JP2007279015A - Sensor and method for detecting cell activity - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a means and a method for acquiring sequentially time-serial information from one cell or from a cell population. <P>SOLUTION: This sensor for detecting cell activity is provided with at least two kinds or more of detecting particles, and detects a substance derived from the activity of the cell. The each detecting particle contains a detecting component indicating optical characteristics, in response to the detecting substance, and the kinds of the detecting particles are different, in response to kinds of the detecting components. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a cell activity detection sensor for detecting information derived from cell activity, and a cell activity detection method using the cell activity detection sensor.

  The omics analysis technology such as genome, transcriptome, proteome, metabolome, and cerome is a technology system that analyzes living phenomena as a whole of genes, transcriptions, proteins, and metabolism, and analyzes the living body itself. Omics technology and ideas are used in the medical and industrial fields from the most basic academic field of describing life phenomena.

  Genome analysis has become possible due to the development of sequence analysis technology using a high-speed DNA sequencer, but recently, as a next-generation genome sequence analysis technology, a large number of DNA probes are classified and fixed on a solid surface. Sequencing by hybridization using DNA microarrays (DNA chips) and high-throughput pyrosequencing using microreactor beads have been studied as real things. In transcriptome analysis, DNA microarrays (DNA probe arrays and techniques for the same purpose for DNA chips) have already been put into practical use and used by many researchers. For proteome analysis, protein array chips in which various proteins are immobilized as probes (generally, antibodies are used as probes) have been used.

  In order to make a DNA microarray or protein array chip, a method of synthesizing oligomers of sequences designed in a large number of compartmented cells using photochemical reaction and lithography often used in the semiconductor industry one by one (Non-patent Document 1) Or a method of implanting DNA probes or protein probes one by one in each compartment (see Non-Patent Document 2).

  Each of these microarrays has a structure in which a plurality of probes are aligned in an array form by dividing the section into a slide glass or silicon plane. In either method, as a sample, cells or tissues are disrupted to extract DNA (for genome analysis), RNA (for transcriptome analysis) or protein (for proteome analysis) present in the cell, In general, a substance that has been reacted and captured on a chip is labeled and detected by some method. In particular, for analysis of DNA or RNA, pretreatment for amplifying the extracted DNA or RNA by PCR or the like is performed, and at this stage, fluorescent labeling is performed on the cDNA to which the sample DNA or RNA is transcribed. Since the protein cannot be amplified, there is a case where the signal is amplified using an enzyme for the label as in the immunoassay.

  For detection, fluorescent samples are introduced onto the substrate by hybridizing a sample polynucleotide (hereinafter simply referred to as a sample polynucleotide) such as a DNA fragment or mRNA fluorescently labeled onto a probe on the chip substrate and cDNA reversely transcribed from this. Detect with a scanner. Alternatively, after hybridizing the sample polynucleotide, a fluorescently labeled oligo complementary to the sample polynucleotide is ligated adjacent to the probe by a ligation reaction, or a fluorescently labeled dNTP substrate is reacted using a DNA polymerase. The phosphor to be introduced onto the substrate is detected. Recently, an electrochemical detection method using a redox reaction has been put into practical use.

  In the case of proteins, an affinity reaction such as an antigen-antibody reaction is used to capture a specific protein on the substrate and then analyzed with a mass spectrometer, or a sandwich reaction is performed with a fluorescently labeled antibody or an enzyme labeled antibody. There are a method for detecting the phosphor and enzyme activity remaining on the surface, and a detection method using electrochemiluminescence. In an example of the electrochemiluminescence method, a protein is captured on the electrode surface with an antigen capturing antibody. A sandwich reaction is performed on the captured protein with a ruthenium complex-labeled antibody (see Non-Patent Document 3). Ruthenium is oxidized on the electrode surface, and emits light when the ruthenium electrons, which are excited when they are reduced by being coupled with the redox reaction of TPA, fall to the ground state. As a detection method aiming at high-sensitivity and quantitative detection, particles of about 700 mm that can be detected with a normal microscope can be used as a label, and the number of particles reacted on the substrate is counted with a counter to determine the target substance. There is a report of an immunoassay for quantitative detection (see Non-Patent Document 4).

  Metabolome and serome analyze the metabolic status and activity status of living organisms and cells. Currently, mass spectrometry is the most realistic method for comprehensively analyzing metabolites. Methods have also been developed for measuring localization of specific substances in cells and changes over time of individual substances. For example, it is a well-known fact that various ion trackers have been developed as materials for measuring ions such as calcium and magnesium and are widely used in ion channel research. This is a technique of measuring the intracellular spatial distribution of ions using a fluorescent compound that coordinates to ions present in the cells. Another example is a method of tracking the localization of a specific protein in cells over time using green fluorescein protein (GFP).

On the other hand, research has been conducted on optically or electrochemically measuring cells while culturing them in a microstructure on a substrate. Since these measure the shape of the cell and change in the cell potential, information can be obtained while the cells are continuously alive (see Patent Documents 1 to 4).
Science 251, 767-773 (1991) Proc. Natl. Acad. Sci. USA 93, 4613-4918 (1996) Clin. Chem., 37, 1534-1539 (1991) Anal. Biochem. 202, 120-125 (1992) JP 2006-42671 A JP 2006-94703 A JP 2006-1112846 A JP 2006-115723 A

  The biggest problem in obtaining information on living organisms, especially cells, using DNA microarrays, protein array chips, or mass spectrometry is the destruction of cells and tissues in order to perform these analysis methods. DNA, RNA, protein, and metabolites must be analyzed. In the first place, it is not enough to obtain information at a certain moment using various microarrays and mass spectrometry in order to describe life phenomena, and it is necessary to grasp how substances change in a continuous time. Moreover, since cells interact with each other, it is necessary to describe the physiological behavior of each cell in consideration of the spatial positional relationship between the cells.

  Of course, even in the conventional method, analysis is performed to destroy a group of cells existing in various spaces every moment and to have spatial and temporal continuity, but the same cell or group of cells is used. Therefore, averaged data obtained by statistical processing is obtained. For this reason, for example, when measuring a specific cell in a multicellular tissue, it is considered to measure how the reaction of a cell at a distant location (hereinafter referred to as a reporter cell) becomes, Obviously it becomes a problem. In other words, even if many sets of stimulator cells and reporter cells are obtained for measurement over time, and the response of the reporter cells after stimulation is measured and measured, it is difficult to accurately control the state of each cell. By repeating this experiment, statistical processing is performed, and important signals are buried in noise. Even if data that is not buried in noise is obtained by statistical processing and the result of seemingly continuous analysis is obtained, the state of the same cell or tissue is not actually chased over time.

  In recent biology, research to investigate how individual cells function while interacting with the outside world and neighboring cells is becoming popular. For this purpose, as described above, Conventional methods of destroying cells and examining their components are often not appropriate. In addition, when one cell is basically required to be handled, such as an embryonic stem cell used for regenerative medicine, the cell cannot be destroyed for analysis. These are used after differentiation induction, but it is necessary to follow and control the cell state from the early stage of division.

  In the method using an ion tracker or GFP, the localization of the ion or specific protein in the cell can be measured over time, but the range of use is limited. This is because ion trackers have limited use for ions such as calcium and magnesium. In the method using GFP or a similar fluorescein protein, a fusion protein in which the fluorescein protein is recombined with the target protein is produced using a gene recombination technique, and the fluorescence is measured. Therefore, the size of the original protein is different and may affect the activity, and there is no guarantee that the same information as the native cell can be obtained. Furthermore, in these methods, since substances that can be measured are limited to inorganic ions or fusionable proteins, it is difficult to trace substances involved in metabolism of sugars, fatty acids, nucleic acids, and proteins, particularly low molecular organic substances. As a method of using mass spectrometry, a method of measuring volatile components in a single screen with a mass spectrometer has been studied as in the case of an expiratory mass spectrogram, but in order to perform measurement at the level of individual cells or cell populations. In terms of the lower limit of measurement, the reality is unavoidable at present.

  An object of the present invention is to provide means and a method for sequentially obtaining information over time from one cell or a cell population (a group of cells having the same function that is supposed to perform a specific function of a tissue). It is another object of the present invention to propose a means and method capable of performing sequential analysis while cells are not exhausted but remain alive.

  The present invention is a cell activity detection sensor comprising at least two types of detection particles and detecting a substance derived from cell activity, wherein the detection particles have optical absorption characteristics corresponding to the substance to be detected. The type of the detection particle is different depending on the type of the detection component. The detection particles can be configured to be identifiable based on their fluorescence characteristics, nuclear magnetic resonance characteristics, magnetic characteristics, optical absorption characteristics, particle size, or a combination thereof, and in particular the detection particles. Such a configuration is useful when there are a large number of types.

  The detection component preferably includes a metal coordinated porphyrin derivative and / or a metal coordinated phthalocyanine derivative. Metal-coordinate porphyrin derivatives and metal-coordinate phthalocyanine derivatives are useful for detecting organic compounds produced by cells. The particle size of the detection particles is preferably 0.1 to 10 μm. Preferably, the detection particles include a gas permeable layer, and gas from the outside of the detection particles passes through the gas permeable layer and reaches the detection component.

      When detection particles are introduced into cells and used, the detection particles are configured to have an outermost surface layer containing a compound having a guanidyl group in order to reduce physical damage to the cells. It is preferable that With such a configuration, the interaction with the phospholipid on the cell membrane on the cell surface can be facilitated, and the detection particles can smoothly enter the cell. Arginine is mentioned as a compound which has a guanidyl group. It is preferable that guanidyl groups are fixed so that there are at least about 6 residues in a narrow range on the outermost surface of the detection particles. When arginine is contained, at least about 6 arginine molecules exist in a narrow range. It is preferable to do this. Of course, an oligomer or polymer having an arginine or guanidyl group may be fixed and used.

  The present invention also relates to a cell activity detection method for detecting a substance derived from cell activity using the cell activity detection sensor, wherein the plurality of detection particles are arranged inside and / or outside the cell. It has an arrangement | positioning process, the measurement process which measures the optical absorption characteristic of the said particle for detection, and the analysis process which analyzes the activity of a cell based on the measurement result of the said measurement process.

  The arrangement step includes, for example, a step of dispersing the plurality of detection particles in cells. Moreover, the arrangement step includes, for example, a step of arranging the plurality of detection particles in the vicinity of the cell in a contact state and / or a non-contact state with the cell.

  Preferably, the analysis step includes a step of acquiring information on the detection substance in correspondence with the position information.

  According to the present invention, a substance produced by the activity of a cell can be detected inside and / or outside the cell, and a living cell can be measured over time. For this reason, it is possible to continuously monitor biochemical changes related to the same cell.

  The present invention is a cell activity detection sensor comprising at least two types of detection particles and detecting a substance derived from cell activity, wherein the detection particles have optical absorption characteristics corresponding to the substance to be detected. It is a sensor for detecting cell activity, which contains the detection component shown, and the type of the detection particle is different depending on the type of the detection component.

  The cell activity detection sensor of the present invention may be detected by arranging the detection particles in the cells, may be detected by arranging the detection particles in the vicinity of the cells, or the detection particles Detection may be carried out by arranging in the cell and in the vicinity of the cell. When placed in the vicinity of the cells, the detection particles may be dispersed on the cells in culture and adsorbed on the surface of the cells, or a plurality of types of detection particles may be fixed on the substrate in advance. It is also possible to adopt a structure in which a plurality of types of detection particles are arranged in the vicinity of the cell by performing cell culture. In order to immobilize multiple types of detection particles on a substrate, different organic compound detection probes are immobilized on each particle of the particle group having mutually distinguishable elements, and the particle group is randomly immobilized on the substrate. By fixing the particles on the substrate so that the organic compounds released by individual cells can be analyzed while culturing the cells at any position in the region where the particles on the substrate are randomly fixed, The problem can be solved.

  The sensor for detecting cellular activity of the present invention is configured to detect a substance derived from the activity of the cell, for example, configured to detect a substance released by the cell due to the activity of the cell. . Examples of substance detection include detection for specifying the type of substance, detection for specifying the quantity, detection of change in the type of released substance, detection of change in the amount of released substance of interest, and the like.

  The kind of the particle for detection respond | corresponds to the kind of the component for a detection contained. As the detection component, a biological substance affinity compound can be used. Examples of the substance to be detected include an organic compound released by a cell. As a detection component for detecting such an organic compound, for example, a metal coordinated porphyrin derivative or a metal coordinated phthalocyanine derivative can be used. Metal-coordinated porphyrin derivatives and metal-coordinated phthalocyanine derivatives are particularly suitable for detecting organic compounds produced by cells. In addition, Solvatochromics known as Richardt's pigment, methyl red for detecting hydrogen ions, xylenol blue, and the like can be used.

  A metal coordinated porphyrin derivative can coordinate a specific molecular group or residue group based on the interaction between a metal coordination site in the central part, a hydrophobic porphyrin residue in the vicinity, a side chain of the porphyrin residue, and a substance. (Nature 406, 710-713 (1990), Angew. Chem. Int. Ed. 44, 4528-4532 (2005)). A metal coordinated porphyrin derivative coordinated with a specific molecular group has a light absorption spectrum different from a metal coordinated porphyrin derivative (hereinafter referred to as a free metal coordinated porphyrin derivative) not coordinated with a specific molecular group. For this reason, the quantity of the specific molecule group which interacted can be measured optically. If the side chain of the porphyrin and the coordination metal are changed, different molecular groups or residue groups will interact. Therefore, by preparing various metal coordinated porphyrin derivatives, various organic compound molecular groups and residues in organic compounds can be obtained. The base group can be detected. Similarly, metal-coordinated phthalocyanine derivatives must be coordinated to specific molecular groups or groups of residues based on the interaction between the metal coordination site in the central part, the hydrophobic porphyrin residue and the side chain of the cyanine residue, and the substance. Can do. Thus, both porphyrin and phthalocyanine have substantially the same compound properties.

  Since these metal-coordinated porphyrin derivatives and metal-coordinated phthalocyanine derivatives are highly hydrophobic and are expected to have an effect on the living body, it may not be preferable to add them directly into the cell or in the vicinity of the cell. In addition, even if added in the cell or in the vicinity of the cell, the proportion of molecules coordinated with biologically related substances such as organic compounds to metal coordinated porphyrin derivatives and metal coordinated phthalocyanine derivatives is low. Detection is difficult because the free metal-coordinated porphyrin derivative or metal-coordinated phthalocyanine derivative is an obstacle to measurement. In order to increase the ratio of metal-coordinated porphyrin derivatives coordinated with bio-related substances, reducing the concentration of metal-coordinated porphyrin derivatives slows the coordination reaction rate of bio-related substances, and in the first place, the number of molecules per volume In some cases, the number will decrease and the lower limit of detection will not be measured. In addition, when detecting multiple types of biological substances, multiple types of metal coordinated porphyrin derivatives and metal coordinated phthalocyanine derivatives must be added at the same time, and the spectra of each other metal coordinated porphyrin derivatives and metal coordinated phthalocyanine derivatives. Will interfere with each other and become a noise component, making it difficult to detect a trace amount.

  Therefore, in the present invention, in order to maintain the concentration of the metal coordination porphyrin derivative or metal coordination phthalocyanine derivative, which is a component for detection, and to enable measurement without interference with each other even when multiple types are used, the metal coordination A porphyrin derivative or a metal-coordinated phthalocyanine derivative is used in the particles. The form in which the metal coordinated porphyrin derivative or the metal coordinated phthalocyanine derivative is contained in the particle is not limited as long as the optical absorption spectrum of the metal coordinated porphyrin derivative can be measured. The form which apply | coated and fixed to the surface of the core of this, the form formed by mixing in the material which comprises the particle for detection, and forming the particle for detection, etc. are mentioned.

  The cell size is generally about 10 to 30 μm in the major axis for animal cells and about 10 to 100 μm in the major axis for plant cells. When the detection particles are used in the cells, the particle size of the detection particles is preferably about 1/10 or less of the major axis of the cells. Therefore, when the detection particles are used in a cell, the particle size of the detection particles is preferably 1000 nm or less. When the number of detection particles arranged in a cell is plural, it is preferable that the particle size is smaller. In the cell activity detection method of the present invention, the optical absorption spectrum of the detection component is measured, and the cell activity is analyzed based on the measurement result. In this case, the lower limit of the particle size of the detection particle is the objective lens under general conditions in which transmitted light or reflected light of the objective lens is wavelength-dispersed by a diffraction grating and an optical absorption spectrum is obtained using a CCD two-dimensional sensor. Is determined by the following resolution determined by the numerical aperture and the wavelength λ.

Resolution = 0.61λ / Numerical aperture The absorption wavelength of the metal-coordinated porphyrin derivative or metal-coordinated phthalocyanine derivative is in the visible light region of 400 to 600 nm. Therefore, when using a lens with a numerical aperture of 0.64, the wavelength is 375 to 572 nm. This is a temporary lower limit that allows the particle size to be analyzed. However, this value is obtained when transmitted light or reflected light is measured with a normal objective lens. It is a generally known fact that the resolution can be further improved by using a near-field microscopic optical system using an evanescence wave. In this case, even if the particle is smaller than the above-mentioned particle size, for example, a particle having a particle size of 50 nm, It is possible to measure the optical absorption spectrum by distinguishing the particles.

  Some metal coordinated porphyrin derivatives and metal coordinated phthalocyanine derivatives have fluorescence. Since the fluorescence spectrum of the metal coordinated porphyrin derivative or metal coordinated phthalocyanine derivative changes due to the interaction with the biological material, it can be used for detection of the biological material. In this case as well, even detection particles having a particle size of about 50 nm can be measured if the fluorescence intensity is sufficient. The smaller the particle size, the larger the amount of particles that can be introduced into the cell, which is advantageous when measuring the spatial distribution of the substance to be detected in the cell. In addition, even when using more types of metal-coordinated porphyrin derivative particles and metal-coordinated phthalocyanine derivative particles, the smaller the particle size, the greater the number of particles that can be introduced per species, so in cells with limited space. This is advantageous in measuring a plurality of types of substances.

  When the number of types of detection particles increases, as described later, for example, the absorption wavelength of the metal coordinated porphyrin derivative or metal coordinated phthalocyanine derivative, and further the fluorescence characteristics of the detection particles used, the nuclear magnetic resonance characteristics, It is preferable to be able to identify the type of detection particles based on magnetic characteristics, optical absorption characteristics (excluding characteristics derived from detection components), particle sizes, or combinations thereof. When making it possible to distinguish from each other by the combination of the absorption wavelength of the metal-coordinated porphyrin derivative or metal-coordinated phthalocyanine derivative and the particle size of the detection particles, the metal coordinated porphyrin derivative with a short absorption wavelength is included in the particles with a small particle size. It is preferable that a metal coordinating porphyrin derivative or a metal coordinating phthalocyanine derivative having a long wavelength is contained in a particle having a large particle size.

  In addition, in order to prevent the case where a metal coordinated porphyrin derivative or metal coordinated phthalocyanine derivative may give toxicity or activity to cells, in order to prevent this, the cytoplasm or cell surface and the metal coordinated porphyrin derivative or metal coordinated phthalocyanine derivative When a metal coordinated porphyrin derivative or a metal coordinated phthalocyanine derivative is applied to the surface of the core of the detection particle so as to prevent direct contact, a gas permeable layer is further formed on the surface. As a material for the gas permeable layer, a copolymer of 2-methacryloyloxyethyl phosphorylcholine and a hydrophobic substrate, which is the same as that for the polyfluorocarbon-based molecular layer or cell layer, is preferably used. Alternatively, a silicon polymer can be used. These gas-permeable thin layers are preferably thin in terms of the reactivity of the metal coordinated porphyrin derivative, which is a component for detection, and are particularly preferable since a good result can be obtained when the thickness is 0.2 μm or less. That is, the particle structure is at least a three-layer structure of a nucleus, an inner shell, and an outer shell, the core is made of a solid of an inorganic compound or an organic polymer, and the inner surface covered with the outer surface of the core is a metal coordinated porphyrin It is preferable that a biomaterial-affinity compound such as a derivative or a metal coordination phthalocyanine derivative is fixed, and the outer shell has a structure made of a gas-permeable substance.

  The material of the part that becomes the core of the detection particle is not particularly limited. For example, polystyrene is preferably used. When making polystyrene particles, metal coordinated porphyrin derivatives and metal coordinated phthalocyanine derivatives are mixed to form nuclei containing metal coordinated porphyrin derivatives, and a silicon polymer or polyfluorocarbon layer is formed on the surface, or 2-methacryloyl By adsorbing a copolymer of oxyethyl phosphorylcholine and a hydrophobic substrate, particles for detection whose surface is covered with a gas-permeable layer can be obtained. Alternatively, since the polystyrene particle surface is hydrophobic, a metal coordinated porphyrin derivative or metal coordinated phthalocyanine derivative is adsorbed to the polystyrene particle surface, and then a silicon elastomer polymer is adsorbed. It can be set as a particle for detection which has a porphyrin derivative layer, a metal coordination phthalocyanine derivative layer, and a gas-permeable layer on the outermost surface. In addition, silicon or glass inorganic materials can be used as the core material of the detection particles. In the case of silicon, the metal coordinated porphyrin derivative or metal coordinated phthalocyanine derivative may be adsorbed directly on the surface of the inorganic particles, or more generally, a silane treatment is performed to provide a hydrophobic organic layer on the surface and a metal is formed there. A coordinated porphyrin derivative or a metal coordinated phthalocyanine derivative may be bound. Adsorption of metal-coordinated porphyrin derivatives and metal-coordinated phthalocyanine derivatives uses a method of evaporating the solvent after applying a metal-coordinated porphyrin derivative or metal-coordinated phthalocyanine derivative dissolved in an organic solvent, and vacuum deposition technology Then, a method of fixing to the particle surface can be taken.

  The detection particles thus prepared are preferably used in a mixture of a plurality of types. For example, it is assumed that a group of particles each containing 256 kinds of metal coordinated porphyrin derivatives and metal coordinated phthalocyanine derivatives are incorporated into cells using a blunt action. For example, about 1 to 10 detection particles enter each cell. Alternatively, some particles remain on the cell surface. These particles are localized or dispersed in the cells, but each particle can be recognized individually. From the relationship between the wavelength and size of light, when measuring using light absorption, it is possible to distinguish and measure particles that are present at intervals of about 400 nm, so there are a large number of particles in which many particles are arranged at such intervals or less. It is preferable not to be taken up into cells.

  In this way, organic compounds produced by cells coordinate with metal-coordinating porphyrin derivatives and metal-coordinated phthalocyanine derivatives according to the properties of the metal-coordinated porphyrin derivatives and metal-coordinated phthalocyanine derivatives. The optical absorption spectrum and fluorescence spectrum of the particles change. By analyzing the spectrum of particles captured as a microscopic image using microspectroscopy, the amount of a specific molecular group (or a specific residue group in the molecule) inside and outside the cell can be measured. Since the information on the position of the particles can also be obtained with a microscopic image, the distribution of specific molecular groups inside and outside the cell can be obtained. Information from the same cell can be obtained continuously by measuring the spectral change while tracking the position of the particles at regular time intervals.

  When there are few types of metal coordinated porphyrin derivatives, metal coordinated phthalocyanine derivatives, etc., an index for identifying the spectrum of the metal porphyrin derivative, metal coordinated phthalocyanine derivative, etc., particularly the absorption maximum of the coordinated metal atom group itself Can be used as However, if the number of types of metal coordinated porphyrin derivatives, metal coordinated phthalocyanine derivatives, and the like to be used increases, it becomes impossible to distinguish them by their own spectrum alone, so another index is required.

  In the present invention, as the index, first, various phosphors are mixed in the particles, and the difference in fluorescence wavelength between the various phosphors is used as an index. Fluorescein-type, rhodamine-type, phthalocyanine-type, BODIPY, etc. can be used as the index phosphor. Although not particularly limited, the wavelength is different from the absorption maximum band of biomaterial-affinity compounds such as metal coordinated porphyrin derivatives Use fluorescent dyes in the region. When a fluorescent substance having the same wavelength as that of a biological substance affinity compound such as a metal coordination porphyrin derivative is used, quenching may occur. It is also possible to create a group of particles with different amounts of two to three different fluorescent dyes so that they can be identified. In any case, particles for detection having a biomaterial affinity compound such as a metal-coordinated porphyrin derivative that can be identified by combining a group of particles indexed with different fluorescent dyes and a different metal-coordinated porphyrin derivative in a one-to-one manner. Obtainable. About eight types of phosphors that can be distinguished from each other can be obtained in a wavelength region of 400 nm to 800 nm. A plurality of laser light sources are used for excitation of these phosphors. By using energy transfer, the number of laser light sources for excitation of a plurality of phosphors can be reduced.

  Further, the present invention is configured such that the type of detection particle can be identified by the nuclear magnetic resonance characteristics of the nucleus, and this may be used as an index. By using micro NMR with a gradient magnetic field of several T / mm, the types of detection particles distributed in the cell are identified.

  Further, the present invention may be configured such that the detection particles themselves are magnetic materials, and the detection types can be identified by the magnetic field characteristics. A SQUID can be used for magnetic field detection.

  Further, the present invention may be configured such that the type of particle for detection can be identified by the shape of the particle, particularly the particle size. By fixing different metal porphyrin derivatives, metal coordinated phthalocyanine derivatives, etc. to particles of different particle sizes, the types of porphyrin derivatives, metal coordinated phthalocyanine derivatives, etc. can be identified using the particles as an index. Since the particle size variation of the particles is expected to be about CV = 2 to 5%, for example, four stages of particle groups that can be distinguished from each other by the particle size can be prepared in the range of 400 nm to 600 nm. In this case, since the particle size to be used is as small as four stages, it is practical to combine with the maximum absorption wavelength of the biomaterial-affinity compound itself such as a metal porphyrin derivative or with an index of another fluorescent dye.

  As described above, as a method for obtaining cell information using particles encapsulating a biological substance affinity compound such as a metal coordinated porphyrin derivative as a cell activity detection sensor, a cell activity detection sensor is arranged outside the cell. There are a method for obtaining information, a method for obtaining information by inserting it into a cell, or a method for using these in combination. When the detection particles constituting the cell activity detection sensor are arranged outside the cells, the particles are added to the cells in culture and adsorbed to the cells, and the particles are fixed on the culture substrate in advance, The method of culturing cells can be taken. In order to insert a detection particle into a cell, a detection particle is inserted using a cell that can utilize phagotosis or endocytosis of the cell. When used for cells having low phagotosis or endocytosis activity, cell permeability is preferably enhanced by immobilizing a compound having a guanidyl group such as arginine on the surface of the detection particles. The compound having a guanidyl group may be covalently bonded to the surface of the particle for detection through a silane coupling reaction or the like. For example, it is blended and coated in a polymer state in a material forming a gas permeable layer. It is effective even if used.

(First embodiment)
The present embodiment relates to a cell activity detection sensor including a plurality of types of detection particles. FIG. 1 is a diagram schematically showing a cross section of detection particles constituting a cell detection sensor. The detection particle 1 of the present embodiment includes a detection layer 3 that includes a core 2 that forms a central portion thereof, and a detection component such as a metal coordination porphyrin derivative or a metal coordination phthalocyanine derivative that is formed on the surface of the nucleus 2. Consists of. The core 2 is a particle having a particle diameter of 400 μm made of polystyrene. The detection layer 3 is formed by adsorbing a detection component on the nucleus 2.

  As a method for adsorbing the detection component to the nucleus 2, a solution in which a detection component such as an arbitrary metal coordination porphyrin derivative or metal coordination phthalocyanine derivative is dissolved in an appropriate solvent is applied to the nucleus 2, and an excess solution is applied. After washing and removing, it can be dried and adsorbed. As for the solvent and the washing solution to be used, optimum ones differ depending on the components to be detected such as a metal coordination porphyrin derivative and a metal coordination phthalocyanine derivative to be adsorbed, but in general, it can be carried out by the following method. First, a metal coordination porphyrin derivative or the like is dissolved in ethanol at a concentration of 1 mM, and 0.5% (w / v) polystyrene particles similarly suspended in ethanol are mixed in an equal volume. After gently stirring for 30 minutes, the polystyrene particles are collected by centrifugation at 10,000 G for 30 minutes. The color of the precipitate changes when a metal coordinated porphyrin derivative or a metal coordinated phthalocyanine derivative is adsorbed. The color tone varies depending on the type of metal coordination porphyrin derivative used and the like, such as a red color tone and a blue color tone. The adsorption state of the metal coordinated porphyrin derivative or the like can be determined by the change in color of the polystyrene particles. When the color change of polystyrene particles is small, it may be determined that the amount of adsorption of metal coordinated polystyrene derivatives, etc. is small, but the cause is that the polystyrene particles used are coated with a surfactant etc. There is. In order to prevent this, it is preferable to wash the polystyrene particles with pure water and ethanol in advance. If the amount of adsorption is still small, reduce the alcohol concentration of the mixture. The precipitated polystyrene particles are rinsed with 20% ethanol, rinsed with pure water, and dried.

The porphyrin derivative compounds, as Nature418,399-402 (2002) porphyrin compound according (Apotaipu metal _{free), H 2 T (2,6-} OHPh) P, H 2 T (3,5-pyrimidyl) P , H ₂ TPP, H ₂ T (2-pyridyl) P, H ₂ T (3-pyridyl) P, H ₂ T (4-pyridyl) P, H ₂ tris (4-pyridyl) P, HH ₂ trans-bis 5,10,15, described in (4-pyridyl) P, H ₂ cis-bis (4-pyridyl) P, H ₂ mono (4-pyridyl) P, Angewandte Chemie Int. Ed. 44, 4528-4532 (2005) 20-tetrakis (2 ', 6'-bis (tert-butyldimethylsilyloxyl) phenyl) porphylyne can be used. Among these apoporphyrin derivatives, the most concerned metals include Fe ³⁺ , Sn ⁴⁺ , Co ³⁺ , Cr ³⁺ , Mn ³⁺ , Co ²⁺ , Cu ²⁺ , Ru ²⁺ , Zn ²⁺ , In addition to most transition metal ions belonging to 3A, 4A, 5A, 6A, 7A, 8, 1B, 2B, such as Ag ²⁺ , europium and ruthenium, Al, Ga, In, Tl, Si, Ge, Sn, Pb, As, Sb, Bi, Se, Te, Pr ions may be available. An arbitrary metal coordination porphyrin derivative is selected from a combination of these apoporphyrin derivatives and various metal (including semi-metal group) coordination compounds. As the phthalocyanine derivative, in addition to so-called phthalocyanine, various structures such as a structure in which a peripheral phenyl group is substituted with naphthol and a side chain added to the phenyl group or naphthol group can be used. Middle metals such as phthalocyanine derivatives include Fe ³⁺ , Sn ⁴⁺ , Co ³⁺ , Cr ³⁺ , Mn ³⁺ , Co ²⁺ , Cu ²⁺ , Ru ²⁺ , Zn ²⁺ , Ag ²⁺ In addition to most transition metal ions belonging to 3A, 4A, 5A, 6A, 7A, 8, 1B, 2B, such as europium and ruthenium, Al, Ga, In, Tl, Si, Ge, Sn, Pb, As , Sb, Bi, Se, Te, Pr may be available. Similar to the porphyrin derivative, an arbitrary metal coordination phthalocyanine derivative is selected from a combination of various metal (including semi-metal group) coordination compounds.

  Here, an example in which a metal porphyrin derivative or a metal coordinated phthalocyanine derivative is fixed to a single particle having a particle diameter of 400 nm has been described. The particles for detection thus prepared can be used as they are added to cells. When a plurality of types of detection particles are used at the same time, they can be identified by the absorption wavelength of the metal coordinated porphyrin derivative, and up to about 5 to 6 types of sensor particles can be used simultaneously for cell measurement.

  When there are more types of detection particles, other indexes may be combined in order to identify the types of detection particles.

  For example, the absorption wavelength of the metal coordinated porphyrin derivative or metal coordinated phthalocyanine derivative used as the detection component is combined with the particle size of the detection particles and the fluorescence index. First, two types of particles having a particle diameter of 405 nm and 450 nm are selected as detection particles constituting the detection sensor. In addition, it is possible to add three kinds of particles by adding 500 nm particles, and to further increase the particle size variation. In the two types of 405 nm and 450 nm, the particle size variation of the particles is SD = 2%, so that they can be separated from each other at 5σ or more.

  Further, two kinds of phosphors such as sulforhodamine 101 (λex = 594 nm, λem = 515 nm) and Cy-5 (λex = 650 nm, λem = 670 nm) are used as index phosphors for detection particles. A combination of particles in which the amount of two kinds of phosphors, sulforhodamine 101 and Cy-5, is changed to 8 stages is used. That is, the relative amount of Cy-5 is 0, 14, 28, 43, 57, 71, 86, 100, while the relative amount of Cy-5 is 0, 14, 28, 43, 57, 71, All combinations 64 particles including 8 types 86,100 are obtained. This makes it possible to identify particles having a particle size index of 2 to 3 and a fluorescence index of 64 and 2 × 64 = 128 to 3 × 64 = 192. Furthermore, the maximum absorption wavelength of the soret band of metal coordinated porphyrin derivatives and other metal coordinated porphyrin derivatives and metal coordinated phthalocyanine derivatives is classified into three stages of 400 nm or less band, 400 to 450 nm band, and 450 nm or more band. . By adding the sore band of the metal coordinated porphyrin derivative, the maximum absorption wavelength of the metal coordinated phthalocyanine derivative, and the like to the indexing, it is possible to identify particle sensors having 384 to 576 stages as a whole. The Soray band of the metal coordinated porphyrin derivative is concentrated in the 400 to 450 nm band, but this band can be further classified into sub bands. In any case, it is possible to discriminate 256 or more detection particles by devising a combination of particle size, fluorescence, and Soray band.

(Second Embodiment)
The present embodiment relates to a cell activity detection sensor including a plurality of types of detection particles. FIG. 2 is a diagram schematically showing a cross section of detection particles constituting a cell activity detection sensor. The detection particle 4 in the present embodiment is different from the detection particle 1 in the first embodiment only in that a gas permeable layer 5 is further provided outside the detection layer 3, and only this point will be described. Description is omitted.

  By providing the gas permeable layer 5 in the detection particle 4 of the present embodiment, it is possible to detect a substance derived from the activity of the cell without the detection component in the detection layer 3 being in direct contact with the cell. It becomes. The detection particles 4 of the present embodiment are suitable mainly for detecting volatile organic compounds.

  A method for producing the detection particle 4 of the present embodiment will be described. The detection layer 3 containing a metal coordinated porphyrin derivative or a metal coordinated phthalocyanine derivative is formed on the surface of polystyrene particles having a particle size of 400 μm by the same method as that for the detection particle 1 of the first embodiment. Next, 1 ml of a 25% ethanol solution of a copolymer of 0.2% 2-methacryloyloxyethyl phosphorylcholine and a hydrophobic substrate is added to the surface of the detection layer 3 and left for 15 minutes. And it wash | cleans and vacuum-drys. By this step, the detection particles 4 in which the surface of the detection layer 3 is covered with the gas permeable layer 5 having a hydrophilic property and gas permeability are obtained. As a copolymer of 2-methacryloyloxyethyl phosphorylcholine and a hydrophobic substrate, those having a large hydrophobic skeleton are suitable.

(Third embodiment)
The present embodiment relates to a cell activity detection sensor including a plurality of types of detection particles having cell membrane permeability. Cell permeability is enhanced by immobilizing a compound having a guanidyl group such as arginine on the surface of the detection particle. The compound having a guanidyl group includes a core 2 shown in FIG. 1 and a detection layer including a detection component such as a metal coordinated porphyrin derivative or metal coordinate phthalocyanine derivative formed on the surface of the nucleus 2 In the example consisting of 3, the guanidyl group is bonded directly to the surface of the nucleus. That is, the guanidyl group is exposed in the core 2 in an island shape, and a metal coordination porphyrin derivative, a metal coordination phthalocyanine derivative, or the like may be fixed to the remaining portion. As a method of exposing guanidyl groups in an island shape, silane coupling agents having guanidyl groups and metal coordination having carboxyl groups in the side chains in advance on submicrometer-sized glass or zirconium or silicon inorganic particles It can be prepared by dehydrating condensation of porphyrin derivative or metal coordination phthalocyanine derivative and the like and 3-aminopropyltrimethoxysilane.

  By performing a silane coupling treatment with a solution in which the compound is mixed, the inorganic particle surface is bonded to the guanidyl group and the metal coordinated porphyrin derivative or the metal coordinated phthalocyanine derivative in a chimeric form. Thereafter, a sensor particle having a guanidyl group and a metal coordinated porphyrin derivative or metal coordinated phthalocyanine derivative on the surface can be obtained by adsorbing a metal coordinated porphyrin derivative or metal coordinated phthalocyanine derivative.

  In the three-layered particle shown in FIG. 3, when forming the outermost gas permeable layer, a copolymer of 2-methacryloyloxyethyl phosphorylcholine and a hydrophobic substrate and a hydrophobic group in which an ethylene group such as a dodecyl group is linked. It is prepared by coating a compound having a structure in which a guanidyl group is present at one end of a sex residue in a mixture of about 10 to 1.

Example 1
In this example, an experimental example will be described in which the distribution of an organic compound in a cell is detected using the cell activity detection sensor of the second embodiment. Here, 64 types of detection particles having different types of metal coordination porphyrin derivatives are used as the cell activity detection sensors. Each detection particle has a gas permeable layer 5 consisting of a surface coating with a copolymer of 2-methacryloyloxyethyl phosphorylcholine and a hydrophobic substrate. In addition, each detection particle is indexed in 128 steps with a fluorescent index of sulfurhodamine 101 and Cy-5 and a particle size index of 405 nm and 450 nm, and the relative amount of sulfohodamine 101 and Cy-5 is 0, 14, 43, 71, 86, 100 and 100, 14, 43, 71, 86, 0 are used, which are selected to be 64 stages by combining 36 kinds and two kinds of particle sizes. . Sn (TPP) Cl ₂ , Co (TPP) Cl, Cr (TPP) Cl, Mn (TPP) Cl, Co (TPP), Cu (TPP), as metal-coordinated porphyrin derivatives fixed to detection particles Ru (TPP), Zn (TPP), Ag (TPP), Fe (TFPP) Cl, and phthalocyanine include Al (III) Phthalocyanine and Zn (II) Phthalocyanine.
In order to detect the distribution of the organic compound in the cell, first, a cell sample is prepared and a cell culture substrate is prepared. A method for preparing cell samples is described. Neural stem cells collected from the human nasal cavity are cultured by the microsphere method. Disrupt the microspheres by hopping to obtain a neural stem cell suspension.

  FIG. 3 is a top view schematically showing a cell culture chip used in this example, and FIG. 4 is a cross-sectional view taken along line AA of FIG. As shown in FIGS. 3 and 4, the cell culture chip 10 includes a substrate 11 and a partition member 12 provided on the substrate 11, and is an internal region surrounded by the partition member 12 on the substrate 11. Is a cell culture tank 13. A plurality of concave dot portions are formed on the bottom surface of the cell culture tank 13 so that cells can be fixed to the respective dot portions.

  Hereinafter, a method for producing the cell culture chip 10 will be described. A glass substrate 11 having a diameter of 40 mm and a thickness of 0.11 mm on which a collagen layer 14 is formed is mounted on a spin coater, and the temperature is maintained at 50 ° C. 1 ml of 1% agarose aqueous solution is applied to the glass substrate 11 and immediately rotated at 50 rpm for 5 seconds, and then rotated at 200 rpm for 15 seconds. The glass substrate 11 is once left at a humidity of 70% or more and a room temperature to promote gelation of agarose. In this state, the thickness of the agarose gel layer 15 is 10 μm or less. A 200 μm thick polyester tape is applied to the outer peripheral portion of the substrate 11 on which the agarose gel layer 15 is formed on the agarose gel layer 15 to form the partition wall member 12. Since the polyester tape is water-repellent, the aqueous solution can be held in the area inside the tape. That is, it functions as the partition member 12.

  Pure water is added to the inner region of the partition member 12, and the surface of the substrate 11 is irradiated with a laser of 1480 nm, and the agarose gel layer 15 of the laser irradiation portion is regularly removed in a dot shape. The removed concave portion becomes the dot portion 16. 3 and 4 show a state in which 6 × 6 dot portions 16 are formed by laser irradiation. In each dot portion 16, the agarose gel layer 15 is removed and the collagen layer 14 is exposed. The size of each dot portion 16 and the distance between the dot portions 16 are not particularly limited, but one neural stem cell is fixed in each dot portion 16, and a neural circuit is connected between the neural stem cells fixed to each dot portion 16. Is a value that can be formed. A region inside the partition member 12 serves as a cell culture tank 13.

  Using the cell culture chip 10 produced as described above, neural stem cells are cultured, and substances derived from the cell activity are detected. The method will be described below.

A predetermined culture solution is added to the cell culture tank 13 of the cell culture chip 10. Next, the cells are sucked up from the neural stem cell suspension prepared as described above with a capillary pipette, and the cells are discharged one by one to each dot portion 16 on the substrate, and the culture is continued at 37 ° C. in a 5% CO ₂ atmosphere. The cells adhere to the surface of the dot portion 16. Alternatively, a neural stem cell suspension with a predetermined concentration may be uniformly applied to the bottom surface of the cell culture tank 13 and cultured. Even in this case, since the cells do not adhere to the surface on which the agarose gel layer 15 is formed, the cells can be adhered to the dot portion 16.

  Adherent neural stem cells differentiate and eventually begin to grow axons and dendrome. At this time, the groove 17 may be formed by irradiating a 1450 nm laser between the adjacent dot portions 16 so as to facilitate the formation of the neural circuit. In FIG. 3, although the illustration of the cells and the neural circuit is omitted, a state in which the groove 17 is formed in a part is shown.

  FIG. 5 is a diagram schematically showing the state of the nerve cell array circuit in the region 18 of FIG. A pair of axons 21e-i and dendromes 22e-i extend from the respective nerve cells 20e-i in the dot portions 16e-i, and the respective axons and introns come into contact with each other to form a gap junction, thereby forming a neuron array. A circuit 23 is formed.

  Next, the particles for detection are added to the medium in the cell culture tank 13, and the culture is further continued for 6 hours. By this step, the detection particles are taken up by each cell. When the supernatant culture medium is replaced with a new one in this state, the detection particles taken into the cells and the detection particles adsorbed on the cell surface are present.

  For the sake of explanation, FIG. 6 shows a cross section in which the detection particles are arranged inside and on the surface of the two cells 20e and 20f that are fixed in the dot portion 16 and form a nerve cell array circuit. It is an image figure. In FIG. 6, three types of detection particles 24, 25, and 26 are simply shown as detection particles.

  Through the above steps, preparation for detecting a substance derived from the activity of neural stem cells is completed. Hereinafter, the detection process will be described.

Returning to FIG. 3, for example, haloperidol is added as a stimulating substance to the terminal cells fixed to the dot portions 16a of the neuron array circuit 23 formed by the cells fixed to the dot portions 16a to 16k. Then, the metal coordinated porphyrin derivative in the detection particle coordinates the haloperidol molecule, and the spectrum changes. FIG. 7 shows a change curve in which the differential absorbance (ΔA) of four kinds of detection particles 50, 51, 52, and 53 among the detection particles for cells added with haloperidol is expected. T ₁ is the point where haloperidol was added, and the horizontal axis is time. The type of metal-coordinated porphyrin derivative is identified from the fluorescence spectrum of each detection particle, and the state of cell activity is identified from the spectrum change due to the coordination of the metal-coordinated porphyrin derivative and the cell-derived organic compound. Although it is possible to identify individual cell-derived organic compounds, even if they cannot be identified, it is possible to capture the activity status of the cells by obtaining the spectrum change pattern of each detection particle.

  When the difference absorbance of each detection component is measured for each cell, the relative value of the difference absorbance expected for the detection component having a large value is shown in FIG. FIG. 8A shows the difference absorbance (vertical axis) regarding a specific detection component for each cell (horizontal axis) in the nerve cell array circuit 23 after addition of haloperidol, and FIG. It is. Note that the horizontal axis indicates the symbol of the dot portion where the cell is fixed. Before the addition of haloperidol, the difference absorbance is all within a certain range, but after the addition of haloperidol, there is a cell position where the difference absorbance reaches a peak.

FIG. 9 is a diagram showing a peak position expected when the peak position of the differential absorbance in the neuron array circuit 23 after addition of haloperidol is evaluated over time. T ₂ are indicates a time obtained by adding haloperidol in FIG. The vertical axis indicates the symbol of the dot portion to which the cell is fixed. As shown in FIG. 9, it is expected that the peak position is obtained as a pattern that reflects and vibrates at both ends of the neuron array circuit 23 composed of 11 cells. In the present neuron array 23, an electrical signal flows in one direction. If such a result is obtained, it is suggested that there is a mechanism for transmitting a signal bidirectionally in the analysis of the substance level. It will be. Moreover, it will be suggested that the influence of haloperidol is not temporarily settled but repeatedly affects cells.

  In this example, haloperidol is added to the neuron array circuit 23, and the influence of haloperidol on the cells is observed by monitoring the spectrum of the detection particles taken into the cells. Similarly, the effects of various compound additions can be detected. Traditionally, when trying to investigate the effects of such compounds on living organisms, it is necessary to add compounds to cultured cells and change the growth rate, or how much compound amount and time it takes for half of the cells to die Or they were administered to individual animals and observed. By using the present invention, the influence on the cells, and further, the cell circuit that can be said to be artificially reconstructed pseudo-tissue as shown this time, in addition to the nerve cell circuit, the cardiomyocyte circuit and the hepatocyte tissue By combining the detection particles with the detection particles, the influence of the compound can be quickly and quantitatively shown. Therefore, by using the present invention, there is a potential element that develops into an excellent method capable of performing drug discovery, particularly toxicity test, in the form of replacing valuable biological specimens.

  The cell activity detection sensor and the cell activity detection method of the present invention are useful for toxicity tests in drug discovery.

The figure which represented typically the cross section of the particle | grains for a detection which comprises the sensor for cell detection of 1st Embodiment. The figure which represented typically the cross section of the particle | grains for a detection which comprises the sensor for cell detection of 2nd Embodiment. The top view which shows typically the cell culture chip | tip used in a 1st Example. AA sectional drawing of FIG. The figure which showed typically the state of the nerve cell array circuit in the area | region 18 of FIG. Sectional drawing which shows the image of the state of the cell by which the particle for detection is arrange | positioned inside and on the surface. The figure which shows the change curve of the particle | grains for detection anticipated when haloperidol is added. (A) is the figure before haloperidol addition, (b) is a figure which shows the relative value of the difference absorbance of each cell after haloperidol addition. The figure which evaluated the peak position where the difference absorbance in a nerve cell array circuit after adding haloperidol was predicted over time.

Explanation of symbols

1, 4, 24, 25, 26 Detection particle 2 Nucleus 3 Detection layer 5 Gas permeable layer 10 Cell culture chip 11 Glass substrate 12 Partition member 13 Cell culture tank 14 Collagen layer 15 Agarose gel layer 16 Dot part 17 Groove 23 Nerve Cell array circuit 20e, 20f, 20g, 20h, 20i cells

Claims (10)

A cell activity detection sensor comprising at least two or more types of detection particles and detecting a substance derived from cell activity,
The detection particles contain a detection component exhibiting optical characteristics according to the substance to be detected,
The cell activity detection sensor, wherein the type of the detection particle is different depending on the type of the detection component.   The sensor for detecting cellular activity according to claim 1, wherein the detection particles can be identified based on their fluorescence characteristics, nuclear magnetic resonance characteristics, magnetic characteristics, optical absorption characteristics, particle diameters, or a combination thereof. .   The cell activity detection sensor according to claim 1, wherein the detection component includes a metal coordinated porphyrin derivative and / or a metal coordinated phthalocyanine derivative.   The cell activity detection sensor according to claim 1, wherein a particle diameter of the detection particles is 0.1 to 10 μm. The detection particles comprise a gas permeable layer;
The cell activity detection sensor according to any one of claims 1 to 4, wherein a gas from outside the detection particles passes through the gas permeable layer and reaches the detection component.   The cell activity detection sensor according to claim 1, wherein the detection particles have an outermost surface layer containing a compound having a guanidyl group. A cell activity detection method for detecting a substance derived from cell activity using the cell activity detection sensor according to any one of claims 1 to 6,
An arrangement step of arranging the plurality of detection particles inside and / or outside the cell;
A cell activity detection method comprising: a measurement step of measuring an optical absorption characteristic of the detection particles; and an analysis step of analyzing a cell activity based on a measurement result of the measurement step.   The cell activity detection method according to claim 7, wherein the arranging step includes a step of dispersing the plurality of detection particles in a cell.   The cell activity detection method according to claim 7 or 8, wherein the arranging step includes a step of arranging the plurality of detection particles in the vicinity of the cell in a contact state and / or a non-contact state. The cell activity detection method according to claim 7, wherein the analysis step includes a step of acquiring information on a detection substance in correspondence with position information.

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