JP2006129798A - Cell chip and method for modifying cell and method for controlling cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a cell chip for researching the functions of cells and for bio-assaying various substances with the cells. <P>SOLUTION: The bulkhead structure chip has a smaller hole than the outer diameter of a cell to change a part of the cell into a semi-permeable membrane. The cell is immobilized at the small hole-having position on one side of the chip, and a cell membrane toxin such as streptolysin O is made to act on one part of the cell from the other side through the small hole to change the cell membrane of the small hole portion into the semi-permeable membrane. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a bioassay of various substances using cells for studying cell functions, a cell chip therefor, and a cell control method and modification method for the purpose of producing substances using cells.

  Along with the progress of the genome project, the movement to understand living organisms at the DNA level, to diagnose diseases and to understand life phenomena has become active. In order to understand life phenomena and investigate the function of genes, it is frequently conducted to examine the expression status of genes. As an effective method, a DNA probe array or a DNA chip in which a large number of DNA probes are classified and fixed on a solid surface (called an oligo chip because it is actually an oligonucleotide derivative) Is also used (Non-patent Document 1 or Non-Patent Document 2). Alternatively, a method is used in which the amount of a product obtained by PCR amplification of mRNA (or cDNA) having a specific sequence contained in a collected sample is examined, the PCR product length, and the base sequence.

  These isolated mRNAs are used not only for analysis purposes but also for various purposes by converting them into cDNA and incorporating them into vectors. As an example, the collected mRNA is incorporated into a vector, cloned, and then complementary strand synthesis is performed with primers having partially different bases using the vector as a template, and used for gene recombination where any genetic modification is performed. .

  Research on bioassays that detect extremely small amounts of substances by using cells themselves as sensors is also active. In particular, investigating the effects of various harmful substances and investigating the efficacy and side effects of drugs are largely dependent on mammals such as mice, but in the future, cell-based assays will also be used from the viewpoint of animal welfare. Transition is a trend of the times.

  From another point of view, there is a great need for controlling the function of cells freely, producing useful substances, and applying them to medical treatment. In fact, attempts have been actively made to control cells by manipulation from outside the cells, such as antisense oligonucleotides and RNAi.

Science 251, 767-773 (1991) Proc. Natl. Acad. Sci. USA 93, 4613-4918 (1996)

  There is no methodologically versatile technique for such cell-based assays and cell function control. For the assay, cells are expressed with a receptor or transporter that captures a specific substance using genetic recombination technology, and this is coupled with an ion pump, an electrode is inserted, and the amount of current flowing through the cell membrane is measured. . The principle of cell function control is that a control substance is inserted into the cell to control cell activity.

  However, the cells are separated from the outside by a stable cell membrane, and it is difficult to quantitatively add or remove any substance into the cells for the convenience of the practitioner. Of course, many technologies have been developed, such as making gold particles into a vehicle, sticking what you want to insert into the cell, and driving it into the cell, but certainty and reproducibility of the amount of substance taken into the cell It can be considered that there is considerable variation.

  It seems that the effective method is to use a technique of making the cell membrane semi-permeable (swmi-contact cell: Molecular Biology of the Cell 11,3073-3087 (2000)), or phagotosis or endocytosis. However, when the cell membrane is made semipermeable, there is a defect that a hole is formed in the entire cell surface, and the intracellular substance (cytoplasm) flows out of the cell. In addition, methods using phagotosis and endocytosis are not responsive and are difficult to use in terms of sequential control of cell functions.

  The point of the present invention is to establish a technique for suppressing the loss of the contents of cells and reliably inserting the target substance into the cell or recovering the intracellular substance, and assaying specific substances or producing useful substances It is to provide a technology that makes it easier.

  The technique closest to the method of improving the conventional technique and performing stable cell control and assay stably is a technique for making the cell membrane semi-permeable. In the present invention, the entire cell is not made semi-permeable, but is limited to a small part of the cell to prevent the cytoplasm from flowing out of the cell, so that the cell can be used stably.

  In order to make a part of cells semipermeable, a chip having a partition wall structure with a hole smaller than the outer diameter of the cell is used. A cell is fixed at a position of the pore on one surface of the chip, and a cell membrane toxin such as streptricin O is allowed to act on a part of the cell through the pore from the other surface, so that the cell membrane of the pore portion Is made semi-permeable. Using this semipermeable membrane portion, the substance is taken in and out of the cell. In addition, by providing electrodes inside and outside the partition wall, ions passing through the cell membrane are measured, or a voltage is applied to the electrodes to force the substance into and out of the cells.

  The present invention only makes a part of the cell membrane semi-permeable. Further, since the semipermeable membrane is normally separated from the cell membrane by the partition wall, the cytoplasm does not diffuse outside the partition wall. It is also possible to observe cells continuously or to inject substances into cells sequentially. Moreover, intracellular substances can be taken in and out only by diffusion in the semipermeable membrane. Since various substances can be added from the outside, it is possible to control protein interactions and gene expression, and actively analyze physiological phenomena occurring in cells. By providing electrodes inside and outside the partition wall, ions passing through the cell membrane can be measured, and substances can be forcibly put in and taken out. Compared to the loading and unloading of substances into cells by patch clamp, this is a much more stable operation.

Example 1
FIG. 1 is a cross-sectional view showing an outline of the relationship between a cell chip according to Example 1 and cells fixed to the pores.

  In FIG. 1, 100 is a cell chip. 20 is a cell. As will be described later, the cells 20 are fixed to the pores 3 of the cell chip 100.

  Reference numeral 1 denotes a cell fixing substrate of the cell chip 100, which is made of, for example, a silicon substrate. The size is, for example, 5 mm in the height direction in the figure and 500 μm in the direction perpendicular to the figure. The thickness is, for example, 100 μm. The protrusion 2 is formed at one end of the cell-fixing substrate 1, and the height is, for example, 5 μm, and the thickness of the protrusion 2 is, for example, 2 μm. A pore 3 is formed at the top of the protrusion 2. This size is, for example, 2 to 5 μmφ. Electrode layers 4 and 5 are formed on both lower surfaces of the cell-fixing substrate 1. The electrode layers 4 and 5 are mostly covered with the insulating layers 6 and 7, but are exposed in the vicinity of the protrusion 2 and in the vicinity of the end portion of the cell fixing substrate 1.

  Reference numeral 10 denotes a back plate, which is attached so as to cover the entire back surface of the protrusion 2 in order to form the buffer chamber 8 on the back surface of the protrusion 2 of the cell fixing substrate 1. The thickness of the back plate 10 is, for example, 100 μm as a whole, but as shown in the figure, protrusions 11 are formed at positions corresponding to the pores 3 on the surface on the cell-fixing substrate 1 side. Protrusions 12 and 13 having through holes at positions are formed at both ends of the protrusion 11. Capillaries 14 and 15 can be attached to the protrusions 12 and 13, and can be connected to a micro syringe pump (not shown). Using the capillaries 14 and 15, as indicated by thick arrows, the buffer is circulated in the buffer chamber 8 or the suction inside the buffer chamber 8 is sucked in excess of the supply of the buffer so that the inside of the buffer chamber 8 is brought into a negative pressure state. can do. The protrusion 11 is provided to disturb the flow of the supplied buffer so as to flow toward the pore 3.

  Since the figure becomes complicated, the illustration of the microscope is omitted, but a droplet of a buffer suitable for cell culture is dropped on the observation glass plate of the microscope, and the protrusion 2 of the cell fixing substrate 1 and Cells 20 are placed facing each other. At this time, the cells 20 are placed in a shape facing the pores 3 of the protrusions 2 while observing with a microscope. 21 is the lipid bilayer of the cell 20 and 22 represents the cytoplasm. In FIG. 1, a broken line surrounding the protrusion 2 and the cell 20 of the cell fixing substrate 1 shows an image of a region immersed in the droplet. The buffer chamber 8 is included in the area surrounded by the broken line. However, as will be described later, the buffer chamber 8 is not connected to the droplet when the cells 20 are fixed to the cell fixing substrate 1. It will be a thing. On the other hand, conductive wires are connected to the electrode layers 4 and 5 exposed at the end of the cell-fixing substrate 1 outside the range of the droplets, and are drawn out and connected to a measuring instrument (not shown) or a computer.

  While observing with a microscope, the cells 20 are brought into contact with the pores 3 of the protrusions 2. At this time, if the electrical conductivity between the electrode layers 4 and 5 is monitored, before the cell 20 contacts the pore 3 of the protrusion 2, the electrode layer 5 exposed in the buffer chamber 8 and The electrode layer 4 exposed in the droplet is short-circuited by the buffer, but after contact, the pore 3 is blocked by the lipid bilayer 21 of the cell 20, so that it is substantially insulated. Since it becomes a state, cell contact can be confirmed. When cell contact is confirmed, the supply of the buffer by the capillaries 14 and 15 connected to the protrusions 12 and 13 is controlled, and the inside of the buffer chamber 8 is set to a negative pressure by performing suction that exceeds the supply of the buffer. The cell 20 is firmly fixed to the pore 3.

Next, streptolysin O is injected into the buffer chamber 8 using a microsyringe pump connected to the capillaries 14 and 15 attached to the protrusions 12 and 13 of the back plate 10. Streptolysin O acts on the part of the lipid bilayer 21 of the cell 20 that is in contact with the pore 3 through the pore 3, and only this part becomes a semipermeable membrane. The semipermeable membrane is a state in which the cytoskeleton remains but a hole is opened in the lipid bilayer 21. The reaction conditions for streptricin O are, for example, Fumi Kano, Y. Sako et al, Reconstraction of Brefeldin A-induced Golgi Tubulation and Fusion with the Endoplasmic Reticulum in Semi-Intact Chinese Hamster, Molecular Biology of the Cell 11,3073-3087. (2000) is modified based on the cell type based on the technology disclosed in (2000). For example, in the case of ovarian cells, it is 10 minutes at 4 ° C. in a 25 mM HEPES buffer solution having a pH of 7.4 containing streptolysin O (60 ng / ml), 115 mM potassium acetate, 2.5 mM MgCl ₂ , 1 mM dithiothreitol, 2 mM EGTA. Then, it is washed with 25 mM HEPES buffer (32 ° C.) having a pH of 7.4 containing 115 mM potassium acetate, 2.5 mM MgCl ₂ , 1 mM dithiothreitol, and 2 mM EGTA. During this time, since the cells 20 are in the buffer of the droplets, the damage to the cells 20 is small.

  Hereinafter, a method of using the thus configured cell chip 100 to which cells having a partial semipermeable membrane are fixed will be described.

  A short RNA, which is a part of specific mRNA, is caused to flow into the buffer chamber 8 from the capillary 14. A part of this RNA is taken into the cell 20 from the pore 3 through the semipermeable membrane. In general, when RNA is introduced into the cell 20, it is rapidly lost upon attack by RNase. However, in the cell chip 100 of Example 1, since fresh RNA is continuously supplied from the capillary 14 into the buffer chamber 8, the RNA in the cell 20 is indicated by a thin arrow in the pore 3, An equilibrium state is formed between the cell 20 and the buffer chamber 8 through the semipermeable membrane. For this reason, a fixed amount of RNA can always be held in the cell. This applies not only to RNA but also to DNA or RNA or derivatives thereof.

  In this way, the current value between the electrodes 4 and 5 is monitored before and after the RNA is put into the cell 20. If the RNA introduced into the cell affects the transporter of the cell double membrane 21, ion channels existing in the cell double membrane 21 are coupled, and fluctuations in the membrane transport of ions appear. A current flows between 5. That is, a substance can be introduced into a cell and its influence on the cell can be monitored. This can be used for measuring the influence of chemical substances and proteins in addition to RNA.

  Alternatively, various chemical substances are added to the droplet on the side where the cell double membrane 21 on the droplet side that is not exposed to streptocricin O is present, and this is introduced into the cell 20 via the transporter, Can affect the cell by the potential difference between the electrodes 4 and 5. That is, a bioassay is possible.

  FIGS. 2A to 2G are diagrams for explaining the outline of the process of forming the cell fixing substrate 1 of Example 1 using semiconductor technology. 2A to 2G are cross-sectional views on the left side and corresponding plan views on the right side.

  First, as shown in FIG. 2A, a silicon substrate 1 having a predetermined crystal axis is prepared, a mask 31 is provided on one surface thereof, and a window 32 from which the mask 31 has been removed is formed at a position where a projection 3 is to be formed. To do. As shown in FIG. 2B, the portion corresponding to the quadrangular pyramid 33 is removed by performing an etching process. Next, as shown in FIG. 2C, the mask 31 is removed, the mask 34 is applied to the other surface of the silicon substrate 1, and a window from which the mask 34 has been removed is formed at the position where the projection 3 is formed. Concave portions 35 and 36 having a triangular cross section are formed. The concave portions 35 and 36 are continuous concave portions corresponding to the quadrangular pyramids 33 as can be seen from the plan view. Next, as shown in FIG. 2D, a mask 37 is provided at a position surrounded by the recesses 35 and 36, and the window 38 is opened. Next, as shown in FIG. 2 (e), the pores 3 are opened in the corresponding portions of the substrate 1 using this window 38. At this time, the substrate 1 in the periphery of the recesses 35 and 36 is also etched to form the protrusions 2. Next, as shown in FIG. 2 (f), an electrode 4 is formed on the surface of the substrate 1 where the protrusions 2 are provided. The electrode 4 is an aluminum vapor deposition layer. Next, as shown in FIG. 2G, almost the entire surface excluding both ends of the electrode 4 is covered with a polyimide insulating layer 6. Next, the electrode 5 is formed on the other surface of the substrate 1. The electrode 5 is a platinum deposition layer, and the entire surface except the both ends of the electrode 5 is covered with a polyimide insulating layer 7. Thus, the cell fixing substrate 1 of the cell chip 100 is formed. Here, detailed data on the semiconductor technology is omitted, but those skilled in the art can easily implement it.

  Similarly, the back plate 10 can also be formed by processing a silicon substrate by semiconductor technology. And the cell chip | tip 100 is comprised by bonding the cell fixed board | substrate 1 and the backplate 10 together.

(Example 2)
In Example 1, a single cell was fixed to the pore part of the cell chip, and only the cell membrane of the pore part was made semi-permeable to explain a cell chip capable of bioassay. However, in multicellular organisms, cells rarely function alone, and function as a whole while emphasizing surrounding cells. Such multicellular systems are thought to exchange information using various chemical substances. In many cases, such chemical substances are in minute amounts, and it is difficult to perform sequential analysis from living cells. In Example 2, a cell chip that enables a bioassay for a group of cells functioning as a whole in cooperation with surrounding cells is proposed. Example 2 is the same as Example 1 in that cells are fixed to the pore part of Example 1 and only the cell membrane of the pore part is made semipermeable to enable bioassay. is there.

  FIG. 3 is a cross-sectional view showing an outline of the relationship between the cell chip according to Example 2 and the cells fixed to the pores.

  In FIG. 3, reference numeral 200 denotes a cell chip according to the second embodiment, which includes a cell fixing substrate 41, a back plate 51, and side wall plates 61 and 62. Note that the side wall plates are also provided in the rear direction and the front direction of the paper not shown. The space enclosed by these forms a buffer chamber 71 as in the first embodiment.

  A pore 43 is formed in the cell fixing substrate 41. These correspond to the cell fixing substrate 1 and the pores 3 of Example 1. Unlike the cell fixation substrate 1 of Example 1, the cell fixation substrate 41 of Example 2 has no protrusions 2 and a large number of pores 3 formed. This is because a large number of cells are arranged on the surface of the cell fixing substrate 41 to form a cell group that functions as a whole while cooperating with each other. In the example of the figure, an example in which four pores 43 are configured is shown, but it is easy to increase this number. For example, the entire size of the cell fixing substrate 41 is about 10 × 10 mm. The thickness is, for example, 100 μm. The pores 43 have a diameter of 2 to 5 μm and are arranged at a pitch of 8 μm. Cells 20 are arranged at the positions of the pores 3 of the cell fixing substrate 41, respectively. That is, the cells 20 are arranged at an 8 μm pitch.

  The back plate 51 has the same size as the cell fixing substrate 41 and is arranged facing the cell fixing substrate 41, for example, 1 mm apart. The cell-fixing substrate 41 and the back plate 51 are held in a predetermined positional relationship by the side wall plates 61 and 62 and the side wall plate in the rear direction and the front direction of the paper not shown, and the space surrounded by these is fixed. The buffer chamber 71 is used. Similar to the first embodiment, protrusions 63 and 64 having through holes are formed on the side wall plates 61 and 62. Capillaries (not shown) can be attached to the protrusions 63 and 64, which can be connected to a microsyringe pump (not shown), and a buffer or the like can be supplied. On the back plate 51, projections 52 are formed at positions corresponding to the pores 43. Similar to the protrusion 11 of the first embodiment, the protrusion 52 disturbs the flow of the buffer supplied to the buffer chamber 71 and flows toward the pore 43. An electrode 53 is provided on the surface of the back plate 51 in the buffer chamber 71. The lead line 54 of the electrode 53 is an insulated wire and is led out of the buffer chamber 71.

  FIGS. 4A to 4D are views for explaining the outline of the process for forming the cell fixing substrate 41 of the second embodiment by using semiconductor technology. 4A to 4C are cross-sectional views on the left side and corresponding plan views on the right side. The plan view of FIG. 4D is the same as that of FIG.

  First, as shown in FIG. 4A, a silicon substrate 1 having a predetermined crystal axis is prepared, a mask 31 is provided on one surface thereof, and a window in which the mask 31 is removed in a concave portion where a pore 3 is to be formed. 32 is formed. As shown in FIG. 4B, the portion corresponding to the quadrangular pyramid 33 is removed by etching. Next, as shown in FIG. 4C, the mask 31 is removed, the mask 34 is applied to the other surface of the silicon substrate 1, and a window 35 from which the mask 34 has been removed is formed at a position where the pores 43 are created. . As shown in FIG. 4D, an etching process is performed to form the pores 43. Thus, the cell fixing substrate 41 of the cell chip 200 is formed. Here, detailed data on the semiconductor technology is omitted, but those skilled in the art can easily implement it.

  Similarly, the back plate 51 and the side wall plates 61 and 62 can also be formed by processing a silicon substrate by semiconductor technology. Then, the cell chip 200 is configured by bonding the cell fixing substrate 41, the back plate 51, and the side wall plates 61 and 62 together.

  As is clear by comparing the plan view of FIG. 2 (g) with the plan view of FIG. 4 (c), the cell chip 100 of Example 1 performs a bioassay on a single cell. Thus, the cell chip 200 of Example 2 can perform bioassay by arranging 4 × 4 cells. When the cell chip 200 is placed in a culture dish and immersed in an appropriate culture solution, for example, when the epithelial cells 20 are cultured on the cell fixing substrate 41 of the cell chip 200, a single layer is necessarily formed on the cell fixing substrate 41. Cell sheet is constructed. FIG. 3 shows, as an image, that the cell sheet cultured with the cell chip 200 is in an area of the culture medium with a broken line, and a single-layer cell sheet is formed on the cell fixing substrate 41 of the cell chip 200. Is shown in a sectional view. In FIG. 3, the pores 43 are provided so as to correspond to the pitch of the cell arrangement. However, if the pores 43 are provided at a sufficient density compared to the cell spacing (eg, 8 μm), for example, at a pitch of 5 μm, Regardless of the cell size, the pores are arranged in almost all cells of the single-layer cell sheet formed on the cell fixing substrate 41. Of course, the cells may be previously cultured on the cell fixing substrate 41 on which the pores 43 corresponding to the size of the cells are arranged, using an agarose microchamber array or the like at regular intervals. Furthermore, arbitrary cells may be arranged to form a cell sheet. In addition, regarding the cell 20 of FIG. 3, 21 is a cell double membrane, 22 is a cytoplasm, 23 is a transporter which exists in a cell double membrane.

  First, also in the second embodiment, the buffer is supplied to the buffer chamber 71 through the protrusions 63 and 64 of the side wall plates 61 and 62 as in the first embodiment while observing with a microscope. When the electrical conductivity between the electrode 55 immersed in the culture solution of the culture dish and the electrode 53 in the buffer chamber 71 is monitored, the cell sheet of the cells 20 is not firmly fixed on the cell fixing substrate 41. Sometimes, the electrode 55 immersed in the culture medium in the culture dish and the electrode 54 exposed in the buffer chamber 8 have a relatively low electrical conductivity due to the buffer. In this state, as shown by the thick arrows, through the through holes of the protrusions 61 and 62, the supplied buffer is controlled, and the suction inside the buffer chamber 71 is reduced to a negative pressure. Thus, the cell sheet is fixed on the cell fixing substrate 41.

  Here, streptolysin O is injected into the buffer chamber 71 as in the first embodiment. Streptolysin O acts on the portion of the lipid bilayer 21 of the cell 20 in contact with the pore 43 through the pore 43, and only this portion becomes a semipermeable membrane. Thereby, the cell chip which has the cell by which only the part corresponding to the pore 43 was made into the semipermeable membrane is obtained.

  Hereinafter, a method of using the thus configured cell chip 200 to which cells having a partial semipermeable membrane are fixed will be described.

  A short RNA, which is a part of a specific mRNA, is caused to flow into the buffer chamber 71 through the through holes of the protrusions 61 and 62 as indicated by thick arrows. A part of this RNA is taken into the cell 20 from the pore 43 through the semipermeable membrane. Normally, when RNA is introduced into the cell 20, it rapidly disappears due to RNase attack. However, in the cell chip 200 of Example 2, since fresh RNA is continuously supplied into the buffer chamber 71 from the through holes of the protrusions 61 and 62, the RNA in the cell 20 has a thin arrow in the pore 43. As shown in FIG. 3, the cell 20 and the buffer chamber 71 form an equilibrium state through the semipermeable membrane. For this reason, a certain amount of RNA can always be held in the cell.

  In this way, the current value between the electrodes 54 and 55 is monitored before and after the RNA is put into the cell 20. If the RNA introduced into the cells constituting the cell sheet has an effect on the transporter 23 of the cell double membrane 21, ion channels existing in the cell double membrane 21 are coupled and change in membrane transport of ions. Appears and a current flows between the electrodes 54 and 55. That is, a substance can be introduced into a cell and its influence on the cell can be monitored. This can be used for measuring the influence of chemical substances and proteins in addition to RNA.

  Alternatively, various chemical substances are added to the culture solution on the side where the cell double membrane 21 on the culture dish side, which is not exposed to streptolysin O, and introduced into the cell 20 via the transporter, Can affect the cell by the potential difference between the electrodes 54 and 55. That is, a bioassay is possible.

  Alternatively, when a chemical substance indicated by a white triangle is added to the culture dish, the chemical substance is taken into the cell via the transporter 23 as indicated by a thin solid triangle. The arrow passing through the transporter 23 is for the image of the passage of chemical substances. The taken-in chemical substance passes through the semipermeable membrane and elutes from the pores 43 into the buffer chamber 71 as black solid triangles. By collecting this through the through hole of the protrusion 62, the reaction of the cell to the chemical substance can be evaluated.

  In this specification, biochemical substances include aminopeptides such as amino acids, dipeptides and tripeptides, polypeptides such as proteins, nucleic acids, RNAs such as mRNA, monosaccharides, disaccharides, oligosaccharides, and polysaccharides. It refers to hormones such as saccharides and steroids, neurotransmitters such as noradrenaline, dopamine and serotonin, other endocrine disruptors, various drugs, and substances related to life phenomena such as potassium, sodium, chloride ions and hydrogen ions. Thus, biochemical substances have a wide variety of properties. Therefore, it is extremely useful to be able to evaluate the effect on these cells by directly acting on the cells.

  For example, if the above-mentioned chip is prepared from cells in which SLC6A1, which is a transporter, is forcibly expressed, it is useful for detecting γ-aminobutyric acid. Those forcibly expressing SLC6A2 can be used for measurement of noradrenaline, and SLC6A4 for serotonin. SLCO3A1 can be used to measure prostaglandins and SLC6A5 can be used to measure glycine.

  Furthermore, when the chip of the present invention is used, the substance can be purified. For example, cells in which a dopamine transporter is forcibly expressed by the above-described method are arranged in an array on the chip as in chip 200 shown in FIG. Each cell has a semipermeable membrane only at the part in contact with the pore. A sample solution containing dopamine or a derivative thereof is added to the chip, and a voltage is applied to the electrodes 54 and 55. Then, the desired dopamine or an analog thereof is recovered through the cell membrane. A completely different structure does not pass through the membrane. Of course, other substances are also recovered through the transporters for the other substances, but the target substance is substantially recovered because of the large difference in the number of related transporters. Similar purification can be performed with amino acids and sugars. In particular, if the transporter is forced to speak what can identify stereoisomerism, for example, only the L form can be purified from synthetic amino acids (DL mixture) with less energy.

  As described above, the present invention can be used not only for assaying chemical substances but also for producing substances such as purification of chemical substances.

It is sectional drawing which shows the outline | summary of the relationship between the cell chip by Example 1, and the cell fixed to the pore part. (A)-(g) is a figure explaining the outline | summary of the process which forms the cell fixing substrate 1 of Example 1 using a semiconductor technology. It is sectional drawing which shows the outline | summary of the relationship between the cell chip by Example 2, and the cell fixed to the pore part. (A)-(d) is a figure explaining the outline | summary of the process which forms the cell fixing substrate 41 of Example 2 using semiconductor technology.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1,41 ... Cell fixed substrate (silicon substrate), 2 ... Protrusion, 3, 43 ... Fine pore, 4, 5 ... Electrode layer, 6, 7 ... Insulating layer, 8, 71 ... Buffer chamber, 10, 51 ... Back plate 11, 12, 13, 63, 64 ... protrusions, 14,15 ... capillaries, 20 ... cells, 21 ... cell lipid bilayers, 31,34,37 ... masks, 32,38 ... windows, 33 ... square pyramids, 35, 36 ... recess, 53 ... electrode, 54 ... lead wire, 61, 62 ... side wall plate, 100, 200 ... cell chip.

Claims (9)

  A cell fixing substrate having one surface as a surface for fixing cells, pores having a smaller diameter than cells provided in the cell fixing portion of the substrate, and fixing cells at the positions of the pores of the cell fixing substrate A cell chip comprising a buffer chamber formed on the back surface of the buffer surface, wherein a liquid such as a buffer can be continuously supplied to the buffer chamber.   When a cell is fixed by adding a droplet to the position of the pore on the cell fixing surface of the cell fixing substrate, the electric conductivity between the droplet and the buffer chamber or the flowing current is The cell chip according to claim 1, further comprising an electrode for measuring.   The cell chip according to claim 2, wherein the electrodes are formed on both surfaces of the cell fixing substrate.   The cell chip according to claim 1 or 2, wherein a plurality of the pores are provided, and an interval between the pores is smaller than an interval between cells fixed to the cell fixing substrate.   A cell fixing substrate having one surface as a surface for fixing cells, pores having a smaller diameter than cells provided in the cell fixing portion of the substrate, and fixing cells at the positions of the pores of the cell fixing substrate A buffer chamber configured on the back surface of the cell surface, and the cells fixed to the cell fixing substrate of the cell chip capable of continuously supplying a liquid such as a buffer are placed in the buffer solution in the buffer chamber. A cell modification method, wherein streptricin O is supplied to the buffer chamber to make the lipid bilayer of the cell at the pore position semi-permeable.   The cell modification method according to claim 5, wherein after the supply of streptricin O, arbitrary DNA or RNA or a derivative thereof is added to the buffer chamber.   A cell fixing substrate having one surface as a surface for fixing cells, pores having a smaller diameter than cells provided in the cell fixing portion of the substrate, and fixing cells at the positions of the pores of the cell fixing substrate A buffer chamber configured on the back surface of the cell surface, and the cells fixed to the cell fixing substrate of the cell chip capable of continuously supplying a liquid such as a buffer are placed in the buffer solution in the buffer chamber. , Supplying streptricin O to the buffer chamber to modify the cell by making the lipid bilayer membrane of the cell at the pore position semi-permeable, and adding an arbitrary chemical substance to the buffer around the modified cell A method for extracting a chemical substance, comprising adding the chemical substance passing through the lipid bilayer membrane of the cell to the buffer chamber from the semipermeable membrane.   A cell fixing substrate having one surface as a surface for fixing cells, pores having a smaller diameter than cells provided in the cell fixing portion of the substrate, and fixing cells at the positions of the pores of the cell fixing substrate A buffer chamber configured on the back side of the surface for the electrode, electrodes are provided in the buffer chamber and on the surface side for fixing the cells, and a liquid such as a buffer can be continuously supplied to the buffer chamber. Place the cells fixed on the cell fixing substrate of the prepared cell chip in a buffer solution, supply streptricin O to the buffer chamber, and make the lipid bilayer of the cell at the pore position semi-permeable A cell chip in which an arbitrary peptide is expressed is prepared by modifying a cell and adding an mRNA encoding an arbitrary membrane protein or a vector encoding the mRNA sequence into the cell from a buffer chamber. Cells chip any chemical substance is added to the periphery of the buffer cell, and detecting a chemical substance that affinity to the membrane proteins in the electrode. A cell fixing substrate having one surface as a surface for fixing cells, pores having a smaller diameter than cells provided in the cell fixing portion of the substrate, and fixing cells at the positions of the pores of the cell fixing substrate Provided with a buffer chamber formed on the back surface of the surface, and electrodes are provided in the buffer chamber and on the surface side for fixing the cells, and a liquid such as a buffer can be continuously supplied to the buffer chamber. Place the cells fixed on the cell fixing substrate of the cell chip in a buffer solution, supply streptricin O to the buffer chamber, and make the lipid bilayer of the cell at the position of the pores semi-permeable A cell chip having a structure in which cells are modified, a voltage is applied between the electrodes, and a chemical substance passing through the fixed cells is continuously collected from the buffer chamber.

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