JP2013040800A - Device and method for detecting substance released from cell, and immobilized enzyme substrate for detecting substance released from cell - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a device and a method for detecting substances released from a cell which detects a concentration distribution of nucleotide-related chemical substances released from the cell, on a cellular tissue, and an immobilized enzyme substrate for detecting substances released from a cell.SOLUTION: A cellular tissue S is placed on a transparent substrate 10 on which GAPDH (glyceraldehyde-3-phosphate dehydrogenase) is immobilized. ATP (Adenosine-5'-triphosphate) on the surface of the cellular tissue S is consumed under the presence of GAP (glyceraldehyde-3-phosphate) and NADto produce NADH (nicotinamide adenine dinucleotide). Evanescent wave L2 is generated on the surface of the transparent substrate 10 by irradiation light from a light source unit 7 provided on a side of the transparent substrate 10 to fluorescently emit a portion near the transparent substrate 10, namely, NADH on the surface of the cellular tissue S. A reaction system is thus designed, so that ATP that is the nucleotide can be detected with fluorescence radiated by a fluorescent substance (NADH) generated by the reaction.

Description

  The present invention relates to a detection apparatus, a detection method, and an immobilized enzyme substrate for detection, which detect a cell-released substance released from a cell tissue. In particular, the present invention relates to a concentration distribution on a cell tissue for nucleotide-related chemical substances released from the cell tissue. The present invention relates to a cell release substance detection device to be detected, a cell release substance detection method, and an immobilized enzyme substrate for cell release substance detection.

  Conventionally, a luciferin / luciferase luminescence system, which is bioluminescence, has been used for general purpose for the quantification of adenosine triphosphate (hereinafter sometimes simply referred to as “ATP”) (Non-patent Document 1). In the presence of ATP and magnesium ions, luciferin, which is a luminescent substrate, is converted to oxyluciferin, which is a luminescent material, by luciferase to emit light. In detecting ATP, generally, a cell extract is collected by crushing a cell tissue of a subject sample, and luciferin and luciferase are added to the cell extract. Then, the amount of ATP is quantified by measuring the amount of luminescence of oxyluciferin generated by consuming ATP in the sample extract.

  Since the quantification of ATP in a sample by the luciferin / luciferase luminescence method has good sensitivity and excellent rapidity, ATP quantification methods other than the luminescence method have hardly been put to practical use.

  Patent Document 1 discloses a technique for detecting a trace amount of ATP in a sample by using both the ATP amplification method and the ATP cycling method. In this technique, first, adenylate kinase and AMP (adenylic acid) are used, ATP in a sample is reacted with AMP to produce two molecules of ADP (adenosine diphosphate), and the resulting two molecules of ADP 2 molecules of ATP are generated by the action of polyphosphate kinase. Since the generated two molecules of ATP again become a substrate that reacts with AMP, by performing the reaction n times, one ATP is amplified to 2n ATP (ATP amplification method). Thereafter, the enzyme used for the amplification is inactivated, and ATP is amplified again by the ATP cycling method. Then, after passing through the ATP cycling method, the amount of ATP in the initial sample is quantified by quantifying ATP in the sample using an enzyme colorimetric method or an electrical quantification method. Thereby, a slight amount of ATP in the initial sample can be quantified.

  Further, the inventors of the present application have proposed a miniaturized optical device for observing and measuring the concentration distribution of a chemical substance released from a living tissue in correspondence with the position on the tissue (Patent Document 2). ). In this optical apparatus, the concentration distribution of chemical substances such as GABA and glutamic acid can be measured using evanescent light.

JP 2006-223163 A JP 2008-139280 A

DeLuca, M., and McElroy, W.D., Kinetics of the firefly luciferase catalyzed reactions.Biochemistry, 26,921-925 (1974).

  However, the techniques disclosed in Patent Document 1 and Non-Patent Document 1 are both techniques for quantifying ATP in a sample extract, and do not directly detect and measure ATP released from cells. . For this reason, there has been a problem that the concentration distribution on the cell tissue (ATP spatial distribution) cannot be grasped. In other words, in the conventional technique, it is impossible to grasp from which cell of the tissue piece ATP is released. In addition, since ATP inside and outside the cell cannot be detected separately, it cannot be determined whether the detected ATP is present in the cell or released from the cell.

  Therefore, the inventors of the present application tried to detect the concentration distribution of ATP released from the cell tissue by combining the observation apparatus using evanescent light disclosed in Patent Document 2 and the luciferin / luciferase luminescence method. However, since the molar extinction coefficient of luciferin was 18, the luminescence obtained was very weak, and the spatial distribution of ATP could not be detected.

  In other words, if the conventional luciferin / luciferase luminescence method is used to detect the ATP concentration distribution on the cell tissue, a highly sensitive photodetector such as a photomultiplier tube is required to detect the luminescence. End up. In other words, a high-cost and large-scale optical device is required, and there is a problem that ATP detection on a cell tissue cannot be easily performed at a low cost.

  Here, if it is possible to perform ATP amplification, it is considered that an expensive high-sensitivity photodetector can be eliminated. For example, if ATP amplification is performed by applying the technique disclosed in Patent Document 1, 50 ATP is required. A step of inactivating the enzyme at a temperature of ℃ or higher is necessary, which may cause cell degeneration. For this reason, a practical technique that can detect ATP released from the cell tissue in association with each position on the cell tissue has not yet been proposed.

  For example, ATP can be used as an index of freshness of fresh food products such as meat and fish because ATP is released from cells having physiological activity but not released from cells that have lost biological activity. However, since ATP is present in a large amount in a cell, the conventional technique for detecting ATP from a cell extract cannot distinguish between ATP inside and outside the cell and cannot accurately determine freshness. It was.

  Furthermore, when the brain is damaged by infarction or lack of oxygen, ATP is released from glial cells for the purpose of protecting nerve cells. Diagnosis of the ischemic state of the brain is very difficult, and even if a skilled surgeon performs craniotomy and observes the state, it is difficult to accurately diagnose and predict the disease state. If it is possible to accurately detect the release state (concentration distribution) of ATP, it is thought that useful knowledge can be obtained for diagnosis of disease state and determination of drug efficacy, but no useful technique for detecting ATP on cellular tissues has been proposed. Under the present circumstances, there is a problem that neither the determination of the cerebral ischemia nor the evaluation of the effectiveness of the drug remains without using the ATP release state as an index.

  The present invention has been made in order to solve the above-described problems, and relates to a nucleotide-releasing chemical substance released from a cell, a cell-releasing substance detection device, a cell-releasing substance detection method, An object of the present invention is to provide an immobilized enzyme substrate for detecting a cell-release substance.

  In order to achieve this object, a cell-released substance detection device according to claim 1 is provided with a phosphorylation reaction for phosphorylating a substrate and a substrate after phosphorylation on a placement surface side on which a cell tissue is placed. A substrate having an enzyme that catalyzes an oxidation reaction that oxidizes, and excitation light that excites a fluorescent material generated from a fluorescent source substance in response to the reaction on the substrate is irradiated toward the cell tissue on the substrate. A light irradiation means, a detection means for detecting fluorescence emitted from the fluorescent material by the excitation light emitted by the light irradiation means, and an image forming means for forming an image based on the fluorescence detected by the detection means. And a concentration distribution of the nucleotide-related chemical substance released from the cell tissue placed on the substrate is detected from an image formed by the image forming means.

  Here, the nucleotide-related chemical substance is a chemical substance that serves as a phosphate supply source for the substrate, and examples thereof include various ribonucleotides and deoxyribonucleotides.

  Furthermore, the substrate having an enzyme may be a substrate in which the enzyme is bonded to the substrate by a chemical bond, such as a substrate in which the enzyme is simply applied or added to the substrate and does not have a chemical bond to the substrate. May be.

  The fluorogenic substance means a raw material molecule that reacts with a specific chemical substance existing in the reaction system to generate a fluorescent substance. Such a fluorescent material is not necessarily limited to one kind of substance, and is a concept including a mixture of two or more kinds of substances.

  The cell-release substance detection apparatus according to claim 2 is the cell-release substance detection apparatus according to claim 1, wherein the enzyme is glyceraldehyde 3-phosphate dehydrogenase.

  The cell-released substance detection device according to claim 3 is the cell-released substance detection device according to claim 1 or 2, wherein the substrate has the enzyme immobilized on the surface on the mounting surface by a chemical bond. .

  The cell emission substance detection apparatus according to claim 4 is the cell emission substance detection apparatus according to any one of claims 1 to 3, wherein the light irradiation means includes a light source unit that makes excitation light incident on a side surface of the substrate. The evanescent light is generated on the mounting surface of the substrate by the excitation light applied to the side surface of the substrate from the light source unit.

  The cell emission substance detection device according to claim 5 is the cell emission substance detection device according to any one of claims 1 to 4, wherein the detection means does not overlap an optical path of excitation light emitted by the light irradiation means. It is arranged.

  The method for detecting a cell-released substance according to claim 6 is a method for detecting a concentration distribution of a nucleotide-related chemical substance released from a cell tissue, wherein a phosphorylation reaction for phosphorylating a substrate and a substrate after phosphorylation are detected. In the state where the substrate and the fluorogenic substance that is converted into the fluorescent substance along with the enzyme reaction coexist on the substrate toward the cell tissue placed on the substrate having the enzyme that catalyzes the oxidation reaction that oxidizes, Detected by the light irradiation step of irradiating excitation light of the fluorescent material, the fluorescence detection step of detecting fluorescence emitted from the fluorescent material excited by the excitation light irradiated by the light irradiation step, and the fluorescence detection step And an image forming process for forming an image based on fluorescence, and the concentration distribution of nucleotide-related chemical substances released from the cell tissue is detected by the image formed by the image forming process. It is intended to.

  Here, the nucleotide-related chemical substance is a chemical substance that serves as a phosphate supply source for the substrate, and examples thereof include various ribonucleotides and deoxyribonucleotides.

  Furthermore, the substrate having an enzyme may be a substrate in which the enzyme is bonded to the substrate by a chemical bond, such as a substrate in which the enzyme is simply applied or added to the substrate and does not have a chemical bond to the substrate. May be.

  The fluorogenic substance means a raw material molecule that reacts with a specific chemical substance existing in the reaction system to generate a fluorescent substance. Such a fluorescent material is not necessarily limited to one kind of substance, and is a concept including a mixture of two or more kinds of substances.

  The cell-release substance detection method according to claim 7 is the cell-release substance detection method according to claim 6, wherein the enzyme is glyceraldehyde 3-phosphate dehydrogenase.

  The cell-released substance detection method according to claim 8 is the cell-released substance detection method according to claim 6 or 7, wherein the enzyme is immobilized on the substrate surface by chemical bonding, and the cell tissue is placed on the substrate surface. Is done.

  The immobilized enzyme substrate for detecting a cell-released substance according to claim 9 is one in which glyceraldehyde 3-phosphate dehydrogenase is supported by a chemical bond on the surface of a light-transmitting substrate having a smooth surface. .

  According to the apparatus for detecting a cell-released substance according to claim 1, cells placed on a substrate having an enzyme that catalyzes a phosphorylation reaction that phosphorylates the substrate and an oxidation reaction that oxidizes the phosphorylated substrate. Excitation light is irradiated toward the tissue by the light irradiation means. When the substrate of the enzyme and the fluorogenic material that generates the fluorescent material accompanying the reaction catalyzed by the enzyme reaction are present on the substrate, the fluorescent material is generated while consuming phosphoric acid in the reaction system. Here, since the phosphate supply source is a nucleotide-related chemical substance released by the cell tissue, fluorescence is emitted if the nucleotide-related chemical substance is released from the cell tissue, and if not, no fluorescence is emitted.

  Therefore, by detecting fluorescence with the detection means and forming a fluorescence image with the image forming means based on the detected fluorescence, detecting the concentration distribution (spatial distribution) of nucleotide-related chemical substances on the cell tissue There is an effect that can be.

  In addition, the phosphorylation reaction and oxidation reaction of the substrate are catalyzed by the enzyme included in the substrate, and the presence of the fluorescent source substance generates a fluorescent substance accompanying the enzyme reaction. That is, in a simple reaction system, the nucleotide-related chemical substance that is a phosphate supply source can be immediately detected by the generated fluorescent substance. Therefore, it is possible to maintain a high reaction efficiency as compared with a complicated reaction system and to obtain a highly reliable detection result.

  According to the cell-released substance detection device according to claim 2, in addition to the effect exhibited by the cell-released substance detection device according to claim 1, since the enzyme is glyceraldehyde 3-phosphate dehydrogenase, Nucleotide-related chemicals can be detected by enzymatic reactions catalyzed by existing natural enzymes. In other words, since the reaction between glyceraldehyde 3-phosphate dehydrogenase and its substrate is a normal reaction system (glycolysis system) in natural cell tissues, a stable enzyme reaction can be realized and There is an effect that an excessive load and an unexpected side reaction can be suppressed.

  According to the cell-released substance detection device according to claim 3, in addition to the effect exhibited by the cell-released substance detection device according to claim 1 or 2, the enzyme is immobilized on the surface of the substrate mounting surface by a chemical bond. Therefore, it is possible to suppress the outflow or uneven distribution of the enzyme, and there is an effect that a uniform enzyme reaction can be realized on the substrate as compared with the case where the enzyme is simply applied or added to the substrate. Furthermore, the enzyme can be restricted from moving into the cell tissue placed on the substrate, and the enzyme reaction can be performed locally in the vicinity of the surface of the cell tissue placed on the substrate. Therefore, there is an effect that the nucleotide-related chemical substance released from the cell (that is, on the cell surface) can be accurately detected. As a result, the nucleotide-related chemical substance released outside the cell can be detected separately from the intracellular adenosine triphosphate.

  According to the cell-released substance detection device according to claim 4, in addition to the effect exhibited by the cell-released substance detection device according to any one of claims 1 to 3, the excitation light applied to the side surface of the substrate from the light source unit, Since it is configured to generate evanescent light on the mounting surface of the substrate, when cell tissue is mounted on the mounting surface, it is present in the vicinity of the mounting surface, that is, the fluorescence existing on the surface portion of the cell tissue. Fluorescence can be emitted limited to a substance. For this reason, there is an effect that the nucleotide-related chemical substance released from the cell (that is, on the cell surface) can be accurately detected by being distinguished from the nucleotide-related chemical substance in the cell.

  Furthermore, since the light source unit makes excitation light incident from the end face of the substrate, it is possible to easily change the incident angle, to facilitate the setting of the optical system, and to improve the handleability of the apparatus. There is.

  According to the cell emission substance detection device according to claim 5, in addition to the effect of the cell emission substance detection device according to any one of claims 1 to 4, the detection means is an optical path of excitation light irradiated by the light irradiation means Since the target signal (fluorescence) can be captured separately from the excitation light, the influence of the excitation light (noise) on the detected fluorescence can be reduced. is there.

  According to the method for detecting a cell-released substance according to claim 6, cell tissue placed on a substrate having an enzyme that catalyzes a phosphorylation reaction that phosphorylates the substrate and an oxidation reaction that oxidizes the phosphorylated substrate. The substrate is irradiated with excitation light of the fluorescent material in the light irradiation step in a state where the substrate and the fluorescent material that is converted into the fluorescent material in accordance with the enzyme reaction coexist on the substrate.

  When there is an enzyme substrate and a fluorogenic substance that generates a fluorescent substance in response to a reaction catalyzed by the enzyme reaction, the fluorescent substance is generated while consuming phosphoric acid in the reaction system by the action of the enzyme on the substrate. Is done. Here, since the phosphate supply source is a nucleotide-related chemical substance released by the cell tissue, fluorescence is emitted if the nucleotide-related chemical substance is released from the cell tissue, and if not, no fluorescence is emitted.

  Then, fluorescence emitted from the fluorescent material excited by the excitation light irradiated in the light irradiation step is detected in the fluorescence detection step, and a fluorescent image is formed in the image forming step based on the fluorescence. Thereby, there is an effect that the concentration distribution (spatial distribution) of the nucleotide-related chemical substance released from the cell tissue (on the cell tissue) can be detected.

  In addition, the phosphorylation reaction and oxidation reaction of the substrate are catalyzed by the enzyme, and the presence of the fluorogenic substance here produces a fluorescent substance accompanying the oxidation reaction. That is, in a simple reaction system, the nucleotide-related chemical substance that is a phosphate supply source can be immediately detected by the generated fluorescent substance. Therefore, it is possible to maintain a high reaction efficiency as compared with a complicated reaction system and to obtain a highly reliable detection result.

  According to the method for detecting a cell-released substance according to claim 7, in addition to the effect exerted by the method for detecting a cell-released substance according to claim 6, the enzyme is glyceraldehyde 3-phosphate dehydrogenase. Nucleotide-related chemicals can be detected by enzymatic reactions catalyzed by existing natural enzymes. In other words, since the reaction between glyceraldehyde 3-phosphate dehydrogenase and its substrate is a normal reaction system (glycolysis system) in natural cell tissues, a stable enzyme reaction can be realized and There is an effect that an excessive load and an unexpected side reaction can be suppressed.

  According to the method for detecting a cell-released substance according to claim 8, in addition to the effect exerted by the method for detecting a cell-released substance according to claim 6 or 7, the enzyme is immobilized on the substrate surface by a chemical bond, Because it is placed on the surface of the substrate, it can suppress the outflow or uneven distribution of the enzyme, and it can realize a uniform enzyme reaction on the substrate compared to simply applying or adding the enzyme on the substrate. effective. Furthermore, the enzyme can be restricted from moving into the cell tissue placed on the substrate, and the enzyme reaction can be performed locally in the vicinity of the surface of the cell tissue placed on the substrate. Therefore, there is an effect that the nucleotide-related chemical substance released from the cell (that is, on the cell surface) can be accurately detected.

  According to the immobilized enzyme substrate for detecting a cell-released substance according to claim 9, glyceraldehyde 3-phosphate dehydrogenase is supported by a chemical bond on the surface of a transparent substrate having a smooth surface. Therefore, cell tissue is placed on the substrate, and further, a substrate for glyceraldehyde 3-phosphate dehydrogenase (glyceraldehyde 3-phosphate) and glyceraldehyde 3-phosphate dehydrogenase are provided. By coexisting on the substrate with a fluorogenic substance that is converted into a fluorescent substance in accordance with the catalytic oxidation reaction, it is possible to emit fluorescence corresponding to nucleotide-related chemical substances released from cell tissues. is there. Therefore, if fluorescence analysis is performed using this substrate, nucleotide-related chemical substances released from cell tissues can be detected.

It is a figure which shows schematic structure of the ATP detection system which is one Embodiment of the cell discharge | release substance detection apparatus of this invention. FIG. 2 is a diagram for explaining in detail a transparent substrate used in the measurement apparatus of FIG. 1, which is an embodiment of the immobilized enzyme substrate for cell release substance detection of the present invention. It is a block diagram which shows the electric constitution of an ATP detection system. It is the figure which showed the flowchart of the ATP measurement process performed with the personal computer of an ATP detection system. It is the figure which showed the observation result of the slice piece of the rat cerebrum in Example 1, FIG. 5 (a) is an optical observation result with normal light, FIG.5 (b) is a fluorescence image of the same cell tissue. . FIG. 6 is a diagram showing temporal changes in the fluorescence intensities at the respective positions a to h shown in FIG.

  Hereinafter, preferred embodiments of the present invention will be described with reference to the accompanying drawings. The present invention detects the concentration distribution of nucleotide-related chemical substances on the cell surface. Detecting the concentration distribution means measuring the spatial distribution of how nucleotide-related chemical substances are distributed in a two-dimensional plane having a certain area. First, the nucleotide-related chemical substance detection principle of the present invention will be described using the nucleotide-related chemical substance as ATP.

  Glyceraldehyde 3-phosphate dehydrogenase (hereinafter sometimes simply referred to as “GAPDH”) is an enzyme involved in glycolysis in vivo and is a substrate glyceraldehyde triphosphate (hereinafter referred to as “GAPDH”). And may be abbreviated simply as “GAP”). GAP becomes 1,3-bisphosphoglycerate by the action of GAPDH. The phosphorylation reaction catalyzed by GAPDH is usually based on inorganic phosphoric acid as a phosphoric acid supply source in vivo.

1,3-bisphosphoglyceric acid is produced by oxidizing the resulting aldehyde following phosphorylation of GAP. This oxidation reaction involves the coenzyme nicotinamide adenine dinucleotide (oxidized form, hereinafter simply abbreviated as “NAD ⁺ ”), and the electron acceptor NAD ⁺ is reduced and reduced nicotinamide adenine. Dinucleotide (hereinafter simply abbreviated as “NADH”). Here, NADH is a fluorescent substance. In the present invention, by using the reaction shown in the following reaction formula catalyzed by GAPDH, ATP to be detected is used as a phosphate supply source for GAP phosphorylation, so that its presence can be detected by fluorescence of NADH. .
GAP + ATP → 1,3-bisphosphoglyceric acid + ADP
NAD ⁺ → NADH

That is, NAD ⁺ is a fluorescent material that is converted into a fluorescent material in a reaction mediated by GAPDH. The fluorescent substance is a substance that reacts with a specific chemical substance and is converted into a fluorescent substance in the enzyme reaction. For example, the above-mentioned NAD ⁺ or nicotinamide adenine dinucleotide phosphate (oxidized type, hereinafter , Simply abbreviated as “NADP ⁺ ”). By designing the reaction system in this way, ATP, which is a ribonucleotide, can be detected by the fluorescence emitted by the fluorescent substance (NADH) generated by the reaction.

In the above reaction system, since the reaction molar amount of ATP and the molar amount of NADH (NADPH) produced are the same, the ATP concentration can be measured by measuring the fluorescence intensity of NADH (NADPH). As the fluorescent material, either NAD ⁺ or NADP ⁺ may be used, or a mixture of both may be used.

  Moreover, the enzyme reaction of GAPDH described above is phosphate-requiring. Accordingly, a buffer solution that does not contain phosphoric acid is used as the buffer solution used in the measurement. Examples of such a buffer include Britton-Robinson buffer, Tris buffer, HEPES buffer, and the like.

  Furthermore, since the reaction described above is an enzymatic reaction that proceeds at normal temperature and pressure, the load on the cell tissue is light. Therefore, the cellular tissue set in the reaction field does not immediately lose its physiological activity, and the time-dependent change in the nucleotide-related chemical substance release state from the cellular tissue can be measured by fluorescence detection.

  In the present invention, the nucleotide-related chemical substance to be measured is, for example, ribonucleotide, deoxyribonucleotide, ribonucleoside of any one of adenosine, guanosine, uridine, and cytidine, or deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine, deoxy. A phosphate group bonded to any deoxyribonucleoside of cytidine. In the present invention, those without a phosphate group are not detected. Therefore, nucleotides can be selectively detected without being affected by nucleosides that are similar structures. For example, adenosine accumulates (releases) in cells that are not physiologically active. On the other hand, ATP (ADP, AMP), which is an analogous structure, is released only from cells having physiological activity. In the present invention, since ATP (ADP, AMP) is detected and adenosine is not detected, the presence / absence and state of the physiological activity of the cell tissue can be detected with high accuracy.

  The enzyme used in the present invention catalyzes a phosphorylation reaction that phosphorylates a substrate and an oxidation reaction that oxidizes the substrate after phosphorylation, for example, a combination of asparagine synthetase and glutamate dehydrogenase And a combination of two enzymes, acetokinase and glucokinase (or hexokinase) and glucose-6-phosphate dehydrogenase, and GAPDH described above. Preferably, GAPDH is used. This is because the reaction system is simple and the reaction rate is faster than other enzymes. When GAPDH is used, the reaction speed is a rapid reaction in the order of m seconds (100 ms or less), so that damage to the cell tissue can be suppressed and the reliability of the obtained detection result can be improved.

  FIG. 1 is a diagram showing a schematic configuration of an ATP detection system 100 which is an embodiment of the cell emission substance detection device of the present invention. The ATP detection system 100 is configured to detect ATP present on the surface of a cell tissue by fluorescence emission using an enzyme reaction of GAPDH. FIG. 1 (a) shows a schematic diagram of the entire system, and FIG. 1 (b) shows an enlarged view of the surface of a transparent substrate 10 which is an immobilized enzyme substrate.

  As shown in FIG. 1 (a), the ATP detection system 100 controls a measuring device 1 provided with a reaction field and an optical system for detecting ATP from a cell tissue, and the measuring device 1. A personal computer (hereinafter simply abbreviated as “PC”) 20 that displays an ATP concentration distribution as an image, and shows how ATP is distributed on a cell tissue, that is, a certain area. The concentration distribution (spatial distribution) on the dimension plane is measured. The measuring device 1 and the PC 20 are connected to each other via a cable 18.

  The measuring apparatus 1 includes a reaction field that generates fluorescence by an enzyme reaction with respect to the cell tissue S to be measured, and irradiates the fluorescent material generated by the reaction with excitation light and detects (images) the generated fluorescence. It is configured. The measuring apparatus 1 includes a light source unit 7, a transparent substrate 10, a condenser lens 11, a photodetector 12, a CCD camera unit 13, and a substrate holding unit 16.

  The light source unit 7 is disposed on the side of the transparent substrate 10 and irradiates light toward the end surface of the transparent substrate 10. The light source unit 7 includes a housing (not shown), and the light source 8 and the incident light side lens 9 are accommodated in the housing. In FIG. 1A, the direction (light path) of light from the light source 8 is indicated by a dashed-dotted arrow L1.

  In the housing of the light source unit 7, an incident light side lens 9 is disposed on the optical path L 1 of the irradiation light from the light source 8, and the irradiation light from the light source 8 is collected by the incident light side lens 9. Thereafter, the light is incident on the end face of the transparent substrate 10. The light source unit 7 is pivotally supported so as to be movable in the vertical direction on the rear side (opposite side to the incident light side lens 9 in the housing). By operating an adjustment dial (not shown), the amount of operation can be reduced. Accordingly, the installation angle of the light source unit 7 can be changed. Thereby, the incident angle to the end surface of the transparent substrate 10 of the irradiation light from the light source 8 can be adjusted.

  The light source 8 is for irradiating the light which excites a fluorescent substance, and the light emitting diode (LED) which irradiates 360 nm excitation ultraviolet light (UV) is used. In the present embodiment, the irradiation light (excitation ultraviolet light) emitted from the light source 8 is monochromatic light and does not require a bandpass filter. The wavelength of the excitation light from the light source 8 is not particularly limited as long as it can excite the fluorescent substance. In general, as the wavelength of a fluorescent substance has a higher absorbance, the transition to an excited state is more likely to occur, and the transition to the excited state is most active when the wavelength of the absorption maximum is irradiated. NADH has an absorption maximum at 340 nm. Therefore, even at a wavelength in the vicinity thereof, NADH has a relatively high absorbance and is good excitation light. In the present embodiment, 360 nm ultraviolet light capable of generating sufficient fluorescence near the absorption maximum of NADH is used as excitation light.

  The light source 8 is not particularly limited as long as it emits excitation light that excites the generated fluorescent substance, and is one selected from the group consisting of an LED, a mercury lamp, a xenon lamp, and a halogen lamp. If it is one. When a mercury lamp, a xenon lamp, or a halogen lamp is used, an appropriate filter is used so that appropriate excitation light is irradiated according to the fluorescent substance to be measured.

  The transparent substrate 10 is a substrate on which the tissue cells S to be measured are placed, and is formed in a rectangular plate shape. It is held by the substrate holder 16 and is disposed above the CCD camera unit 13. The transparent substrate 10 is a transparent substrate and serves as an optical waveguide for light emitted from the light source 8. The transparent substrate 10 is made of a highly refractive material, and crystals such as quartz, glass (heavy flint glass), sapphire, and acrylic resins such as PMMA (polymethyl methacrylate) can be used, but are not limited thereto. Is not to be done. Alternatively, the substrate surface may be dip coated with an LB film over the entire length of the waveguide. When the transparent substrate 10 generates a large amount of heat due to excitation light irradiation, the transparent substrate 10 is made of quartz, glass, or the like having high heat resistance. Further, the material of the transparent substrate 10 is preferably a material that does not absorb the wavelength of the excitation light. Quartz is used for the transparent substrate 10 of this embodiment.

  The front surface and the back surface of the transparent substrate 10 have a high degree of smoothness. For example, the surface roughness is formed to be equal to or less than the wavelength of incident light. In the measuring apparatus 1, the light incident from the end face of the transparent substrate 10 is designed to be totally reflected at the interface of the transparent substrate 10 and emitted from the end face opposite to the incident side. For this reason, an evanescent wave L <b> 2 is generated on the surface of the transparent substrate 10.

  The substrate holding unit 16 holds the transparent substrate 10 substantially horizontally, and includes two support columns 16a erected at predetermined intervals along the optical path direction L1, and a ceiling mounted on the two support columns 16a. A pair of plates 16b are provided on the front side and the back side of the measuring apparatus 1. The top plate 16b is a plate-like member formed with a width that comes into contact with the surface of the transparent substrate 10 in a narrow range near the end of the surface. By placing the transparent substrate 10 on a pair of top plates 16b, the opposite end portions on the two sides are supported and held in a state of being laid on four columns.

  FIG. 1B shows an enlarged surface portion I of the transparent substrate 10. As shown in FIG. 1B, an evanescent wave L2 is generated on the surface of the transparent substrate 10 due to total reflection. The evanescent wave L2 is light that is generated during total reflection and oozes out on the opposite side of the totally reflected interface. The evanescent wave is light that can change the penetration depth from several tens of nanometers to several hundreds of nanometers according to the total reflection angle. That is, since the evanescent wave L2 exists only in the vicinity of the surface of the transparent substrate 10, when the cell tissue S is placed on the transparent substrate 10, optical information on the extremely shallow portion of the surface of the cell tissue S irradiated with the evanescent wave L2 is obtained. Can be acquired.

Here, the surface of the transparent substrate 10 serves as a reaction field for enzyme reaction, and GAPDH is immobilized on the surface of the transparent substrate 10. In the measurement, GAP and NAD ⁺ are supplied to the surface of the transparent substrate 10. For this reason, if ATP is released from the cell tissue S set on the transparent substrate 10, an enzymatic reaction that consumes ATP phosphate occurs due to the action of GAPDH, and 1,3 bisphosphoglycerate is generated from GAP. At the same time, NAD ⁺ is reduced to produce NADH.

  Since the generated NADH is a fluorescent material, it is excited by the evanescent wave L2 to emit fluorescence. That is, by detecting the fluorescence excited by the evanescent wave L2, it is possible to detect ATP present on the cell surface, that is, released from the cell.

  The cellular tissue S to be measured is a biological sample, and examples thereof include a tissue piece, a cell, and a micro organism obtained from an organism by incision or puncture, but are not limited thereto.

  In the present embodiment, the measuring apparatus 1 is configured to detect the fluorescent light generated from the generated fluorescent light toward the lower side of the transparent substrate 10 (direction indicated by a two-dot chain line arrow L3).

  Returning to FIG.

  The condenser lens 11 is disposed on the opposite side of the light source unit 7 with the transparent substrate 10 interposed therebetween. The excitation ultraviolet light that is incident on the end surface of the transparent substrate 10 from the light source unit 7, propagates along the surface direction in the transparent substrate 10, and is emitted from the end surface on the opposite side to the incident side is condensed to the photodetector 12. This is a lens for input. The condensing lens 11 is movably held and can change an angle with respect to the end surface of the transparent substrate 10 by an operator's operation.

  The photodetector 12 is installed behind the condenser lens 11 on the optical path of the excitation ultraviolet light L1. The photodetector 12 detects incident light incident through the condenser lens 11 with a photodiode. The detection signal from the detector 11 is output to the PC 20, and the operator can adjust the incident angle of the light source unit 7 based on the detection result displayed on the PC 20.

  The CCD camera unit 13 is disposed below the transparent substrate 10 and receives fluorescence emitted downward from the cell tissue S and performs imaging (acquisition of optical information) of the cell tissue S. is there. The CCD camera unit 13 is configured as a CCD camera having a pinhole function. The pinhole function referred to here means a camera function that can introduce a light from a hole (light introduction hole) having a diameter of 0.05 to 0.5 mm and can be miniaturized with a focal length of 20 mm or less. The CCD camera unit 13 includes an imaging lens 14 and a CCD 15. The imaging lens 14 is disposed below a light introduction hole provided on the upper surface of the main body, and a CCD 15 is disposed below the imaging lens. The fluorescence emitted from the cell tissue S is taken in from the light introduction hole, and an image is formed on the surface of the CCD 15 via the imaging lens 14.

  The CCD 14 is a photoelectric conversion element, and mainly includes a light receiving unit (sensor) for sensing light, a vertical transfer unit (vertical register) for vertically transferring photoelectrically converted charges, and a horizontal transfer unit (horizontal register) for horizontally transferring. It is a general thing consisting of. The extracted charge is converted into a voltage by the output circuit.

In the measuring apparatus 1 of the present embodiment, as described above, the CCD camera unit 13 receives the fluorescence in the direction of the optical path L3 emitted from the tissue cell S and disposed below the transparent substrate 10. That is, the CCD camera unit 13 is disposed at a position that does not overlap with the optical path of the excitation light L1 (direction orthogonal to the optical path of the excitation light), and receives the fluorescence of the optical path L3 emitted downward from the tissue cell S. . For this reason, it is possible to separate the excitation light propagating through the optical path L1 while totally reflecting the inside of the transparent substrate 10, and to input the fluorescence, which is optical information from the tissue cell S, to the CCD 15, such as a dichroic mirror and a cut filter. Can be made unnecessary. The image signal acquired by the CCD camera unit 13 is output from the measuring apparatus 1 to the PC 20 via the cable 18.

  In this embodiment, a CCD camera is used for fluorescence detection. However, the present invention is not limited to this as long as close-up photography can be performed at an infinite focus. For example, a fluorescence microscope or a CMOS camera having a pinhole function can be used. . By adopting a camera structure having a pinhole, the objective lens in front of the coupling lens (before the optical path direction) can be eliminated, and the camera body can be reduced in size accordingly. If it is not necessary to reduce the size of the apparatus, a camera structure including an objective lens may be used instead of the pinhole.

  The PC 20 is a computer that controls the measurement apparatus 1 and includes a liquid crystal display (hereinafter simply referred to as “LCD”) 27 that is a display device and a keyboard 28 that is an input device. The operator can cause the measurement apparatus 1 to perform measurement by inputting a command from the keyboard. The imaged image data (fluorescence image) of the cell tissue S is displayed on the LCD 27. Here, in this embodiment, a reaction field is formed so as to detect fluorescence of ATP released on the surface of the cell tissue S (contact surface with the transparent substrate 10), and the LCD 27 has a concentration distribution of ATP. Shown in two-dimensional fluorescence image.

  Further, the PC 20 not only causes the measurement apparatus 1 to perform ATP measurement, but also causes the LCD 27 to display a signal acquired by the photodetector 12 of the measurement apparatus 1 based on a command input from the operator's keyboard 28. Can be done. For this reason, the operator can adjust the optical path of the measuring apparatus 1 while viewing the display result displayed on the LCD 27.

  FIG. 2 is a diagram for explaining in detail the transparent substrate 10 used in the measuring apparatus 1, which is an immobilized enzyme substrate for detecting adenosine triphosphate of the present embodiment.

  FIG. 2A is a partial cross-sectional view schematically showing the configuration of the transparent substrate 10. In FIG. 2A, the upper side of the drawing is illustrated as being the front side of the transparent substrate 10. In addition, the enzyme (GAPDH) is schematically shown in an elliptical shape. As shown in FIG. 2A, the enzyme (GAPDH) is uniformly arranged on the entire surface of the transparent substrate 10, that is, the surface on which the cell tissue S is placed, and is transparent by chemical bonding. It is fixed to the surface of the substrate 10.

FIG. 2B is a partially enlarged view of the minute region II in FIG. In FIG. 2B, the horizontal direction of the paper is the vertical direction (direction perpendicular to the plane of the transparent substrate 10 (thickness direction)). As shown in FIG. 2 (b), GAPDH forms a Schiff base on the terminal side and is bonded to the silane compound, and is fixed to the surface of the transparent substrate 10 that is quartz (SiO ₂ ) through the silane compound. It has become. The silane compound is a so-called silane coupling agent, and forms a covalent bond with the surface of the inorganic compound (quartz) by a dehydration condensation reaction.

As the silane compound forming the Schiff base, for example, an aminosilane compound is selected. Specifically, N-2- (aminoethyl) -3-aminopropylmethyldimethoxysilane, N-2- (aminoethyl) -3 -Aminopropyltrimethoxysilane, N-2- (aminoethyl) -3-aminopropyltriethoxysilane, 3-aminopropyltrimethoxysilane (APTMS), 3-aminopropyltriethoxysilane (APTES), 3-triethoxy Hydrochloride of silyl-N- (1,3-dimethyl-butylidene) propylamine, N-phenyl-3-aminopropyltrimethoxysilane, N- (vinylbenzyl) -2-aminoethyl-3-aminopropyltrimethoxysilane And ethyldimethylaminopropylcarbodiimide (EDC). In this embodiment, 3-aminopropyltrimethoxysilane is used.

  The end of the enzyme, which is a protein, has an amino group, which can react with an aminosilane compound bonded to the transparent substrate 10 to form a Schiff base, thereby forming a bond.

  When the cell tissue S is placed on the transparent substrate 10 by immobilizing the enzyme on the surface of the transparent substrate 10 in this way, the enzyme exists between the transparent substrate 10 and the cell tissue S.

  FIG. 3 is a block diagram showing an electrical configuration of the ATP detection system 100 described above. The ATP detection system 100 includes a measuring apparatus 1 and a PC 20, both of which are connected to each other by a cable 18.

  As described above, the measuring device 1 is a device for detecting ATP released from the cell tissue S, and stores a CPU 2 that is an arithmetic device, and various control programs and fixed value data that are executed by the CPU 2. ROM 3 as a non-volatile memory, RAM 4 as a memory for temporarily storing various data, input / output port 6, light source unit 7, photodetector 12, CCD camera unit 13, The interface 17 is provided.

  The CPU 2, ROM 3, and RAM 4 are connected to each other via a bus line 5, and the bus line 5 is also connected to an input / output port 6. The input / output port 6 is connected to a light source unit 7, a photodetector 12, a CCD camera unit 13, and an interface 17.

  The CPU 2 decodes the command transmitted from the PC 20 and controls each part of the measuring apparatus 1 according to the command. When the command transmitted from the PC 20 is a light source lighting command, the CPU 2 causes the light source unit 7 to turn on the light source 8. If the command transmitted from the PC 20 is a measurement execution command for instructing execution of ATP measurement, the CCD camera unit 13 is caused to execute an operation for acquiring an imaging signal.

  The CCD camera unit 13 includes a CCD 15 and an A / D converter 16 and takes a fluorescent image of the cell tissue S, and its operation is controlled by a controller (not shown). Each pixel signal (imaging signal) acquired by the CCD 15 is amplified by an amplifier, converted into a voltage value, and further converted into digital data by an A / D converter 16. A group of pixel data of all the pixels of the CCD 15 constitutes one frame of image data. The controller executes imaging for the designated number of frames at the designated time lapse, and outputs the obtained image data to the PC 20 via the interface 17.

  In the present embodiment, the CCD camera unit 13 is provided with a color filter, and the image data is formed of color image data (RGB data).

  As described above, the PC 20 controls each process in the measurement apparatus 1, and includes a CPU 21, a ROM 22, a RAM 23, a hard disk drive (hereinafter referred to as “HDD”) 24, and an input / output, which are arithmetic devices. A port 26, an LCD 27 as a display device, a keyboard 28 as an input device, and an interface 29 are provided.

  The CPU 21, ROM 22, RAM 23, and HDD 24 are connected to each other via a bus line 25, and the bus line 25 is also connected to an input / output port 26. The input / output port 26 is connected to the LCD 27, the keyboard 28, and the interface 29.

  The CPU 21 is an arithmetic unit that executes programs stored in the ROM 22 and the HDD 24. Although not shown, the CPU 11 includes a timer. The timer is for measuring a predetermined time, and the stable time of the light source 8 is measured in the ATP measurement process described later.

  The ROM 22 is a non-volatile memory that stores various control programs executed by the CPU 21 and fixed value data.

  The RAM 23 is a rewritable memory for temporarily storing various data and the like, and has a work area for temporarily storing variables and the like when the CPU 21 executes various programs. The RAM 23 includes a setting value memory 23a, a measurement value memory 23b, and an image memory 23c.

  The set value memory 23a is a memory for storing measurement conditions. The ATP detection system 100 can arbitrarily set measurement conditions to be executed by the measurement apparatus 1 based on an input from the operator. The measurement conditions input by the operator are a measurement time (exposure time), a time lapse, the number of photographing frames, an irradiation light amount (applied voltage) of the light source 8, and an amplifier gain. When a measurement condition is input from the operator in an ATP measurement process to be described later, the value of the input item is written in the set value memory 23a, and the previously stored value is updated. When the ATP measurement process described later is executed, the CPU 21 refers to the set value memory 23a and instructs the measurement apparatus 1 to execute measurement under the stored measurement conditions.

  The measurement value memory 23b is a memory for storing image data transmitted from the measurement apparatus 1, and is a buffer memory that functions as a reception buffer. When image data is received from the measuring apparatus 1 after the measurement execution command is transmitted from the PC 20 to the measuring apparatus 1, the received image data is written into the measured value memory 23b. When one frame of image data is stored, the stored image data for one frame is written into the image memory 23c. The image data written in the image memory 23c is erased from the measurement value memory 23b. Note that, during the measurement time, the measurement apparatus 1 repeatedly captures an imaging signal, so that the PC 20 continuously receives image data.

  The image data received by the PC 20 is stored in the measurement value memory 23b and then written in the HDD 24, and image data for the set number of frames is held in the PC 20.

  The image memory 23 c is a memory for storing image data to be displayed on the LCD 27. In the image memory 23c, each pixel data constituting the image data is stored in association with the coordinates (x, y). The coordinates are information indicating a position by an X coordinate (x) indicating a position on the X axis on the LCD 27 and a Y coordinate (y) indicating a position on the Y axis orthogonal to the X axis. When the image data for one frame is stored in the measurement value memory 23b, the CPU 21 writes each pixel data in the image memory 23c in association with predetermined coordinates. When displaying on the LCD 27, image data is read from the image memory 23c, and each pixel data is output to the LCD 27 based on the corresponding coordinates. Thereby, the imaged image data of the tissue cell S is displayed at a predetermined position on the display screen of the LCD 27.

  When the image data for one new frame is stored in the measurement value memory 23b, the image data for one frame is written in the image memory 23c, and the image data stored first is updated.

  The HDD 24 is a rewritable large-capacity memory, and is a non-volatile memory that holds stored data even after the power is turned off. In addition to an operating system (not shown), the HDD 24 includes a measurement device control program 24a for controlling the measurement device 1 and an initial value memory 24b. The ATP measurement processing program shown in the flowchart of FIG. 4 is stored in the HDD 24 as a part of the measurement apparatus control program 24a. Various programs stored in the HDD 24 are loaded into the RAM 23 as necessary and executed by the CPU 21.

  The initial value memory 24b includes measurement conditions (measurement time (exposure time), time lapse, number of frames taken, amount of light emitted from the light source 8 (applied voltage), amplifier gain), and other necessary conditions when the measurement apparatus 1 executes ATP measurement. This is a memory that stores a large value (for example, the stabilization time of the light source 8) by default. When the ATP measurement process is started in the PC 20, the measurement conditions stored in the initial value memory 24b are written and stored in the set value memory 23a. Therefore, when an arbitrary measurement condition is not input by the operator, measurement is executed by the measurement apparatus 1 in accordance with the measurement condition stored by default in the initial value memory 24b. Further, when the measurement condition is input by the operator, the measurement is executed on the input item based on the input value.

  Next, the operation of the ATP detection system 100 configured as described above will be described with reference to the flowchart of FIG. FIG. 4 is a flowchart showing an ATP measurement process executed by the PC 20. The ATP measurement process is a process for causing the measurement apparatus 1 to measure the concentration distribution of ATP in the cell tissue S based on an input operation by the operator. The ATP measurement process is started when the operator requests the PC 20 to perform ATP measurement by a predetermined input operation.

  As shown in FIG. 4, in this ATP measurement process, first, a light source lighting command is transmitted to the measuring apparatus 1 (S1). Thereby, in the measuring apparatus 1, the light source 8 is turned on, and the irradiation light (excitation ultraviolet light) is incident on the end face of the transparent substrate 10. The CPU 21 sets a time (stable time) necessary for stabilizing the light source 8 in the timer at the timing when the light source lighting command is transmitted to the measuring apparatus 1.

  Next, the value of the initial value memory 24b (measurement conditions stored by default) is written to the set value memory 23a (S2), and various commands for executing processing based on input from the operator are displayed. The screen is displayed on the LCD 27 (S3). Thus, when a command displayed on the main screen is input by a predetermined input operation, the PC 20 executes processing according to the input command. Therefore, it is confirmed whether the command is input by the operator (S4). If there is no command input (S4: No), the input of the command is waited. On the other hand, if a command is input (S4: Yes), what is the input command is confirmed (S5), and if the input command is an end command (S5: end command), a measurement end request is received. Therefore, a light source turn-off command is transmitted to the measuring apparatus 1. Thereby, in the measuring apparatus 1, the light source 8 is turned off. Thereafter, in order to end the process in the PC 20, an end process for clearing RAM, saving data, erasing the output screen, etc. is executed (S7), and this ATP measurement process is ended.

  If the input command is a measurement execution command as a result of checking in the process of S5 (S5: measurement execution command), it is an ATP measurement execution request. It is confirmed whether the time (stable time) has elapsed (S8). If the stable time has not elapsed, it is determined that the light source 8 is not stable (S8: No), and the stable time is reached. Wait for further processing until it elapses. On the other hand, if the stable time has elapsed, it is determined that the light source 8 has been stabilized (S8: Yes), and measurement conditions (measurement time (exposure time), time lapse, photographing) stored in the measurement execution command and the set value memory 23a. The number of frames, the amount of light emitted from the light source 8 (applied voltage), and the amplifier gain) are output to the measuring apparatus 1 (S9). Thereby, in the measuring apparatus 1, the designated light quantity is emitted from the light source 8, and the image pickup signal is captured by the CCD 15 for the designated time. When the measurement execution command is output to the measurement device, the CPU 21 sets the measurement time stored in the set value memory 23a to the timer.

  Subsequently, the PC 20 checks whether measurement data, that is, image data has been received from the measurement apparatus 1 (S10). If it has not been received (S10: No), the PC 20 waits for reception, and if received ( (S10: Yes), the received image data is stored in the measured value memory 23b (S11). Then, it is confirmed whether reception of one frame of image data is completed (S12). If it is not completed (S12: No), the process proceeds to S11 until reception of one frame of image data is completed. The processes of S11 and S12 are repeated. If the reception of one frame of image data has been completed as a result of the confirmation in S12 (S12: Yes), the image data stored in the measurement value memory 23b is associated with predetermined coordinates and the image memory 23c. (S13), and then the image data stored in the image memory 23c is output to the LCD 27 to update the display screen (S14). Thereby, the fluorescence image of the cell tissue S imaged by the measuring apparatus 1 is displayed at a predetermined position on the main screen.

  At the timing when the image data is displayed on the LCD 27, the image data stored in the image memory 23c is written and stored in a predetermined area of the HDD 24 by steps not shown.

  Thereafter, it is confirmed whether the measurement time has ended by the timer value (S15). If the measurement time has not ended (S15: No), the process proceeds to S10 until the measurement time ends. , S10 to S15 are repeated. On the other hand, as a result of checking in the process of S15, if the measurement time has ended (S15: Yes), the process proceeds to the process of S4. When the process proceeds to S4 after the process of S15, the image data stored in the image memory 23c is continuously displayed.

  Furthermore, if the input command is a condition setting command as a result of checking in the process of S5 (S5: condition setting command), the measurement condition input screen is displayed because the measurement condition change request is received from the operator. (S16). This input screen is provided with an input field for inputting an arbitrary measurement condition based on an input from the keyboard by the operator, and is configured so that the operator can input a command indicating completion of the input of the measurement condition. Yes. The CPU 21 monitors whether or not a command indicating completion of input of measurement conditions is input on this input screen (S17). If no command indicating completion of input of measurement conditions is input (S17: No), the CPU 21 waits for the input. On the other hand, if the command indicating the completion of the input of the measurement condition has been input (S17: Yes), the measurement condition input in the input field is written in the set value memory 23a (S18), and the corresponding stored previously. Update the value. Then, after executing each process (S19), the process proceeds to S4.

  In each process (S19) executed after the process of S18, a process of switching the display screen of the LCD 27 to the main screen is executed. Further, for example, when the value input in the input field exceeds the range that can be executed by the measuring apparatus 1, a process of displaying an error message is executed.

  In addition, as a result of checking in the process of S5, if the input command is other commands other than the end command, the measurement execution command, and the condition setting command (S5: other), the process corresponding to the input command is performed. The process is executed in the process (S19), and the process proceeds to the process of S4. Other commands include commands for executing calibration of the measuring apparatus 1. When calibration is requested, data detected by the photodetector 12 of the measuring apparatus 1 is displayed on the LCD 27. As a result, the operator can adjust the incident angle of the light source 8 while visually checking the detection data displayed on the LCD 27.

  As described above, according to the ATP detection system 100 of the present embodiment, the concentration distribution of ATP released from the cell tissue can be detected in association with the tissue structure. For this reason, it can be clarified how the cell tissue releases ATP and from which site ATP is released.

  For example, the inflammatory reaction in the brain is often progressive, but according to the present ATP detection system 100, the position where ATP is released and its change over time can be detected for the inflammatory reaction in the brain. Based on the obtained detection result, diagnosis and treatment performed by a doctor can be facilitated smoothly.

  Furthermore, by using the ATP detection system 100, the ATP release status, temporal changes, and spatial patterns can be observed, so that development, screening, and treatment of drugs having a neuroprotective effect can be performed. In addition, freshness can be evaluated by inspecting fresh food products with the ATP detection system 100.

  In this embodiment, “ATP” is used as the nucleotide-related chemical substance. However, as a matter of course, any nucleotide-related chemical substance serving as a phosphate supply source can be used on the cell tissue using this system. Of nucleotides can be detected. According to this, for example, a phosphate component and a nucleotide component contained in food can be measured.

In the above-described embodiment, the ATP detection system 100 is configured to perform measurement by placing the cell tissue S obtained by puncturing or cutting on the transparent substrate 10 installed outside (in vitro). However, a transparent substrate 10 is formed on a part of a probe-like optical fiber, and a catheter for supplying a GAP or NAD ⁺ , or a catheter provided with a CCD at a position facing the transparent substrate 10 via the optical fiber, It is good also as a structure which measures in the state inserted in.

  In the above embodiment, the substrate holding unit 16 has been described as being fixed. However, a transfer motor is provided, and the transparent substrate 10 can be moved to any position in the vertical and horizontal directions in accordance with a position control command input from the PC 20. You may do it. According to this, it becomes easy to finely adjust the positional relationship between the incident light from the light source 8, the transparent substrate 10, and the cell tissue S to be measured, and high-accuracy measurement can be realized.

  Further, in the above embodiment, the excitation light is incident on the end surface of the transparent substrate 10 and the CCD camera unit 13 that is a detector is disposed below the transparent substrate 10. However, the present invention is not limited to this. As a method for entering the excitation light, a prism method, a grating method, or the like may be used. Furthermore, it is good also as a structure which detects the fluorescence radiated | emitted from the cell tissue S above the transparent substrate 10, and is good also as a structure which detects what propagated the transparent substrate 10 which is a waveguide.

  In addition, the light source unit 7 may be disposed directly opposite the end face of the transparent substrate 10 and the light source 8 may be installed so that excitation light is incident from the side. Even in such a configuration, evanescent light can be generated on the transparent substrate 10 and ATP on the surface of the cell tissue S can be detected. In addition, no equipment such as a lens for adjusting the optical path is required, and the entire apparatus can be downsized.

The cell-release substance detection method described in the claims includes a series of steps of detecting ATP performed using the ATP detection system 100 described in the above embodiment, that is, fixing GAPDH in the above embodiment. In the state where GAP and NAD ⁺ are supplied to the surface of the transparent substrate 10 toward the cell tissue S placed on the transparent substrate 10 that has been converted to light, excitation light is emitted from the light source unit 7 and emitted from the cell tissue S. This corresponds to a series of steps of detecting the fluorescence to be detected by the CCD camera unit 13 and forming and displaying an image on the PC 20 based on the detected fluorescence.

EXAMPLES Next, although this invention is demonstrated further in detail based on an Example, this invention is not limited to these Examples. As an example, with the ATP measurement system 100 described above, rat cerebrum was used as the cell tissue to be measured, and the ATP concentration distribution on the tissue cells was measured.
Example 1

  A newborn rat (Normal P23) immediately after birth was prepared, disinfected with 70% ethanol, and decapitated under sterilization. In order not to damage the cerebellum, the scalp and skull were cut from the medullary side and incised. Thereafter, the whole brain was lifted and extracted with a bend tweezers, and blood and the like were washed in physiological saline. Thereafter, the whole brain was transferred onto a petri dish, the medulla and diencephalon were separated with a scalpel knife, and the cerebrum was removed. The arachnoid membrane and blood vessels attached to the cerebrum were peeled off with a tapered tweezers and washed again with physiological saline, and then the isolated cerebrum was sliced to 400 μm with a rotary slicer or cryostat.

The slice piece (cell tissue S) was scooped with a wide-mouth dropper, and after excess moisture was absorbed, it was immersed in a HEPES buffer immediately before measurement, and then placed on the transparent substrate 10 of the measuring apparatus 1. Next, GAP and NAD ⁺ were added, and 10 mM after the start of measurement, 1 mM glutamic acid was dropped on a slice piece on the transparent substrate 10 to capture a fluorescent image. Glutamate is known to damage brain cells, and ischemia is simulated by administration of glutamate.

  Prior to taking a fluorescent image, the ATP measurement process was started in the PC 20 and the light source 8 was turned on. The measurement conditions were set with an exposure time of 300 s, a maximum gain, a time plus 1.0 s, and a number of frames of 130.

  In this example, the light source 8 was an LED having an ultraviolet wavelength of 365 nm (Nichia NSHU550B (ultraviolet LED)) and was observed with an inverted fluorescence microscope equipped with a CCD camera below the transparent substrate 10. Further, as the transparent substrate 10, a quartz glass plate (25 mm × 25 mm × 1 mm) having GAPD fixed on the surface thereof was used.

  The results are shown in FIG. 5 and FIG. FIG. 5A is a diagram showing a state in which a slice of a cerebrum prepared by the above procedure is observed with visible light. FIG.5 (b) is the figure which showed the result of having observed the slice piece shown to Fig.5 (a) by fluorescence, and is a fluorescence image of the sample piece after glutamic acid administration. FIG. 6 is a diagram showing temporal changes in the fluorescence intensities at the respective positions a to h shown in FIG.

  The optical image shown in FIG. 5A and the fluorescent image shown in FIG. 5B correspond to each other, and in each figure, the left side of the drawing is the outside of the cerebral cortex. The cerebral cortex is a six-layered structure, and in FIG. 5, the layers are laminated in the order of one to six layers from the lower left to the upper right of the drawing. In FIG.5 (b), a white part is a fluorescence emission location.

FIG. 5A and FIG. 5B show that strong fluorescence is generated in the first and second layers of the cerebral cortex. Also, the eight graphs corresponding to the respective positions a to h shown in FIG.
The horizontal axis represents time (seconds), and the vertical axis represents fluorescence intensity. The fluorescence intensity is a relative value with a maximum value of 4096. As can be seen from FIG. 5 and FIG. 6, it is shown that the fluorescence intensity at the positions a, b, and c of the sample piece is clearly increased.

  In the cerebral cortex, many glial cells are distributed in the first and second layers, and in the sample piece in which cerebral ischemia is induced by glutamic acid administration, strong fluorescence is generated in the first and second layers. Was in good agreement with the physiological findings. That is, the above results show that ATP was released from damaged cell tissue. When the cell-released substance detection device of the present invention is used, nucleotide-related substances released from cells on the cell tissue. It was shown that the density distribution can be detected by imaging.

7 Light source unit (light irradiation means)
10 Transparent substrate (substrate, immobilized enzyme substrate for cell-release substance detection)
15 CCD (detection means)
100 ATP detection system (cell release substance detection device)
S13, S14 (part of image forming means)

Glyceraldehyde 3-phosphate dehydrogenase (hereinafter sometimes simply referred to as “GAPDH”) is an enzyme involved in glycolysis in vivo and is a substrate glyceraldehyde 3 -phosphate ( Hereinafter, it is simply abbreviated as “GAP”). GAP becomes 1,3-bisphosphoglycerate by the action of GAPDH. The phosphorylation reaction catalyzed by GAPDH is usually based on inorganic phosphoric acid as a phosphoric acid supply source in vivo.

In this example, the light source 8 was an LED having an ultraviolet wavelength of 365 nm (Nichia NSHU550B (ultraviolet LED)) and was observed with an inverted fluorescence microscope equipped with a CCD camera below the transparent substrate 10. . Further, the transparent substrate 10, using a quartz glass plate with a fixed GAPD H on the surface (25mmx25mmx1mm).

Claims (9)

A substrate having an enzyme that catalyzes a phosphorylation reaction that phosphorylates the substrate and an oxidation reaction that oxidizes the substrate after phosphorylation on the placement surface side on which the cell tissue is placed;
A light irradiating means for irradiating a cellular tissue on the substrate with excitation light for exciting a fluorescent material generated from the fluorogenic material with a reaction on the substrate;
Detection means for detecting fluorescence emitted from the fluorescent material by the excitation light irradiated by the light irradiation means;
Image forming means for forming an image based on the fluorescence detected by the detecting means,
An apparatus for detecting a released substance of a cell, wherein the concentration distribution of a nucleotide-related chemical substance released from a cell tissue placed on the substrate is detected from an image formed by the image forming means.   The cell-release substance detection device according to claim 1, wherein the enzyme is glyceraldehyde triphosphate dehydrogenase.   The cell-released substance detection device according to claim 1 or 2, wherein the substrate is one in which the enzyme is immobilized on the surface of the mounting surface by chemical bonding. The light irradiation means includes a light source unit that makes excitation light incident on a side surface of the substrate,
The cell according to any one of claims 1 to 3, wherein evanescent light is generated on a mounting surface of the substrate by excitation light applied to a side surface of the substrate from the light source unit. Emission substance detection device.   5. The cell emission substance detection apparatus according to claim 1, wherein the detection means is disposed at a position that does not overlap an optical path of excitation light emitted by the light irradiation means. A method for detecting a concentration distribution of nucleotide-related chemical substances released from cellular tissue, comprising:
A phosphor and a fluorescent substance accompanying the enzyme reaction toward the cell tissue placed on the substrate having an enzyme that catalyzes a phosphorylation reaction that phosphorylates the substrate and an oxidation reaction that oxidizes the phosphorylated substrate. A light irradiation step of irradiating excitation light of the fluorescent material in a state where the fluorescent material to be converted into the substrate coexists on the substrate;
A fluorescence detection step for detecting fluorescence emitted from the fluorescent material excited by the excitation light irradiated by the light irradiation step;
An image forming step of forming an image based on the fluorescence detected by the fluorescence detection step,
A method for detecting a cell-released substance, comprising detecting a concentration distribution of a nucleotide-related chemical substance released from a cell tissue from an image formed by the image forming step.   The cell-release substance detection method according to claim 6, wherein the enzyme is glyceraldehyde triphosphate dehydrogenase.   The method for detecting a cell-released substance according to claim 6 or 7, wherein the enzyme is immobilized on the surface of the substrate by chemical bonding, and the cell tissue is placed on the surface of the substrate.   An immobilized enzyme substrate for detecting a cell-releasing substance, characterized in that glyceraldehyde triphosphate dehydrogenase is supported by a chemical bond on the surface of a transparent substrate having a smooth surface.

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