In the present invention, “sample liquid” means a liquid containing a sample.

  If the above-mentioned test chip is used, it is not necessary to prepare a reagent solution in advance, and at the same time as the reagent dissolves in the sample liquid, the reaction between the sample and the reagent starts, so that the reagent activity is maintained sufficiently high. Can be used for reaction with the specimen. In addition, since it is not necessary to prepare a reagent solution in advance, there is no possibility that impurities such as microorganisms are mixed in the reaction field with the preparation or dispensing of the reagent solution, and the inspection can be performed with such high accuracy. In addition, since preparation and dispensing of the reagent solution are not necessary, the test can be performed easily.

  According to the present invention, in the above-described test chip, the reagent stored in the second well is a luminescent reagent that emits light in the presence of ATP, and a solid extraction reagent that extracts ATP in the microorganism is stored. A third well and a third opening for opening the third well to the atmosphere are further formed in the main body, and the third well is between the first well and the second well. The inspection chip which constitutes a part of the flow path is provided.

  In such a test chip (hereinafter referred to as “microorganism detection chip”), the sample liquid is introduced into the first well, and then the sample liquid is circulated through the flow path and stored in the third well. When the sample liquid is stored in the third well, the solid extraction reagent stored in the third well is dissolved in the sample liquid, and ATP in the microorganism is extracted in the third well. After the sample liquid is stored in the third well and a sufficient time has passed for extraction of ATP, the sample liquid is circulated through the flow path between the third well and the second well and stored in the second well. Let When the sample liquid is stored in the second well, the solid luminescent reagent stored in the second well is dissolved in the sample liquid, reacts with ATP in the sample liquid in the second well, and emits light. Produce. Distribution of the sample liquid introduced into the first well into the flow path and accommodation in the other well are performed by adjusting the pressure applied to the sample liquid through the first, third, and second openings. Such a chip for detecting microorganisms can be used for detecting microorganisms in a specimen using ATP as an index by combining with a photodetector. In the present invention, “luminescent reagent” means a reagent that reacts with ATP to generate luminescence, and “extraction reagent” means a reagent that extracts ATP in a microorganism. Any reagent may be composed of one kind of substance or may be composed of two or more kinds of substances.

  As the above-mentioned chip for detecting a microorganism, a chip in which an ATP-degrading enzyme is immobilized on the inner wall surface of the flow path between the first well and the third well is preferable. If such a chip for microorganism detection is used, ATP other than ATP (hereinafter referred to as “background ATP”) in the microorganism is decomposed and removed by the ATP-degrading enzyme before the sample solution and the extraction reagent are mixed. Therefore, microorganisms in the specimen are detected with such high accuracy. In the present invention, “ATP-degrading enzyme is immobilized” means that the ATP-degrading enzyme is bound to the solid surface by physical adsorption, chemical bonding, etc., and the solid surface is brought into contact with water or an aqueous solution. , ATP-degrading enzyme does not desorb from the solid surface.

  The microbe detection chip further includes a fourth well containing a solid amplification reagent for amplifying ATP, and a fourth opening for opening the fourth well to the atmosphere. Preferably, the fourth well forms a part of the flow path between the third well and the second well. In such a microbe detection chip, the sample liquid is accommodated in the third well, and after sufficient time has passed for extraction of ATP, the sample liquid is placed in the flow path between the third well and the fourth well. Circulate and accommodate in the fourth well. When the sample liquid is stored in the fourth well, the solid amplification reagent stored in the fourth well is dissolved in the sample liquid, and reacts with ATP in the sample liquid in the fourth well. ATP in the liquid is amplified. After the sample liquid is accommodated in the fourth well and sufficient time has passed for amplification of ATP, the sample liquid is circulated through the flow path between the fourth well and the second well and accommodated in the second well. Let When the sample liquid is stored in the second well, the solid luminescent reagent stored in the second well is dissolved in the sample liquid, reacts with ATP in the sample liquid in the second well, and emits light. Produce. The flow of the sample liquid introduced into the first well into the flow path and the accommodation into the other wells are performed by adjusting the pressure applied to the sample liquid through the first, third, fourth, and second openings. To do. If such a microorganism detection chip is used, the ATP in the sample liquid is amplified after the ATP in the microorganism is extracted by the extraction reagent and before the sample liquid and the luminescent reagent are mixed. Microorganisms are detected with such high sensitivity. In the present invention, the “amplification reagent” means a reagent for amplifying ATP, and it may be composed of one kind of substance or two or more kinds of substances.

  As the above-mentioned microbe detection chip, there is also a chip in which a light reflecting portion for reflecting light generated in the second well is provided on a part of the inner wall surface of the second well so as to surround the reagent. preferable. If such a chip for detecting microorganisms is used, the light detector is arranged so that the light reflected by the light reflecting part is incident on the light receiving part of the light detector, so that the light is incident on the light receiving part of the light detector. Since the amount of light can be increased, microorganisms in the specimen can be detected with such high sensitivity. In the present invention, “reflecting light” means reflecting at least part of light.

  As the above-mentioned microbe detection chip, it is also preferable that the second opening is covered with a breathable light reflecting film that reflects light generated in the second well. If such a microbe detection chip is used, the photodetector is arranged so that the light reflected by the light reflecting film is incident on the light receiving portion of the photodetector, thereby entering the light receiving portion of the photodetector. Since the amount of light can be increased, microorganisms in the specimen can be detected with such high sensitivity. Note that the flow of the sample liquid introduced into the first well into the flow path and the storage in the other wells are performed by adjusting the pressure applied to the sample liquid through the opening. It must be breathable.

  As the inspection chip (including the microorganism detection chip), all except the first opening (the first opening and the second opening when the second opening is covered with the light reflecting film) are used. It is preferable that the opening is covered with a filter. By using such an inspection chip, it is possible to prevent impurities such as microorganisms from entering all the wells except the first well, so that the inspection can be performed with such high accuracy.

  ADVANTAGE OF THE INVENTION According to this invention, the chip | tip for a test | inspection which makes it possible to use a reagent for reaction with a test substance is provided, maintaining the activity of a reagent high enough.

  Preferred embodiments of the present invention will be described below with reference to the drawings. In the description of the drawings, the same reference numerals are used for the same or equivalent elements, and duplicate descriptions are omitted.

(First embodiment)
A first embodiment of the present invention will be described with reference to FIGS. FIG. 1 is a plan view of the inspection chip according to the first embodiment. FIG. 2 is a cross-sectional view taken along line II-II in FIG.

  The inspection chip 10 according to the first embodiment includes a main body 1, and the main body 1 includes three layers of substrates 1 a to 1 c. Substrate 1a-1c should just be comprised with the material which has translucency. Examples of such materials include resins such as polycarbonate (PC), polyethylene terephthalate (PET), polydimethylsiloxane (PDMS), polyimide, polystyrene (PS), and polymethyl methacrylate (PMMA), quartz glass, and Pyrex (registered trademark). ) Glass such as glass.

  On the substrate 1a, a specimen liquid introduction well (first well) 2, a light emitting well (second well) 3, and an extraction well (third well) 42 are formed. Of the surface of the substrate 1a, the surface opposite to the substrate 1b with respect to the substrate 1a has a specimen liquid introduction well opening (first opening) 2a for opening the specimen liquid introduction well 2 to the atmosphere, and light emission. A light emitting well opening (second opening) 3a for opening the well 3 to the atmosphere and an extraction well opening (third opening) 42a for opening the extraction well 42 to the atmosphere are formed. . Each well is formed by a side wall surface extending in a cylindrical shape from the edge of the opening toward the substrate 1b side, and a bottom surface connected to an end of the side wall surface. Here, the bottom surfaces of the sample liquid introduction well 2 and the extraction well 42 are each recessed in a direction away from the end of the side wall surface. Specifically, the bottom surface has an opening area when a well (excluding a columnar portion formed by a side wall surface) is cut in a virtual plane parallel to the surface of the substrate 1a. It is formed in a tapered shape (conical shape or truncated cone shape) so as to become smaller toward.

The luminescent well 3 contains a solid luminescent reagent (solid reagent) 5. Examples of the luminescent reagent 5 include a reagent composed of firefly luciferin, firefly luciferase, and divalent metal ions (Mg ^{2+ and the} like). ATP reacts with firefly luciferin and firefly luciferase in the presence of oxygen and divalent metal ions to cause light emission by firefly luciferin.

  The extraction well 42 contains a solid extraction reagent 6. Examples of the extraction reagent 6 include lytic enzymes (lysozyme, chitinase, chitosanase, etc.), cationic surfactants (benzalkonium chloride, benzethonium chloride, cetyltrimethylammonium chloride, etc.), nonionic surfactants (polyoxyethylene, etc.) Alkyl ether, polyoxyethylene alkylphenyl ether, polyoxyethylene sorbitan ester, sorbitan fatty acid ester, etc.). In addition to the lytic enzyme and the like, the extraction reagent 6 may contain, for example, a nonionic surfactant, a chelating agent (such as ethylenediaminetetraacetic acid), or a saccharide (such as sucrose, glucose, or fructose).

  The solid reagent is preferably freeze-dried when it contains an enzyme for both the luminescent reagent 5 and the extraction reagent 6. The lyophilized reagent can maintain its original activity for a long period (for example, more than half a year).

  A light reflecting portion 11 is provided on the cylindrical side wall surface of the luminescent well 3 over the entire circumference of the side wall surface, and the light reflecting portion 11 surrounds the solid luminescent reagent 5. The light reflecting portion 11 is not provided on the bottom surface of the light emitting well 3. The light emitting well opening 3 a is covered with the light reflecting film 12. Of the light generated in the light emitting well 3, the light directed toward the side wall surface or the light emitting well opening 3 a is reflected by the light reflecting portion 11 or the light reflecting film 12 and goes out of the light emitting well 3 from the bottom surface. Although the light reflection film 12 has air permeability, this allows the light emitting well 3 to be pressurized or depressurized. The light reflecting film 12 also functions as a filter that prevents impurities such as microorganisms from entering the light emitting well 3. The light reflection part 11 should just be comprised with the material which reflects light. Examples of such a material include metals such as aluminum, nickel, titanium, chromium, gold, copper, and iron. Moreover, the light reflection film | membrane 12 should just be comprised with the air permeable material which reflects light. Examples of such a material include polytetrafluoroethylene, polyvinylidene fluoride, and the like. The average pore diameter of the light reflecting film 12 is preferably 0.1 to 1 μm in order to prevent impurities such as microorganisms from entering the light emitting well 3.

  The extraction well opening 42 a is covered with the filter 13. This prevents impurities such as microorganisms from entering the extraction well 42 and allows the light emitting well 3 to be pressurized or depressurized. The average pore diameter of the filter 13 is preferably 0.1 to 1 μm in order to prevent impurities such as microorganisms from entering the extraction well 42.

  The sample liquid introduction well 2 and the extraction well 42 are connected by a flow path 41. Specifically, one end of the channel 41 is connected to the end of the tapered bottom surface of the sample liquid introduction well 2, and the other end of the channel 41 is connected to the side wall surface of the extraction well 42. Accordingly, the sample liquid in the sample liquid introduction well 2 can flow into the extraction well 42 via the flow path 41. The extraction well 42 and the light emitting well 3 are connected by a flow path 43. Specifically, one end of the channel 43 is connected to the end of the tapered bottom surface of the extraction well 42, and the other end of the channel 43 is connected to the side wall surface of the light emitting well 3. Therefore, the sample liquid in the extraction well 42 can flow into the light emitting well 3 through the flow path 43. The extraction well 42 constitutes the flow path 4 together with the flow path 41 and the flow path 43. That is, the channel 4 connects the sample liquid introduction well 2 and the light emitting well 3.

  Moreover, since the flow path 41 is formed in a meandering shape, it is sufficiently long compared to the case where it is linear. An ATP-degrading enzyme is immobilized on the inner wall surface of the channel 41 (not shown in the figure), so that ATP in the liquid flowing through the channel 41 is decomposed and removed. Examples of the ATP degrading enzyme include apyrase, adenosine phosphate deaminase, luciferase and the like.

  Next, a method for detecting microorganisms in a specimen using the test chip 10 will be described.

  First, the sample liquid is introduced into the sample liquid introduction well 2. Further, the photodetector 21 having the light receiving portion 21a is installed so that the light receiving portion 21a faces the light emitting well 3 and contacts the surface of the substrate 1c. Further, the pressure ports 22 and 23 are installed so as to cover the sample liquid introduction well opening 2a and the extraction well opening 42a, respectively. In FIG. 2, the photodetector 21, the light receiving unit 21 a, and the pressure ports 22 and 23 are indicated by two-dot chain lines.

  Next, the sample liquid introduced into the sample liquid introduction well 2 is circulated through the flow path 41 and accommodated in the extraction well 42 by pressurizing the inside of the sample liquid introduction well 2 with the pressure port 22. When the sample liquid is stored in the extraction well 42, the solid extraction reagent 6 stored in the extraction well 42 is dissolved in the sample liquid, and ATP in the microorganism is extracted in the extraction well 42.

  Finally, after a sufficient time for extraction of ATP has elapsed, the inside of the extraction well 42 is pressurized by the pressure port 23, whereby the sample liquid accommodated in the extraction well 42 is circulated through the flow path 43 to emit light. 3 to accommodate. At this time, in order to prevent the back flow of the sample liquid and the pressure loss in the extraction well 42, the inside of the sample liquid introduction well 2 is simultaneously pressurized by the pressure port 22. When the sample liquid is accommodated in the luminescent well 3, the solid luminescent reagent 5 is dissolved in the sample liquid and reacts with ATP in the sample liquid in the luminescent well 3 to generate luminescence. The light emitted from the luminescent reagent 5 enters the light receiving portion 21 a and is detected by the photodetector 21. In this way, microorganisms in the specimen are detected.

  If the test chip 10 is used, it is not necessary to prepare a reagent solution in advance, and at the same time as the reagent dissolves in the sample liquid, the reaction between the sample and the reagent starts, so that the reagent activity is maintained sufficiently high. Can be used for reaction with the specimen. In addition, since it is not necessary to prepare a reagent solution in advance, there is no possibility that impurities such as microorganisms are mixed in the reaction field with the preparation or dispensing of the reagent solution, and the microorganism can be detected with such high accuracy. In addition, since preparation and dispensing of the reagent solution are not necessary, microorganisms can be detected easily.

  Further, in the test chip 10, the flow path 41 is formed in a meandering shape and is sufficiently long, so that the ATP decomposition is fixed to the inner wall surface of the flow path 41 while the sample liquid flows through the flow path 41. The background ATP in the sample liquid is almost completely decomposed and removed by the enzyme. Therefore, if the inspection chip 10 is used, microorganisms can be detected with such high accuracy.

  Further, in the test chip 10, the light emitted from the luminescent reagent 5 toward the side wall surface of the luminescent well 3 or the luminescent well opening 3 a is reflected by the light reflecting portion 11 or the light reflecting film 12 to be emitted from the luminescent well. 3 is emitted from the bottom surface of the light emitting well 3 to the outside of the light emitting well 3, and then enters the light receiving portion 21a. Therefore, if the inspection chip 10 is used, the amount of light incident on the light receiving portion 21a increases, and microorganisms can be detected with such high sensitivity.

  Further, in the testing chip 10, the light emitting well opening 3 a is covered with the light reflecting film 12, and the extraction well opening 42 a is covered with the filter 13, so that impurities such as microorganisms enter the light emitting well 3 and the extraction well 42. Is prevented from mixing. Therefore, if the inspection chip 10 is used, microorganisms can be detected with such high accuracy.

  In the test chip 10, the flow path 41 is connected to the end of the tapered bottom surface of the sample liquid introduction well 2, so that the sample liquid stored in the sample liquid introduction well 2 flows out to the flow path 41. When the operation is performed, the amount of the sample liquid remaining in the well is sufficiently reduced. In addition, since the flow path 43 is connected to the end of the tapered bottom surface of the extraction well 42, when an operation for causing the sample liquid stored in the extraction well 42 to flow out to the flow path 41 is performed, The amount of the remaining sample liquid is sufficiently reduced. Thus, since the amount of the sample liquid remaining in the sample liquid introduction well 2 and the extraction well 42 is sufficiently reduced, the emission intensity corresponding to the amount of the sample in the introduced sample liquid can be obtained by using the test chip 10. Measurement can be performed with high accuracy, and microorganisms can be detected with high accuracy and high reproducibility. Here, the angle formed by the tapered bottom surfaces of the sample liquid introduction well 2 and the extraction well 42 and the surface of the substrate 1a is not particularly limited, but may be, for example, 30 to 60 degrees.

  In the test chip 10, the flow path 41 is connected to the side wall surface of the extraction well 42, so that the sample liquid that has flowed through the flow path 41 is contained in the solid extraction reagent 6 stored in the extraction well 42. Flows into the extraction well 42 from above, and the dissolution of the solid extraction reagent 6 proceeds from both the upper and lower directions. That is, on the one hand, the upper part of the solid extraction reagent 6 is dissolved by the sample liquid flowing out from the flow path 41 and directly hitting the solid extraction reagent 6, and on the other hand, by the sample liquid stored in the extraction well 42. The lower part of the solid extraction reagent 6 is dissolved. Therefore, if the test chip 10 is used, the dissolution rate of the extraction reagent 6 increases accordingly, and microorganisms can be detected quickly.

  Further, when the inside of the sample liquid introduction well 2 is pressurized and the sample liquid is transferred to the extraction well 42, if the flow path 41 is connected to the bottom surface of the extraction well 42, for example, the air is supplied to the sample liquid introduction well 42. 2 is introduced into the sample liquid stored in the extraction well 42 through the flow path 41, bubbles are generated in the sample liquid, and the bubbles prevent the flow of the sample liquid from the extraction well 42 to the flow path 43. There is a fear. On the other hand, in the test chip 10, since the flow path 41 is connected to the side wall surface of the extraction well 42, generation of bubbles in the sample liquid as described above is achieved by optimizing the amount of the sample liquid. Is prevented. That is, when the channel 41 is connected to a portion of the side wall surface of the extraction well 42 that is above the liquid surface of the sample liquid stored in the extraction well 42, the generation of bubbles as described above is prevented. The Therefore, when the test chip 10 is used, by optimizing the amount of the sample liquid, the sample liquid is smoothly transferred from the extraction well 42 to the light emitting well 3 through the flow path 43, and the microorganisms are detected smoothly. be able to.

  In addition, when the channel 41 is connected to a portion of the side wall surface of the extraction well 42 that is above the liquid surface of the sample liquid stored in the extraction well 42, the backflow of the stored sample liquid is ensured. Is prevented. Further, when the sample liquid is allowed to flow into the flow path 43, it is not necessary to pressurize the inside of the sample liquid introduction well 2 in order to prevent the back flow of the sample liquid. Therefore, if the test chip 10 is used, microorganisms can be detected easily with high accuracy by optimizing the amount of the sample liquid.

  Similarly, since the flow path 43 is connected to the side wall surface of the luminescent well 3, the dissolution of the solid luminescent reagent 5 proceeds from both the upper and lower directions. Therefore, if the test chip 10 is used, the dissolution rate of the luminescent reagent 5 increases accordingly, and microorganisms can be detected quickly.

  Next, a method for manufacturing the inspection chip 10 will be described. The inspection chip 10 can be manufactured as follows, for example.

  First, a resist is applied on a silicon wafer and patterned (exposure and development) into a shape corresponding to the flow paths 41 and 43, and then the surface portion of the silicon wafer is etched. In this way, convex portions corresponding to the flow paths 41 and 43 are formed on the silicon surface. Then, a mold having projections corresponding to the sample solution introduction well 2, the light emitting well 3, and the extraction well 42 is prepared, and the resist is removed from the silicon wafer and the mold so that both projections face each other. To face each other. Thereafter, uncured PDMS is injected into the gap formed by the silicon wafer and the mold facing each other.

  Next, the PDMS is cured (125 ° C., 20 minutes), and the PDMS is peeled off to obtain a cured PDMS having the shape of the sample liquid introduction well 2, the light emitting well 3, the extraction well 42, and the flow paths 41 and 43. It is done. The PDMS cured product is processed (such as drilling) so that each well is connected to the flow path.

  Next, another uncured PDMS is applied onto the glass wafer and temporarily cured (50 ° C., 10 minutes). The PDMS cured product is pasted on the temporarily cured PDMS, and the temporarily cured PDMS is cured (125 ° C., 20 minutes).

  Finally, an ATP degrading enzyme is immobilized on the inner wall surface of the channel 41. Immobilization of ATP-degrading enzyme can be performed, for example, as follows. First, after the ATP-degrading enzyme solution was circulated through the channel 41, all the open parts (the sample liquid introduction well 2, the luminescent well 3, and the extraction well 42) were sealed, and about 5 hours in the refrigerator (about 4 ° C.). put. Next, a buffer is circulated through the flow path 41 to remove easily desorbed ATP-degrading enzymes. Finally, the inside of the flow path 41 is replaced with air.

  In order to determine whether the ATP-degrading enzyme is immobilized on the inner wall surface of the flow path 41 and the ATP resolution of the immobilized enzyme, for example, after the ATP solution is circulated through the flow path 41, The accommodated reacted solution is recovered, firefly luciferin and firefly luciferase are added thereto, and the amount of luminescence may be measured with a luminometer. If the amount of luminescence is reduced compared to the original ATP solution, it can be determined that the ATP-degrading enzyme is immobilized. Further, the ATP resolution can be determined based on the amount of decrease in the light emission amount.

  The test chip 10 is preferably used together with a thermostat capable of adjusting the temperature of the sample liquid in the well and the channel. Since the reaction between the specimen and the reagent is exothermic or endothermic, the reaction may become unstable due to a temperature change unless the temperature of the specimen liquid is adjusted. Further, when the temperature changes, the liquid expands and unnecessary bubbles are generated, which may cause clogging of the flow path (excluding the well portion). If used together with a thermostatic device, such troubles can be prevented.

(Second Embodiment)
A second embodiment of the present invention will be described with reference to FIGS. FIG. 3 is a plan view of the inspection chip according to the second embodiment. FIG. 4 is a cross-sectional view taken along line IV-IV in FIG.

  In the testing chip 20 according to the second embodiment, an amplification well (fourth well) 45 constituting a part of the flow path 43 is further formed on the substrate 1a, and the substrate 1a among the surfaces of the substrate 1a. An inspection well chip is provided in that an amplification well opening (fourth opening) 45a is formed on the surface opposite to the substrate 1b, and a solid amplification reagent 7 is stored in the amplification well 45. 10 and different.

  The amplification well 45 is formed by a side wall surface extending in a cylindrical shape from the edge of the amplification well opening 45a toward the substrate 1b side, and a bottom surface connected to the end of the side wall surface. Here, the bottom surface of the amplification well 45 is recessed in a direction away from the end of the side wall surface. Specifically, the bottom surface has an opening area when a well (excluding a columnar portion formed by a side wall surface) is cut in a virtual plane parallel to the surface of the substrate 1a. It is formed in a tapered shape (conical shape or truncated cone shape) so as to become smaller toward.

  As described above, the amplification well 45 contains the solid amplification reagent 7. Examples of the amplification reagent 7 include a reagent composed of adenosine monophosphate (AMP), polyphosphate, adenylate kinase (ADK), and polyphosphate kinase (PPK). ATP reacts with AMP in the presence of adenylate kinase to yield two molecules of ADP, and the two molecules of ADP react with polyphosphate in the presence of polyphosphate kinase to yield two molecules of ATP. ATP is amplified by repeating this reaction. The solid amplification reagent 7 is preferably lyophilized when it contains an enzyme. The lyophilized reagent can maintain its original activity for a long period (for example, more than half a year).

  The amplification well opening 45 a is covered with the filter 14. The filter 14 is for preventing impurities such as microorganisms from entering the amplification well 45. As the filter 14, the same filter as the filter 13 may be used.

  The extraction well 42 and the amplification well 45 are connected by a flow path 44. Specifically, one end of the channel 44 is connected to the end of the tapered bottom surface of the extraction well 42, and the other end of the channel 44 is connected to the side wall surface of the amplification well 45. Accordingly, the sample liquid in the extraction well 42 can flow into the amplification well 45 through the flow path 44. Further, the amplification well 45 and the light emitting well 3 are connected by a flow path 46. Specifically, one end of the channel 46 is connected to the end of the tapered bottom surface of the amplification well 45, and the other end of the channel 46 is connected to the side wall surface of the light emitting well 3. Accordingly, the sample liquid in the amplification well 45 can flow into the light emitting well 3 through the flow path 46. The amplification well 45 constitutes a flow path 43 together with the flow path 44 and the flow path 46. That is, the flow path 43 connects the amplification well 45 and the light emitting well 3.

  As the ATP-degrading enzyme immobilized on the inner wall surface of the channel 41, one that also serves as an ADP-degrading enzyme is preferable. Here, ATP-degrading enzyme "also serves as ADP-degrading enzyme" means that ATP-degrading enzyme, that is, a substance having ATP-degrading enzyme activity (for example, protein) further has ADP-degrading enzyme activity, The ATP degrading enzyme active site and the ADP degrading enzyme active site may be different. If the ATP-degrading enzyme also serves as an ADP-degrading enzyme, the background ATP and ADP (ADP) mixed in the sample liquid before the sample liquid and the extraction reagent 6, the amplification reagent 7, and the luminescent reagent 5 are mixed. Hereinafter, “background ADP” is decomposed and removed. When ATP is amplified by the amplification reagent 7, the ADP in the sample liquid is converted to ATP and amplified. Therefore, in addition to the background ATP, the background ADP is mixed with the sample liquid and the amplification reagent 7. If it is decomposed and removed before being processed, microorganisms can be detected with such high accuracy. An example of an ATP-degrading enzyme that also serves as an ADP-degrading enzyme is apyrase.

  Next, a method for detecting microorganisms in a specimen using the test chip 20 will be described.

  First, the sample liquid is introduced into the sample liquid introduction well 2. Further, the photodetector 21 having the light receiving portion 21a is installed so that the light receiving portion 21a faces the light emitting well 3 and contacts the surface of the substrate 1c. Further, the pressure ports 22, 23 and 24 are installed so as to cover the sample liquid introduction well opening 2a, the extraction well opening 42a and the amplification well opening 45a, respectively. In FIG. 4, the photodetector 21, the light receiving unit 21a, and the pressure port 24 are indicated by a two-dot chain line (the pressure ports 22 and 23 are not shown in FIG. 4).

  Next, the sample liquid introduced into the sample liquid introduction well 2 is circulated through the flow path 41 and accommodated in the extraction well 42 by pressurizing the inside of the sample liquid introduction well 2 with the pressure port 22. When the sample liquid is stored in the extraction well 42, the solid extraction reagent 6 stored in the extraction well 42 is dissolved in the sample liquid, and ATP in the microorganism is extracted in the extraction well 42.

  Next, after a sufficient time for extraction of ATP has elapsed, the inside of the extraction well 42 is pressurized by the pressure port 23, whereby the sample liquid accommodated in the extraction well 42 is circulated through the flow path 44 and amplified well. 45. At this time, in order to prevent the back flow of the sample liquid and the pressure loss in the extraction well 42, the inside of the sample liquid introduction well 2 is simultaneously pressurized by the pressure port 22. When the sample solution is stored in the amplification well 45, the solid amplification reagent 7 stored in the amplification well 45 is dissolved in the sample solution, and reacts with ATP in the sample solution in the amplification well 45, and in the sample solution. ATP is amplified.

  Finally, after a sufficient time has passed for amplification of ATP, the inside of the amplification well 45 is pressurized by the pressure port 24, whereby the sample liquid accommodated in the amplification well 45 is circulated through the flow path 46 and the light emitting well. 3 to accommodate. At this time, in order to prevent the back flow of the sample liquid and the pressure loss in the amplification well 45, the insides of the sample liquid introduction well 2 and the extraction well 42 are simultaneously pressurized by the pressure ports 22 and 23, respectively. When the sample liquid is accommodated in the light emitting well 3, the solid luminescent reagent 5 accommodated in the light emitting well 3 is dissolved in the sample liquid, and reacts with ATP in the sample liquid in the light emitting well 3 to generate luminescence. . The light emitted from the luminescent reagent 5 enters the light receiving portion 21 a and is detected by the photodetector 21. In this way, microorganisms in the specimen are detected.

  In the test chip 20, the amplification well opening 45 a is covered with the filter 14, thereby preventing impurities such as microorganisms from entering the amplification well 45. Therefore, if the inspection chip 20 is used, microorganisms can be detected with such high accuracy. As the filter 14, the same filter as the filter 13 may be used.

  Further, in the test chip 20, the flow path 44 is connected to the end of the tapered bottom surface of the extraction well 42, so that an operation for causing the sample liquid stored in the extraction well 42 to flow out to the flow path 44 is performed. In this case, the amount of the sample liquid remaining in the well is sufficiently reduced. In addition, since the flow path 46 is connected to the end of the tapered bottom surface of the amplification well 45, when an operation for causing the sample liquid stored in the amplification well 45 to flow out into the flow path 46 is performed, The amount of the remaining sample liquid is sufficiently reduced. As described above, the amount of the sample liquid remaining in the extraction well 42 and the amplification well 45 is sufficiently reduced. Therefore, if the test chip 20 is used, the emission intensity corresponding to the amount of the sample in the introduced sample liquid is highly accurate. In addition, microorganisms can be detected with high accuracy and high reproducibility. Here, the angle formed by the tapered bottom surfaces of the extraction well 42 and the amplification well 45 and the surface of the substrate 1a is not particularly limited, but may be, for example, 30 to 60 degrees.

  In the test chip 20, since the flow path 44 is connected to the side wall surface of the amplification well 45, the sample liquid flowing through the flow path 44 is a solid amplification reagent 7 stored in the amplification well 45. Flows into the amplification well 45 from above, and the dissolution of the solid amplification reagent 7 proceeds from both the upper and lower directions. That is, on the one hand, the upper part of the solid amplification reagent 7 is dissolved by the sample liquid flowing out from the flow path 44 and directly hitting the solid amplification reagent 7, and on the other hand, by the sample liquid stored in the amplification well 45. The lower part of the solid amplification reagent 7 is dissolved. Therefore, if the test chip 20 is used, the dissolution rate of the amplification reagent 7 increases accordingly, and microorganisms can be detected quickly.

  In addition, when the inside of the extraction well 42 is pressurized and the specimen liquid is transferred to the amplification well 45, if the flow path 44 is connected to the bottom surface of the amplification well 45, for example, air flows from the extraction well 42 to the flow path. 44, the gas is introduced into the sample liquid stored in the amplification well 45, bubbles are generated in the sample liquid, and the bubbles may hinder the flow of the sample liquid from the amplification well 45 to the flow path 46. On the other hand, in the test chip 20, since the flow path 44 is connected to the side wall surface of the amplification well 45, the generation of bubbles in the sample liquid as described above is achieved by optimizing the amount of the sample liquid. Is prevented. That is, when the flow path 44 is connected to a portion of the side wall surface of the amplification well 45 that is above the liquid surface of the sample liquid stored in the amplification well 45, the generation of bubbles as described above is prevented. The Therefore, if the test chip 20 is used, by optimizing the amount of the sample liquid, the sample liquid is smoothly transferred from the amplification well 45 to the light emitting well 3 through the flow path 46, and the microorganisms are detected smoothly. be able to.

  In addition, when the flow path 44 is connected to a portion of the side wall surface of the amplification well 45 that is above the liquid surface of the sample liquid stored in the amplification well 45, the backflow of the stored sample liquid is ensured. Is prevented. Further, when the specimen liquid is allowed to flow out into the flow path 46, it is not necessary to pressurize the inside of the specimen liquid introduction well 2 or the extraction well 42 in order to prevent the backflow of the specimen liquid. Therefore, if the test chip 20 is used, microorganisms can be easily detected with high accuracy by optimizing the amount of the sample liquid.

  Similarly, since the flow path 46 is connected to the side wall surface of the luminescent well 3, the dissolution of the solid luminescent reagent 5 proceeds from both the upper and lower directions. Therefore, if the test chip 20 is used, the dissolution rate of the luminescent reagent 5 increases accordingly, and microorganisms can be detected quickly.

  If the test chip 20 is used, the background ATP in the sample liquid is decomposed and removed by the ATP-degrading enzyme, the ATP in the microorganism is extracted by the extraction reagent 6, and then the sample liquid and the luminescent reagent 5 are mixed. Since ATP in the sample solution is amplified before the detection, microorganisms can be detected with such high sensitivity. If the ATP-degrading enzyme also serves as the ADP-degrading enzyme, the background ADP is decomposed and removed in addition to the background ATP, so that microorganisms can be detected with higher accuracy.

(Third embodiment)
A third embodiment of the present invention will be described with reference to FIGS. FIG. 5 is a plan view of an inspection chip according to the third embodiment. 6 is a cross-sectional view taken along line VI-VI in FIG.

  In the testing chip 30 according to the third embodiment, a bead storage well 48 that constitutes a part of the flow path 41 is further formed on the substrate 1a, and the substrate 1b with respect to the substrate 1a on the surface of the substrate 1a This is also different from the inspection chip 10 in that a bead storage well opening 48a is further formed on the opposite surface. In addition, the test chip 30 is also characterized in that instead of the ATP-degrading enzyme being immobilized on the inner wall surface of the flow path 41, the beads 8 having the ATP-degrading enzyme immobilized are housed in the bead housing well 48. This is different from the inspection chip 10.

  The ATP-degrading enzyme immobilized on the beads 8 decomposes and removes ATP in the liquid stored in the bead storage well 48. As the ATP degrading enzyme, the same ATP degrading enzyme as that immobilized on the inner wall surface of the flow channel 41 in the test chip 10 according to the first embodiment can be used, and the ATP degrading enzyme can also be used. preferable.

  The bead storage well opening 48 a is covered with the filter 15. The filter 15 is for preventing contamination of impurities such as microorganisms into the bead storage well 48. As the filter 15, the same filter as the filter 13 may be used.

  The bead storage well 48 is formed by a side wall surface extending in a cylindrical shape from the edge of the bead storage well opening 48a toward the substrate 1b and a bottom surface connected to the end of the side wall surface. . Here, the bottom surface of the bead storage well 48 is recessed in a direction away from the end of the side wall surface. Specifically, the bottom surface has an opening area when a well (excluding a columnar portion formed by a side wall surface) is cut in a virtual plane parallel to the surface of the substrate 1a. It is formed in a tapered shape (conical shape or truncated cone shape) so as to become smaller toward.

  The sample liquid introduction well 2 and the bead storage well 48 are connected by a flow path 47. Specifically, one end of the channel 47 is connected to the end portion of the tapered bottom surface of the sample liquid introduction well 2, and the other end of the channel 47 is connected to the side wall surface of the bead storage well 48. Therefore, the sample liquid in the sample liquid introduction well 2 can flow into the bead storage well 48 via the flow path 47. The bead storage well 48 and the extraction well 42 are connected by a flow path 49. Specifically, one end of the channel 49 is connected to the end of the tapered bottom surface of the bead storage well 48, and the other end of the channel 49 is connected to the side wall surface of the extraction well 42. Accordingly, the sample liquid in the extraction well 42 can flow into the extraction well 42 via the flow path 49. The connection portion between the bead storage well 48 and the flow path 49 is covered with the filter 16. Here, the filter 16 may be any filter that allows permeation of microorganisms and prevents the beads 8 from flowing out. The average diameter of the beads 8 is preferably larger than that of the microorganism. Therefore, the average diameter of the beads 8 is preferably 10 μm or more, and the average pore diameter of the filter 16 is preferably less than 10 μm. The bead storage well 48 constitutes a channel 41 together with the channel 47 and the channel 49. That is, the channel 41 connects the sample liquid introduction well 2 and the extraction well 42.

  Next, a method for detecting microorganisms in a specimen using the test chip 30 will be described.

  First, the sample liquid is introduced into the sample liquid introduction well 2. Further, the photodetector 21 having the light receiving portion 21a is installed so that the light receiving portion 21a faces the light emitting well 3 and contacts the surface of the substrate 1c. Further, the pressure ports 22, 26 and 23 are installed so as to cover the sample liquid introduction well opening 2a, the bead storage well opening 48a and the extraction well opening 42a, respectively. In FIG. 6, the photodetector 21, the light receiving unit 21a, and the pressure ports 26 and 24 are indicated by two-dot chain lines (the pressure port 22 is not shown in FIG. 6).

  Next, the sample liquid introduced into the sample liquid introduction well 2 is circulated through the flow path 47 and stored in the bead storage well 48 by pressurizing the inside of the sample liquid introduction well 2 with the pressure port 22. When the sample liquid is stored in the bead storage well 48, the background ATP in the sample liquid reacts with the ATP-degrading enzyme immobilized on the beads 8 and is decomposed in the bead storage well 48.

  Next, after a sufficient time has elapsed for decomposition of the background ATP, the inside of the bead storage well 48 is pressurized by the pressure port 26, whereby the sample liquid stored in the bead storage well 48 is circulated through the flow path 49. And stored in the extraction well 42. At this time, in order to prevent the backflow of the sample liquid and the pressure loss in the bead storage well 48, the inside of the sample liquid introduction well 2 is simultaneously pressurized by the pressure port 22. When the sample liquid is stored in the extraction well 42, the solid extraction reagent 6 stored in the extraction well 42 is dissolved in the sample liquid, and ATP in the microorganism is extracted in the extraction well 42.

  Finally, after a sufficient time for extraction of ATP has elapsed, the inside of the extraction well 42 is pressurized by the pressure port 23, whereby the sample liquid accommodated in the extraction well 42 is circulated through the flow path 43 to emit light. 3 to accommodate. At this time, in order to prevent the back flow of the sample liquid and the pressure loss in the extraction well 42, the inside of the sample liquid introduction well 2 and the bead storage well 48 are simultaneously pressurized by the pressure ports 22 and 26, respectively. When the sample liquid is accommodated in the luminescent well 3, the solid luminescent reagent 5 is dissolved in the sample liquid and reacts with ATP in the sample liquid in the luminescent well 3 to generate luminescence. The light emitted from the luminescent reagent 5 enters the light receiving portion 21 a and is detected by the photodetector 21. In this way, microorganisms in the specimen are detected.

  In the test chip 30, the background ATP in the sample liquid is almost completely decomposed and removed by the ATP-degrading enzyme immobilized on the beads 8 while the sample liquid is stored in the bead storage well 48. Therefore, if the inspection chip 30 is used, microorganisms can be detected with such high accuracy.

  Further, in the test chip 30, the beads 8 having the ATP-degrading enzyme immobilized are accommodated in the bead storage well 48, so that a sufficient amount of ATP-degrading enzyme is immobilized in the chip. It is not necessary to form the flow path 41 in a meandering manner as in the chip 10. Therefore, in the inspection chip 30, the chip can have the same background ATP removal capability as the inspection chip 10, and the chip can be made smaller than the inspection chip 10.

  Further, in the test chip 30, the bead storage well opening 48 a is covered with the filter 15, thereby preventing impurities such as microorganisms from entering the bead storage well 48. Therefore, if the inspection chip 30 is used, microorganisms can be detected with such high accuracy.

  In the test chip 30, the flow path 47 is connected to the end of the tapered bottom surface of the sample liquid introduction well 2, so that the sample liquid stored in the sample liquid introduction well 2 flows out into the flow path 47. When the operation is performed, the amount of the sample liquid remaining in the well is sufficiently reduced. Further, since the flow channel 49 is connected to the end of the tapered bottom surface of the bead storage well 48, when an operation for causing the sample liquid stored in the bead storage well 48 to flow out to the flow channel 49 is performed, the well The amount of the sample liquid remaining in the inside is sufficiently reduced. As described above, the amount of the sample solution remaining in the sample solution introduction well 2 and the bead storage well 48 is sufficiently reduced. Therefore, if the test chip 30 is used, the emission intensity corresponding to the amount of the sample in the introduced sample solution. Can be measured with high accuracy, and microorganisms can be detected with high accuracy and reproducibility. Here, the angle formed by the tapered bottom surfaces of the sample liquid introduction well 2 and the bead storage well 48 and the surface of the substrate 1a is not particularly limited, but may be, for example, 30 to 60 degrees.

  Further, in the test chip 30, the flow path 47 is connected to the side wall surface of the bead storage well 48, so that the sample liquid that has circulated through the flow path 47 is above the beads 8 stored in the bead storage well 48. Then, it flows into the bead storage well 48, and the contact between the beads 8 and the sample liquid proceeds from both the upper and lower directions. That is, on the one hand, ATP in the sample liquid that flows out from the flow path 47 and directly hits the beads 8 reacts with the ATP-degrading enzyme immobilized on the upper part of the beads 8 and is stored in the amplification well 45 on the other hand. ATP in the sample solution reacts with the ATP-degrading enzyme immobilized on the lower part of the solid amplification reagent 7. Therefore, if the test chip 30 is used, the reaction rate between the ATP-degrading enzyme immobilized on the beads 8 and ATP in the sample liquid increases, and microorganisms can be detected quickly.

  In addition, when the inside of the sample liquid introduction well 2 is pressurized and the sample liquid is transferred to the bead storage well 48, if the flow path 47 is connected to the bottom surface of the bead storage well 48, for example, the air The sample is introduced into the sample liquid stored in the bead storage well 48 from the introduction well 2 through the flow path 47, bubbles are generated in the sample liquid, and the bubbles are supplied from the bead storage well 48 to the flow path 49. May interfere with the outflow. On the other hand, in the test chip 30, the flow path 47 is connected to the side wall surface of the bead storage well 48. Therefore, by optimizing the amount of the sample solution, the bubbles in the sample solution as described above can be obtained. Occurrence is prevented. That is, when the flow path 47 is connected to a portion of the side wall surface of the bead storage well 48 that is above the liquid surface of the sample liquid stored in the bead storage well 48, the generation of bubbles as described above occurs. Is prevented. Therefore, if the test chip 30 is used, by optimizing the amount of the sample liquid, the sample liquid is smoothly transferred from the bead storage well 48 to the extraction well 42 through the flow path 49, and the microorganisms are detected smoothly. can do.

  When the flow path 47 is connected to a portion of the side wall surface of the bead storage well 48 that is above the liquid level of the sample liquid stored in the bead storage well 48, the backflow of the stored sample liquid is caused. It is surely prevented. Further, when the specimen liquid is allowed to flow out into the flow path 49, it is not necessary to pressurize the inside of the specimen liquid introduction well 2 in order to prevent the backflow of the specimen liquid. Therefore, if the test chip 30 is used, microorganisms can be detected with high accuracy and simply by optimizing the amount of the sample liquid.

  Similarly, since the flow path 43 is connected to the side wall surface of the luminescent well 3, the dissolution of the solid luminescent reagent 5 proceeds from both the upper and lower directions. Therefore, if the test chip 30 is used, the dissolution rate of the luminescent reagent 5 increases accordingly, and microorganisms can be detected quickly.

(Fourth embodiment)
A fourth embodiment of the present invention will be described with reference to FIGS. FIG. 7 is a plan view of an inspection chip according to the fourth embodiment. FIG. 8 is a cross-sectional view taken along line VIII-VIII in FIG.

  In the testing chip 40 according to the fourth embodiment, a waste liquid storage well 51 and an auxiliary liquid storage well 52 are further formed on the substrate 1a, and the surface of the substrate 1a opposite to the substrate 1b with respect to the substrate 1a. In addition, it differs from the inspection chip 10 according to the first embodiment in that a waste liquid storage well opening 51a and an auxiliary liquid storage well opening 52a are formed. The inspection chip 40 is also different from the inspection chip 10 in that the extraction well 42 does not constitute a part of the flow path 4. The test chip 40 is also different from the test chip 10 in that the flow path 4 does not have a meandering portion and that both ends of each flow path are connected to the bottom surface of the well.

  The auxiliary liquid storage well 52 is formed by a side wall surface extending in a cylindrical shape from the edge of the auxiliary liquid storage well opening 52a toward the substrate 1b side, and a bottom surface connected to the end of the side wall surface. . Here, the bottom surface of the auxiliary liquid containing well 52 is recessed in a direction away from the end of the side wall surface. Specifically, the bottom surface has an opening area when a well (excluding a columnar portion formed by a side wall surface) is cut in a virtual plane parallel to the surface of the substrate 1a. It is formed in a tapered shape (conical shape or truncated cone shape) so as to become smaller toward.

  The sample liquid introduction well 2 and the waste liquid storage well 51 are connected by a flow path 53. Specifically, one end of the flow path 53 is connected to the end of the tapered bottom surface of the sample liquid introduction well 2, and the other end of the flow path 53 is connected to the side wall surface of the waste liquid containing well 51. Accordingly, the liquid in the sample liquid introduction well 2 can flow into the waste liquid storage well 51 via the flow path 53. The connection portion between the sample liquid introduction well 2 and the flow path 53 is covered with a filter 17. The filter 17 is for capturing microorganisms to be detected. Therefore, the average pore diameter of the filter 17 is preferably 0.1 to 1 μm.

  The auxiliary liquid storage well 52 and the sample liquid introduction well 2 are connected by a flow path 54. Specifically, one end of the flow channel 54 is connected to the end portion of the tapered bottom surface of the auxiliary liquid containing well 52, and the other end of the flow channel 54 is connected to the side wall surface of the sample liquid introduction well 2. Therefore, the liquid in the auxiliary liquid storage well 52 can flow into the sample liquid introduction well 2 via the flow path 54.

  The auxiliary liquid storage well 52 and the extraction well 42 are connected by a flow path 55. Specifically, one end of the channel 55 is connected to the end of the tapered bottom surface of the auxiliary liquid containing well 52, and the other end of the channel 55 is connected to the side wall surface of the extraction well 42. Accordingly, the liquid in the auxiliary liquid storage well 52 can flow into the extraction well 42 via the flow path 55.

  The extraction well 42 and the sample liquid introduction well 2 are connected by a flow path 56. Specifically, one end of the flow channel 56 is connected to the end of the tapered bottom surface of the extraction well 42, and the other end of the flow channel 56 is connected to the side wall surface of the sample liquid introduction well 2. Accordingly, the liquid in the extraction well 42 can flow into the sample liquid introduction well 2 via the flow path 56.

  Further, the sample liquid introduction well 2 and the light emitting well 3 are connected by a flow path 4. Specifically, one end of the flow path 4 is connected to the end of the tapered bottom surface of the sample liquid introduction well 2, and the other end of the flow path 4 is connected to the side wall surface of the light emitting well 3. Accordingly, the liquid in the sample liquid introduction well 2 can flow into the light emitting well 3 through the flow path 4.

  Next, a method for detecting microorganisms in a specimen using the test chip 40 will be described.

  First, the sample liquid is introduced into the sample liquid introduction well 2 and the auxiliary liquid is stored in the auxiliary liquid storage well 52. Here, examples of the auxiliary liquid include pure water and LP (constituted with polysorbate 80, lecithin, and peptone) diluted liquid. It is preferable that ATP and microorganisms mixed in the auxiliary liquid are previously removed by autoclaving or the like.

  Further, the photodetector 21 having the light receiving portion 21a is installed so that the light receiving portion 21a faces the light emitting well 3 and contacts the surface of the substrate 1c. In addition, the pressure port is installed so as to cover the specimen liquid introduction well opening 2a, the waste liquid storage well opening 51a, the auxiliary liquid storage well opening 52a, the extraction well opening 42a, and the light emitting well opening 3a. In FIG. 8, the photodetector 21, the light receiving unit 21 a, and the pressure ports 22, 23, and 27 are indicated by two-dot chain lines (the pressure ports 22, 23, and 27 are the sample liquid introduction well opening 2 a, respectively). The extraction well opening 42a and the light emission well opening 3a are covered).

  Next, the sample liquid introduced into the sample liquid introduction well 2 is circulated through the flow path 53 and stored in the waste liquid storage well 51 by pressurizing the inside of the sample liquid introduction well 2 with the pressure port 22. At this time, in order to prevent the inflow of the sample liquid into the other wells and the pressure loss in the sample liquid introduction well 2, the inside of the auxiliary liquid storage well 52, the extraction well 42, and the light emitting well 3 are simultaneously pressurized with the pressure port. When the sample liquid flows out from the sample liquid introduction well 2 and is accommodated in the waste liquid accommodation well 51, the microorganisms in the sample liquid remain in the sample liquid introduction well 2 by the filter 17, and other components (background ATP) ) Flows out from the sample liquid introduction well 2 and is stored in the waste liquid storage well 51.

  Next, the auxiliary liquid stored in the auxiliary liquid storage well 52 is circulated through the flow channel 54 and stored in the sample liquid introduction well 2 by pressurizing the inside of the auxiliary liquid storage well 52 with a pressure port. At this time, in order to prevent the auxiliary liquid from flowing into the extraction well 42, the inside of the extraction well 42 is simultaneously pressurized with the pressure port 23.

  Next, the auxiliary liquid stored in the sample liquid introduction well 2 is circulated through the flow path 53 and stored in the waste liquid storage well 51 by pressurizing the inside of the sample liquid introduction well 2 with the pressure port 22. At this time, in order to prevent the inflow of the sample liquid into the other wells and the pressure loss in the sample liquid introduction well 2, the inside of the auxiliary liquid storage well 52, the extraction well 42, and the light emitting well 3 are simultaneously pressurized with the pressure port. When the auxiliary liquid flows out from the sample liquid introduction well 2 and is stored in the waste liquid storage well 51, components other than the microorganisms (including background ATP) in the sample liquid are washed away by the filter 17, and the waste liquid storage well 51. Is housed.

  Next, the auxiliary liquid stored in the auxiliary liquid storage well 52 is circulated through the flow path 55 and stored in the extraction well 42 by pressurizing the inside of the auxiliary liquid storage well 52 with a pressure port. At this time, in order to prevent the auxiliary liquid from flowing into the sample liquid introduction well 2, the inside of the sample liquid introduction well 2 is simultaneously pressurized by the pressure port 22. When the auxiliary liquid is stored in the extraction well 42, the solid extraction reagent 6 stored in the extraction well 42 is dissolved in the auxiliary liquid.

  Next, the auxiliary liquid in which the extraction reagent 6 is dissolved is pressurized in the extraction well 42 through the pressure port 23, and is circulated through the flow path 56 to be stored in the sample liquid introduction well 2. At this time, in order to prevent the inflow of the auxiliary liquid into the auxiliary liquid containing well 52 and the pressure loss in the extraction well 42, the inside of the auxiliary liquid containing well 52 is simultaneously pressurized with the pressure port. When the auxiliary liquid in which the extraction reagent 6 is dissolved is stored in the sample liquid introduction well 2, ATP in the microorganism is extracted in the sample liquid introduction well 2.

  Finally, after a sufficient time has passed for extraction of ATP, the inside of the sample liquid introduction well 2 is pressurized by the pressure port 22, whereby the auxiliary liquid accommodated in the sample liquid introduction well 2 is circulated through the flow path 4. And accommodated in the light emitting well 3. At this time, in order to prevent the inflow of the sample liquid into the other wells and the pressure loss in the sample liquid introduction well 2, the interiors of the waste liquid storage well 51, the auxiliary liquid storage well 52, and the extraction well 42 are simultaneously pressurized with the pressure port. . When the auxiliary liquid is accommodated in the luminescent well 3, the solid luminescent reagent 5 is dissolved in the auxiliary liquid and reacts with the ATP in the auxiliary liquid in the luminescent well 3 to generate luminescence. The light emitted from the luminescent reagent 5 enters the light receiving portion 21 a and is detected by the photodetector 21. In this way, microorganisms in the specimen are detected.

  As described above, in the test chip 40, components other than microorganisms (including background ATP) in the sample liquid are washed away from the sample liquid introduction well 2 by the filter 17 and stored in the waste liquid storage well 51. . Therefore, if the test chip 40 is used, light emission caused by impurities such as microorganisms in the sample liquid can be prevented, and microorganisms can be detected with such high accuracy.

  In the test chip 40, the flow paths 53 and 4 are connected to the end of the tapered bottom surface of the sample liquid introduction well 2, so that the sample liquid stored in the sample liquid introduction well 2 is passed through the flow path 53. Alternatively, when the operation of flowing out to 4 is performed, the amount of the sample liquid remaining in the well is sufficiently reduced. Further, since the flow paths 54 and 55 are connected to the end of the tapered bottom surface of the auxiliary liquid storage well 52, an operation for causing the sample liquid stored in the auxiliary liquid storage well 52 to flow out to the flow path 54 or 55. When the test is performed, the amount of the sample liquid remaining in the well is sufficiently reduced. Further, since the flow path 56 is connected to the end of the tapered bottom surface of the extraction well 42, when an operation of flowing the sample liquid stored in the extraction well 42 into the flow path 56 is performed, The amount of the remaining sample liquid is sufficiently reduced. As described above, the amount of the sample liquid remaining in the sample liquid introduction well 2, the auxiliary liquid storage well 52, and the extraction well 42 is sufficiently reduced. Therefore, if the test chip 40 is used, the amount of the sample in the introduced sample liquid. Can be measured with high accuracy, and microorganisms can be detected with high accuracy and good reproducibility. Here, the angle formed by the tapered bottom surfaces of the sample liquid introduction well 2, the auxiliary liquid storage well 52, and the extraction well 42 and the surface of the substrate 1a is not particularly limited, and may be, for example, 30 to 60 degrees.

  In the inspection chip 40, the flow paths 55 and 4 are connected to the side walls of the extraction well 42 and the light emitting well 3, respectively. Therefore, the liquid flowing through the flow path 55 or 4 is extracted from the extraction well 42 or the light emission, respectively. The solid reagent contained in the well 3 flows into the well from above, and the dissolution of the solid extraction reagent 6 and the luminescent reagent 5 proceeds in both the upper and lower directions. Therefore, if the test chip 40 is used, the dissolution rate of the extraction reagent 6 and the luminescent reagent 5 increases accordingly, and microorganisms can be detected quickly.

  In the test chip 40, the flow paths 54 and 56 are each connected to the side wall surface of the sample liquid introduction well 2, and the flow path 55 is connected to the side wall surface of the extraction well 42. By optimizing the amount of the auxiliary liquid, generation of bubbles in the liquid stored in the sample liquid introduction well 2 and the extraction well 42 is prevented. Therefore, if the test chip 40 is used, by optimizing the amounts of the sample liquid and the auxiliary liquid, the liquid can be smoothly transferred from the sample liquid introduction well 2 to the waste liquid containing well 51 or the light emitting well 3, and the extraction well. The sample is transferred from 42 to the sample liquid introduction well 2, and microorganisms can be detected smoothly.

  Further, in the test chip 40, since the flow path for the liquid to flow into the well is connected to the side wall surface of the well, the stored liquid is optimized by optimizing the amount of the sample liquid and the auxiliary liquid. Is prevented reliably. Further, when the liquid stored in the well is allowed to flow out, it is not necessary to pressurize the inside of another well in order to prevent the liquid from flowing backward. Therefore, if the test chip 40 is used, microorganisms can be detected with high accuracy and simplicity by optimizing the amounts of the sample liquid and the auxiliary liquid.

(Fifth embodiment)
A fifth embodiment of the present invention will be described with reference to FIGS. FIG. 9 is a plan view of an inspection chip according to the fifth embodiment. 10 is a cross-sectional view taken along line XX in FIG.

  In the testing chip 50 according to the fifth embodiment, an ATP decomposition well 57 is further formed on the substrate 1a, and an ATP decomposition well is opened on the surface of the substrate 1a opposite to the substrate 1b with respect to the substrate 1a. It differs from the test chip 40 according to the fourth embodiment in that a mouth 57a is formed and a solid ATP-degrading enzyme 9 is housed in the ATP-decomposing well 57. The inspection chip 50 is also different from the inspection chip 40 in that the waste liquid containing well 51 is connected to the flow path 61 branched from the middle of the flow path 4. Further, in the test chip 50, the flow path 4 has a serpentine portion (flow path 4a), and an antibody that specifically binds to the microorganism to be detected is immobilized on the inner wall surface of this portion. However, it is different from the inspection chip 40.

  Examples of ATPase 9 include apyrase, adenosine phosphate deaminase, luciferase and the like. The solid ATP-degrading enzyme 9 is preferably lyophilized. The lyophilized reagent can maintain its original activity for a long period (for example, more than half a year).

  The ATP decomposition well opening 57 a is covered with the filter 18. The filter 18 is for preventing contamination of impurities such as microorganisms into the ATP decomposition well 57. As the filter 18, the same filter as the filter 13 may be used.

  The ATP decomposition well 57 is formed by a side wall surface extending in a cylindrical shape from the edge of the ATP decomposition well opening 57a toward the substrate 1b and a bottom surface connected to the end of the side wall surface. Here, the bottom surface of the ATP decomposition well 57 is recessed in a direction away from the end of the side wall surface. Specifically, the bottom surface has an opening area when a well (excluding a columnar portion formed by a side wall surface) is cut in a virtual plane parallel to the surface of the substrate 1a. It is formed in a tapered shape (conical shape or truncated cone shape) so as to become smaller toward.

  Further, as described above, the flow path 4 is connected to the side wall surface of the waste liquid storage well 51 via the flow path 61 branched from the middle thereof. The channel 4 is formed on the side of the light emitting well 3 with respect to the connection part between the channel 4 and the channel 61 and the channel 4a formed in a meandering manner on the sample solution introduction well 2 side. And a flow path 4b.

  The sample liquid introduction well 2 and the waste liquid storage well 51 are connected by flow paths 4 a and 61. Specifically, the flow paths 4 a and 61 are connected to each other at one end, and the other end of the flow path 4 a is connected to the end of the tapered bottom surface of the sample liquid introduction well 2. The end is connected to the side wall surface of the waste liquid storage well 51. Therefore, the liquid in the sample liquid introduction well 2 can flow into the waste liquid storage well 51 through the flow paths 4a and 61.

  The auxiliary liquid storage well 52 and the ATP decomposition well 57 are connected by a flow path 58. Specifically, one end of the channel 58 is connected to the end of the tapered bottom surface of the auxiliary liquid containing well 52, and the other end of the channel 58 is connected to the side wall surface of the ATP decomposition well 57. Therefore, the liquid in the auxiliary liquid storage well 52 can flow into the ATP decomposition well 57 via the flow path 58.

  The ATP decomposition well 57 and the sample liquid introduction well 2 are connected by a flow path 59. Specifically, one end of the channel 59 is connected to the end of the tapered bottom surface of the ATP decomposition well 57, and the other end of the channel 59 is connected to the side wall surface of the sample liquid introduction well 2. Accordingly, the liquid in the ATP decomposition well 57 can flow into the sample liquid introduction well 2 through the flow path 59.

  The extraction well 42 and the sample liquid introduction well 2 are connected by a flow path 56. Specifically, one end of the flow channel 56 is connected to the end of the tapered bottom surface of the extraction well 42, and the other end of the flow channel 56 is connected to the side wall surface of the sample liquid introduction well 2. Accordingly, the liquid in the extraction well 42 can flow into the sample liquid introduction well 2 via the flow path 56.

  Next, a method for detecting microorganisms in a specimen using the test chip 50 will be described.

  First, the sample liquid is introduced into the sample liquid introduction well 2 and the auxiliary liquid is stored in the auxiliary liquid storage well 52. Here, examples of the auxiliary liquid include pure water and LP diluted liquid. ATP and microorganisms mixed in the auxiliary liquid are preferably removed in advance by autoclaving.

  Further, the photodetector 21 having the light receiving portion 21a is installed so that the light receiving portion 21a faces the light emitting well 3 and contacts the surface of the substrate 1c. Further, the pressure port covers the specimen liquid introduction well opening 2a, the waste liquid storage well opening 51a, the auxiliary liquid storage well opening 52a, the ATP decomposition well opening 57a, the extraction well opening 42a, and the light emitting well opening 3a. Install in. In FIG. 10, the photodetector 21, the light receiving unit 21 a, and the pressure ports 22, 28, and 27 are indicated by two-dot chain lines (the pressure ports 22, 28, and 27 are the sample solution introduction well opening 2 a, respectively). ATP decomposition well opening 57a and light emitting well opening 3a are covered).

  Next, the sample liquid introduced into the sample liquid introduction well 2 is circulated through the flow paths 4 a and 61 by being pressurized inside the sample liquid introduction well 2 by the pressure port 22, and is accommodated in the waste liquid accommodation well 51. At this time, in order to prevent the inflow of the sample liquid into other wells and the pressure loss in the sample liquid introduction well 2, the pressure in the auxiliary liquid storage well 52, the extraction well 42, the ATP decomposition well 57 and the light emitting well 3 is simultaneously increased. Pressurize at the port. When the sample liquid flows through the flow paths 4a and 61 and is stored in the waste liquid storage well 51, only the microorganisms to be detected in the sample liquid are bound to the antibody immobilized on the flow path 4a. The components (including background ATP) are stored in the waste liquid storage well 51.

  Next, the auxiliary liquid stored in the auxiliary liquid storage well 52 is circulated through the flow path 58 and stored in the ATP decomposition well 57 by pressurizing the inside of the auxiliary liquid storage well 52 with a pressure port. At this time, in order to prevent the inflow of the auxiliary liquid into the sample liquid introduction well 2 and the extraction well 42 and the pressure loss of the auxiliary liquid storage well 52, the inside of the sample liquid introduction well 2 and the extraction well 42 are simultaneously connected to the pressure port 22 and Pressurize with 23. When the auxiliary liquid is stored in the ATP decomposition well 57, the solid ATP degrading enzyme 9 stored in the ATP decomposition well 57 is dissolved in the auxiliary liquid.

  Next, the auxiliary liquid in which the ATP-degrading enzyme 9 is dissolved is circulated through the flow path 59 by being pressurized inside the ATP-decomposing well 57 through the pressure port 28 and is stored in the sample liquid introducing well 2. At this time, in order to prevent the inflow of the auxiliary liquid into the auxiliary liquid containing well 52 and the pressure loss in the ATP decomposition well 57, the inside of the auxiliary liquid containing well 52 is simultaneously pressurized with the pressure port. When the auxiliary liquid in which the ATP-degrading enzyme 9 is dissolved is accommodated in the sample liquid introduction well 2, the background ATP in the sample liquid introduction well 2 is decomposed.

  Next, the auxiliary liquid stored in the sample liquid introduction well 2 is circulated through the flow paths 4 a and 61 by being pressurized inside the sample liquid introduction well 2 by the pressure port 22, and is stored in the waste liquid storage well 51. At this time, in order to prevent the inflow of the sample liquid into other wells and the pressure loss in the sample liquid introduction well 2, the pressure in the auxiliary liquid storage well 52, the extraction well 42, the ATP decomposition well 57 and the light emitting well 3 is simultaneously increased. Pressurize at the port. When the auxiliary liquid flows through the flow paths 4a and 61 and is stored in the waste liquid storage well 51, the background ATP in the flow paths 4a and 61 is decomposed by the ATP-degrading enzyme in the auxiliary liquid, and the detection target Components other than microorganisms (including background ATP) are washed away from the sample liquid introduction well 2 and the flow paths 4a and 61 and accommodated in the waste liquid accommodation well 51.

  Next, the auxiliary liquid stored in the auxiliary liquid storage well 52 is circulated through the flow channel 54 and stored in the sample liquid introduction well 2 by pressurizing the inside of the auxiliary liquid storage well 52 with a pressure port. At this time, in order to prevent the inflow of the auxiliary liquid into the extraction well 42 and the ATP decomposition well 57 and the pressure loss in the auxiliary liquid storage well 52, the pressure ports 23 and 28 are respectively connected to the inside of the extraction well 42 and the ATP decomposition well 57 at the same time. Pressurize with.

  Next, the auxiliary liquid stored in the sample liquid introduction well 2 is circulated through the flow paths 4 a and 61 by being pressurized inside the sample liquid introduction well 2 by the pressure port 22, and is stored in the waste liquid storage well 51. At this time, in order to prevent the inflow of the sample liquid into other wells and the pressure loss in the sample liquid introduction well 2, the pressure in the auxiliary liquid storage well 52, the extraction well 42, the ATP decomposition well 57 and the light emitting well 3 is simultaneously increased. Pressurize at the port. When the auxiliary liquid flows through the flow paths 4a and 61 and is stored in the waste liquid storage well 51, among the components in the auxiliary liquid, components other than the microorganisms to be detected (including background ATP and ATP-degrading enzyme). Is further washed away from the sample liquid introduction well 2 and the flow paths 4 a and 61 and is stored in the waste liquid storage well 51.

  Next, the auxiliary liquid stored in the auxiliary liquid storage well 52 is circulated through the flow path 55 and stored in the extraction well 42 by pressurizing the inside of the auxiliary liquid storage well 52 with a pressure port. At this time, in order to prevent the flow of the auxiliary liquid into the sample liquid introduction well 2 and the ATP decomposition well 57 and the pressure loss in the auxiliary liquid storage well 52, the inside of the sample liquid introduction well 2 and the ATP decomposition well 57 are simultaneously pressurized. Pressurize at ports 22 and 28. When the auxiliary liquid is stored in the extraction well 42, the solid extraction reagent 6 stored in the extraction well 42 is dissolved in the auxiliary liquid.

  Next, the auxiliary liquid in which the extraction reagent 6 is dissolved is pressurized in the extraction well 42 through the pressure port 23, and is circulated through the flow path 56 to be stored in the sample liquid introduction well 2. At this time, in order to prevent the inflow of the auxiliary liquid into the auxiliary liquid containing well 52 and the pressure loss in the extraction well 42, the inside of the auxiliary liquid containing well 52 is simultaneously pressurized with the pressure port.

  Finally, the auxiliary liquid stored in the sample liquid introduction well 2 is circulated through the flow path 4 (4a and 4b) by pressurizing the inside of the sample liquid introduction well 2 through the pressure port 22, and is stored in the light emitting well 3. Let At this time, in order to prevent the flow of the sample liquid into the other wells and the pressure loss in the sample liquid introduction well 2, the inside of the waste liquid storage well 51, the auxiliary liquid storage well 52, the ATP decomposition well 57 and the extraction well 42 are simultaneously formed. Pressurize at the pressure port. When the auxiliary liquid in which the extraction reagent 6 is dissolved flows through the flow path 4a, ATP in the microorganism to be detected that is bound to the antibody immobilized in the flow path 4a is extracted into the auxiliary liquid. When the auxiliary liquid is accommodated in the luminescent well 3, the solid luminescent reagent 5 is dissolved in the auxiliary liquid and reacts with the ATP in the auxiliary liquid in the luminescent well 3 to generate luminescence. The light emitted from the luminescent reagent 5 enters the light receiving portion 21 a and is detected by the photodetector 21. In this way, microorganisms in the specimen are detected.

  Since the test chip 50 includes an antibody as described above, it can be used for detection of a specific microorganism in the sample.

  As described above, in the test chip 50, the components other than the microorganism to be detected (including background ATP and ATP-degrading enzyme) among the components in the sample liquid and the auxiliary liquid include the sample liquid introduction well 2 and the flow. It is washed away from the path 4 a and stored in the waste liquid storage well 51. Therefore, if the inspection chip 50 is used, the detection target microorganism can be detected with such high accuracy.

  Further, in the test chip 50, the ATP decomposition well opening 57 a is covered with the filter 18, thereby preventing impurities such as microorganisms from entering the ATP decomposition well 57. Therefore, if the inspection chip 50 is used, the detection target microorganism can be detected with such high accuracy.

  Further, in the test chip 50, since the flow path 4a is connected to the end of the tapered bottom surface of the sample liquid introduction well 2, the sample liquid stored in the sample liquid introduction well 2 flows out to the flow path 4a. When the operation is performed, the amount of the sample liquid remaining in the well is sufficiently reduced. Further, since the flow paths 54, 55 and 58 are respectively connected to the end portions of the tapered bottom surface of the auxiliary liquid storage well 52, the sample liquid stored in the auxiliary liquid storage well 52 is passed through the flow paths 54, 55. Alternatively, when the operation of allowing the sample liquid to flow out to 58 is performed, the amount of the sample liquid remaining in the well is sufficiently reduced. Further, since the channel 59 is connected to the end of the tapered bottom surface of the ATP decomposition well 57, when an operation for causing the sample liquid stored in the ATP decomposition well 57 to flow out to the channel 59 is performed, the well The amount of the sample liquid remaining in the inside is sufficiently reduced. Further, since the flow path 56 is connected to the end of the tapered bottom surface of the extraction well 42, when an operation of flowing the sample liquid stored in the extraction well 42 into the flow path 56 is performed, The amount of the remaining sample liquid is sufficiently reduced. As described above, the amount of the sample liquid remaining in the sample liquid introduction well 2, the auxiliary liquid containing well 52, the ATP decomposition well 57, and the extraction well 42 is sufficiently reduced. Therefore, if the test chip 50 is used, the introduced sample liquid is used. The emission intensity corresponding to the amount of the sample in the sample can be measured with high accuracy, and the detection target microorganism can be detected with high accuracy and high reproducibility. Here, the angle formed by the tapered bottom surfaces of the specimen liquid introduction well 2, the auxiliary liquid containing well 52, the ATP decomposition well 57, and the extraction well 42 and the surface of the substrate 1a is not particularly limited, but may be, for example, 30 to 60 degrees. That's fine.

  Further, in the inspection chip 50, the flow paths 58, 55 and 4 are connected to the side wall surfaces of the ATP decomposition well 57, the extraction well 42 and the light emitting well 3, respectively. The liquid flows into the well from the upper side of the solid reagent contained in the ATP decomposition well 57, the extraction well 42 or the luminescent well 3, respectively, and the solid ATP degrading enzyme 9, the extraction reagent 6 and the luminescent reagent 5 are dissolved. Will proceed from both directions. Therefore, if the test chip 50 is used, the dissolution rate of the ATP-degrading enzyme 9, the extraction reagent 6 and the luminescent reagent 5 is increased accordingly, and the detection target microorganism can be detected quickly.

  In the test chip 50, the flow paths 59, 54, and 56 are each connected to the side wall surface of the sample liquid introduction well 2, the flow path 58 is extracted to the side wall surface of the ATP decomposition well 57, and the flow path 55 is extracted. Since it is connected to the side wall surface of the well 42, by optimizing the amount of the sample liquid and the auxiliary liquid, the bubbles in the liquid stored in the sample liquid introduction well 2, the ATP decomposition well 57, and the extraction well 42 are reduced. Occurrence is prevented. Therefore, if the test chip 50 is used, the amount of the sample liquid and the auxiliary liquid is optimized, so that the liquid is smoothly transferred from the sample liquid introduction well 2 to the waste liquid containing well 51 or the light emitting well 3 and ATP decomposition. It is transferred from the well 57 or the extraction well 42 to the sample liquid introduction well 2, and the microorganisms to be detected can be detected smoothly.

  Further, in the test chip 50, since the flow path for the liquid to flow into the well is connected to the side wall surface of the well, the stored liquid is optimized by optimizing the amount of the sample liquid and the auxiliary liquid. Is prevented reliably. Further, when the liquid stored in the well is allowed to flow out, it is not necessary to pressurize the inside of another well in order to prevent the liquid from flowing backward. Therefore, if the test chip 50 is used, the microorganisms to be detected can be detected easily with high accuracy by optimizing the amounts of the sample liquid and the auxiliary liquid.

  The present invention is not limited to the first to fifth embodiments described above.

  For example, in the test chips 10, 30, 40, and 50 according to the first and third to fifth embodiments, the solid light-emitting reagent 5 is stored in the light-emitting well 3. The amplified luminescence reagent may be accommodated. Here, the “amplified luminescence reagent” means a reagent that amplifies ATP and simultaneously reacts with the amplified ATP to generate luminescence. Examples of such amplified luminescence reagent include AMP, polyphosphoric acid, Examples include reagents comprising ADK, PPK, firefly luciferin, firefly luciferase, and divalent metal ions.

  Further, in the test chips 30, 40 and 50 according to the third to fifth embodiments, the well in which the solid amplification reagent is stored is not formed in the main body, but the test according to the second embodiment. Similar to the chip 20, the amplification well 45 is formed as a part of the flow path 4 (the flow path 43 in the test chip 30) in the main body, and the amplification well 45 contains the solid amplification reagent 7. Also good.

  In the test chip 20 according to the second embodiment, the solid amplification reagent 7 is accommodated in the amplification well 45, but another amplification well is formed in the main body portion, and the solid amplification reagent is formed. May be accommodated in one amplification well, and the remaining portion may be accommodated in the other amplification well. That is, if the amplification reagent is a reagent composed of AMP, polyphosphoric acid, ADK and PPK, another amplification well is formed in the main body. For example, solid AMP and polyphosphoric acid are formed in one amplification well. Solid ADK and PPK may be stored in the other amplification well.

  Further, in the testing chip 50 according to the fifth embodiment, the flow path 4a is formed in a meandering shape, and an antibody that specifically binds to the microorganism to be detected is immobilized on the inner wall surface thereof. The bead storage well may be formed as a part of the flow path 4 (4a and 4b), and the bead storage well may store the beads on which the antibody is immobilized. In this case, the flow path 4 may not have a meandering portion.

  In the inspection chips 40 and 50 according to the fourth and fifth embodiments, the flow path connected to the auxiliary liquid accommodation well 52 is not specially treated, but is connected to the auxiliary liquid accommodation well 52. An ATP-degrading enzyme may be immobilized in the channel. Further, the connecting portion between the auxiliary liquid containing well 52 and each flow path is covered with the same filter as the filter 16, and the auxiliary liquid containing well 52 contains the beads 8 on which the ATP-degrading enzyme is immobilized. Good.

  Further, in the inspection chips 10, 20, 30, 40, and 50 according to the first to fifth embodiments, the side wall surface of the well is cylindrical, but the shape of the side wall surface of the well is a quadrangular column or the like. May be.

  In the inspection chips 10, 20, 30, 40, and 50 according to the first to fifth embodiments, the flow path is formed on the substrate 1a among the three layers of the substrates 1a to 1c. The intermediate layer substrate 1b among the three-layer substrates 1a to 1c may be formed. Further, the main body 1 of the inspection chip may be composed of, for example, a two-layer substrate, and the grooves constituting the flow path may be formed on both of the two-layer substrates.

  In the inspection chips 10, 20, 30, 40, and 50 according to the first to fifth embodiments, the flow path for the liquid to flow into the well is connected to the side wall surface of the well. The flow path for the liquid to flow in may be connected to the bottom surface of the well.

  Hereinafter, based on an Example and a comparative example, this invention is demonstrated more concretely. However, the present invention is not limited to the following examples.

Example 1
First, the configuration of the inspection chip used in this example will be described with reference to FIGS. FIG. 11 is a plan view of the inspection chip used in Example 1. FIG. 12 is a cross-sectional view taken along line XII-XII in FIG.

  As shown in FIGS. 11 and 12, the inspection chip 100 used in Example 1 includes a main body portion 1 including three layers of substrates 1 a to 1 c. The substrates 1a and 1b are each a PDMS substrate, and the substrate 1c is a glass substrate. The main body 1 includes a specimen liquid introduction well (first well) 2, a light emitting well (second well) 3, a specimen liquid introduction well opening (first opening) 2 a, and a light emitting well opening ( Secondly, an opening 3a and a flow path 4 that connects the sample liquid introduction well 2 and the light emitting well 3 are formed.

  The sample liquid introduction well 2 is formed by a side wall surface extending in a cylindrical shape from the edge of the sample liquid introduction well opening 2a toward the substrate 1b, and a bottom surface connected to an end of the side wall surface. . Here, the bottom surface of the sample fluid introduction well 2 is recessed in a direction away from the end of the side wall surface. Specifically, the bottom surface has an opening area when a well (excluding a columnar portion formed by a side wall surface) is cut in a virtual plane parallel to the surface of the substrate 1a. It is formed in a tapered shape (conical shape or truncated cone shape) so as to become smaller toward. The light emitting well 3 is formed by a side wall surface extending in a cylindrical shape from the edge of the light emitting well opening 3a toward the substrate 1b side, and a flat bottom surface connected to the end of the side wall surface. . Further, the sample liquid introduction well 2 and the light emitting well 3 are connected by a flow path 4. Specifically, one end of the channel 4 is connected to the end of the tapered bottom surface of the sample liquid introduction well 2, and the other end of the channel 4 is connected to the flat bottom surface of the light emitting well 3.

The luminescent well 3 contains a solid luminescent reagent (solid reagent) 5. Solid luminescent reagent 5 is firefly luciferase (manufactured by Wako Pure Chemical Industries, Ltd.) (5 ng), firefly luciferin (manufactured by Wako Pure Chemical Industries, Ltd.) (0.3 nmol), Tris-HCl (10 nmol), MgCl ₂ (2 nmol). And a reagent obtained by freeze-drying a mixture of trehalose (manufactured by Wako Pure Chemical Industries, Ltd.) (10 mg). Further, a light reflecting portion 11 made of aluminum is provided on the cylindrical side wall surface of the light emitting well 3 over the entire circumference of the side wall surface, and the light reflecting portion 11 surrounds the solid luminescent reagent 5. .

  Using such a test chip 100, the amount of luminescence generated by the reaction between ATP and the luminescent reagent was measured as follows.

  First, 200 μL of 1 nM ATP solution was introduced into the sample solution introduction well 2 with a micropipette. In addition, the photodetector 21 was installed so that the light receiving portion 21a faces the light emitting well 3 and is in contact with the surface of the substrate 1c. As the photodetector 21, a photomultiplier tube (Hamamatsu Photonics, H7155) was used. Moreover, the pressure port 22 connected to the pressure generator (Naganokeiki, PC20) was installed so as to cover the specimen liquid introduction well opening 2a.

  Next, by applying a pressure of 30 kPa to the inside of the sample liquid introduction well 2 through the pressure port 22, the ATP solution was accommodated in the light emission well 3 through the flow path 4, and the light emission intensity was measured by the photodetector 21.

(Comparative Example 1)
A test chip having the same configuration and size as the test chip 100 is prepared except that no reagent is stored in the light-emitting well, and using this, the ATP and the luminescent reagent are mixed as follows. The amount of luminescence generated by the reaction was measured.

  First, 100 μL of 2 nM ATP solution was introduced into the sample solution introduction well with a micropipette. Further, in the same manner as in Example 1, the photodetector and the pressure port were installed in the inspection chip.

  On the other hand, a solid luminescent reagent having the same composition as the luminescent reagent 5 used in Example 1 was dissolved in 100 μL of purified water, and the resulting aqueous solution was allowed to stand for 10 minutes.

  After introducing an aqueous solution that has been allowed to stand for 10 minutes into the light emitting well, a pressure of 30 kPa is applied to the inside of the sample liquid introducing well at the pressure port, so that the ATP solution is accommodated in the light emitting well through the flow path, and the light emission intensity is detected. The activity deterioration rate of the reagent (the emission intensity decrease rate with respect to the emission intensity measured in Example 1) was determined. The results are shown in Table 1.

(Comparative Example 2)
Except that the aqueous solution of the luminescent reagent was allowed to stand for 30 minutes, the reagent activity deterioration rate (the luminescence intensity reduction rate with respect to the luminescence intensity measured in Example 1) was determined in the same manner as in Comparative Example 1. The results are shown in Table 1.

  From Example 1 and Comparative Examples 1 and 2, it was shown that when the test chip of the present invention was used, the test could be performed while maintaining the reagent activity sufficiently high even during the reaction. Further, it was shown that the use of the test chip of the present invention eliminates the need for preparation and dispensing of the reagent solution, and thus the test can be performed simply.

  The test chip of the present invention can be used for tests performed using a protein-containing reagent, particularly for detection of microorganisms (bacteria, fungi, etc.) in foods and drinks using a luciferin-luciferase reaction.

It is a top view of the inspection chip concerning a 1st embodiment. It is sectional drawing along the II-II line of FIG. It is a top view of the inspection chip concerning a 2nd embodiment. FIG. 4 is a sectional view taken along line IV-IV in FIG. 3. It is a top view of the chip for inspection concerning a 3rd embodiment. It is sectional drawing along the VI-VI line of FIG. It is a top view of the chip | tip for a test | inspection which concerns on 4th Embodiment. It is sectional drawing along the VIII-VIII line of FIG. It is a top view of the inspection chip concerning a 5th embodiment. It is sectional drawing along the XX line of FIG. 2 is a plan view of an inspection chip used in Example 1. FIG. It is sectional drawing along the XII-XII line | wire of FIG.

Explanation of symbols

DESCRIPTION OF SYMBOLS 10,20,30,40,50 ... Test | inspection chip, 1 ... Main-body part, 1a-1c ... Board | substrate, 2 ... Sample liquid introduction well, 2a ... Sample liquid introduction well opening, 3 ... Luminescence well, 3a ... Luminescence well Open port 4, 4a, 4b ... flow path, 5 ... luminescent reagent, 6 ... extraction reagent, 7 ... amplification reagent, 8 ... bead, 9 ... ATP-degrading enzyme, 11 ... light reflection part, 12 ... light reflection film, 13 DESCRIPTION OF SYMBOLS -18 ... Filter, 21 ... Photodetector, 21a ... Light-receiving part, 22-24, 26-28 ... Pressure port, 41 ... Channel, 42 ... Extraction well, 42a ... Extraction well open port, 43, 44 ... Channel 45 ... amplification well, 45a ... amplification well opening, 46,47 ... flow path, 48 ... bead storage well, 48a ... bead storage well opening, 49 ... flow path, 51 ... waste liquid storage well, 51a ... waste liquid storage well Open mouth, 52 ... well for containing auxiliary liquid, 2a ... auxiliary fluid receiving well mouth opening, 53-56 ... passage, 57 ... ATP degradation wells, 57a ... ATP degradation well mouth opening, 58,59,61 ... passage, 100 ... testing chip.

Claims (8)

A first well for introducing a sample liquid containing the sample;
A second well containing a solid reagent that reacts with the specimen;
A first opening for opening the first well to the atmosphere;
A second opening for opening the second well to the atmosphere;
A test chip, wherein a flow path connecting the first well and the second well is formed in a main body portion. The reagent contained in the second well is a luminescent reagent that emits light in the presence of ATP;
A third well containing a solid extraction reagent for extracting ATP in the microorganism;
A third opening for opening the third well to the atmosphere is further formed in the main body,
The inspection chip according to claim 1, wherein the third well constitutes a part of the flow path between the first well and the second well.   The test chip according to claim 2, wherein an ATP-degrading enzyme is immobilized on an inner wall surface of the flow path between the first well and the third well. A fourth well containing a solid amplification reagent for amplifying ATP;
A fourth opening for opening the fourth well to the atmosphere is further formed in the main body,
4. The inspection chip according to claim 2, wherein the fourth well constitutes a part of the flow path between the third well and the second well. 5.   The light reflecting portion for reflecting the light generated in the second well is provided on a part of the inner wall surface of the second well so as to surround the reagent. The test | inspection chip as described in any one of -4.   6. The inspection chip according to claim 1, wherein all of the openings except the first opening are covered with a filter.   The said 2nd opening is covered with the air permeable light reflection film which reflects the light produced in the said 2nd well, The Claim 1 characterized by the above-mentioned. Inspection chip. The inspection chip according to claim 7, wherein all the openings except the first opening and the second opening are covered with a filter.

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