JP2006187261A - Method and device for introducing aimed substance into cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new technique for introducing an aimed substance into a cell. <P>SOLUTION: The new technique(method) comprises the following procedure: A to-be-inserted object, having a charged part and containing the aimed substance, is adsorbed to a needle structure body with an electrically conductive part, the needle structure body is then introduced into a cell followed by applying voltage to the electrically conductive part to release the aimed substance into the cell. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a technique for introducing a substance to be introduced into a cell.

There are several techniques for introducing substances into cells. Among them, the introduction techniques at the level of individual cells are currently the microinjection method and several thousand V / cm for cell suspensions. There is known an electroporation method in which a sample to be injected, such as DNA, is introduced into a cell through a small hole that is generated in a cell membrane for a short time when a high voltage is applied with a pulse of several tens of microseconds (for example, Non-Patent Document 1). reference.).
“Methods in Enzymology”, Vol. 65, 1980, p. 816-825

  In the electroporation method, since it is difficult to control the diameter and life of the pores, it is difficult to quantitatively inject the sample into the cells.

  On the other hand, in the current microinjection method, since the target substance to be introduced is introduced into cells by air pressure using a hollow glass capillary, the following drawbacks remain.

  (1) The pressure in the cell rises at the time of introduction, and the cell is easily damaged.

  (2) Since the size of the glass capillary is not sufficiently small with respect to the cell, damage to the cell is great when the capillary tip is inserted into the cell.

  (3) Cell membrane fragments and the like are clogged in the capillary, and injection failure tends to occur. If the size of the glass capillary is made smaller than the present state, the possibility of an increase in the rate of injection failure will increase.

  (4) The introduction target substance cannot be repeatedly injected into the same cell in a short time. In new research areas in particular, there is a great need for technology that repeatedly injects target substances for introduction in order to improve research speed.

  (5) A very small amount of control cannot be performed. For example, it is important to check whether Gleevec has been introduced into cells for the study of cases in which Gleevec does not work in chronic myelogenous leukemia. However, there is a need for a technique that can be inserted into cells by controlling the amount introduced, but this is difficult with the current technology.

  (6) It is difficult to introduce a large number of introduction target substances mainly due to the above (1). The above (2), (4), and (5) are also a cause of this difficulty. If it is about several tens of kinds, it can be introduced if a mixed solution of the introduction target substance is prepared in advance, but a higher level, for example, thousands, tens of thousands, or more kinds of introduction target substances, It is extremely difficult to introduce at the same time or divided into many times.

  (7) Cannot be introduced quantitatively. At best, the concentration of the solution to be injected can be adjusted, and the amount introduced cannot be controlled.

  An object of the present invention is to provide a novel technique for introducing a substance to be introduced into a cell that solves the above problems. Still other objects and advantages of the present invention will become apparent from the following description.

  According to one aspect of the present invention, an insertion object having a charged portion and containing a target substance to be introduced is adsorbed to a needle-like structure having a conductive portion, and the needle-like structure is inserted into a cell. By applying a voltage to the cell, a method for introducing the substance to be introduced into the cell is provided, in which the substance to be introduced is released into the cell.

  According to the embodiment of the present invention, the damage to cells is small and the operation is easy, introduction of a large number of introduction target substances, introduction of a large number of introduction target substances into the same cell, introduction of a small amount of substances, and accurate control of the introduction amount. It is possible to introduce a substance into a cell that can be any or all of them.

  The object to be inserted is formed by attracting a substance to be introduced to a substance having a charged part, and the substance to be introduced and the substance having a charged part have a hydrophobic part and are charged via two hydrophobic parts. The target substance is attracted to the substance having a portion to become an insertion object, the insertion object contains a nucleotide body, the amount of the insertion object to be adsorbed on the needle-like structure or the amount released in the cell, or both The concentration, pH, ion concentration, salt concentration, temperature, type of solvent, concentration of the substance that changes the state of the substance to be introduced, and needle-like structure in the liquid containing the insertion object Adsorption area on the body; concentration, pH, ion concentration, salt concentration, temperature, type of solvent, concentration of the substance that changes the state of the substance to be introduced, and needle-like structure in the liquid containing cells Adsorption by the insertion object discharge area; and at least one parameter selected from the group consisting of the voltage applied to the conductive part, the voltage application time, the retention time in the cell of the conductive part, the surface potential of the conductive part, the material and the structure Controlling the amount or release amount or adsorption amount and release amount, controlling the discharge area of the insertion object on the needle-like structure by inserting the needle-like structure into the cell for a desired length, needle Inserting the needle-like structure into the cell for the desired length using the position where the tip of the needle-like structure is in contact with the cell, visual observation, change in light reflection, or force applied to the needle-like structure Detecting the insertion start point, the needle-like structure after releasing the target substance into the cell, washing with acid, washing with alkali, washing with organic solvent, ultrasonic washing, voltage applied to the conductive part It is preferred that comprises the cleaning and performing at least one cleaning selected from the group consisting of at.

  According to another aspect of the present invention, a needle-like structure having a charged part and a conductive part capable of adsorbing an insertion target containing an introduction target substance, and for inserting the needle-like structure into a cell A needle-like structure drive unit, a container containing a cell-containing liquid, and a voltage application unit for applying a voltage to the conductive unit and releasing the target substance into the cell. A target substance introduction device is provided.

  According to the embodiment of the present invention, damage to cells is small and any or all of introduction of a large number of target substances to be introduced, introduction of a large number of target substances to be introduced into the same cell, introduction of a small amount of substance, and accurate control of the amount of introduction. It is possible to introduce a substance into a cell with a simple operation.

  The object to be inserted is formed by attracting the substance to be introduced to the substance having a charged part, the substance to be introduced and the substance having a charged part have a hydrophobic portion, and the object to be inserted has two hydrophobic properties. It is made by attracting the target substance to the substance having a charged part through the part, the insertion target contains a nucleotide body, and the insertion target adsorbed on the needle-like structure is released into the cell. Having an insertion target amount control unit for controlling the amount, the insertion target amount control unit in the liquid containing cells, the insertion target discharge area on the needle-like structure, the voltage applied to the conductive portion, the voltage application time, The discharge amount is controlled by at least one parameter selected from the group consisting of the retention time of the conductive part in the cell and the surface potential of the conductive part, and the needle-like structure driving part moves the needle-like structure into the cell. Length to insert The needle-like structure driving unit has a function of adjusting the length of insertion into the cell from the position where the tip of the needle-like structure is in contact with the cell as an insertion start point. , The needle-like structure drive unit detects the insertion start point by the change of light reflection or the force applied to the needle-like structure, the conductive part is silicon, silicide, gold, platinum, tungsten, molybdenum and their alloys Further, it is preferable to be made of a material selected from the group consisting of those plated.

  The present invention provides a novel technique for introducing a substance to be introduced into a cell.

  Embodiments of the present invention will be described below with reference to the drawings, examples and the like. In addition, these figures, Examples, etc. and description illustrate the present invention, and do not limit the scope of the present invention. It goes without saying that other embodiments may belong to the category of the present invention as long as they match the gist of the present invention. In the drawings, the same reference numeral represents the same element.

  In the method for introducing a substance to be introduced into a cell according to the present invention, an object containing a substance to be introduced into a cell and having a charged part is adsorbed to a needle-like structure having a conductive part, and the needle-like structure is introduced. By inserting the structure into the cell and applying a voltage to the conductive portion, the substance to be introduced into the cell is electrically released into the cell. In the present invention, “substance to be introduced into a cell” is referred to as an introduction target substance, and “an object including a substance to be introduced into a cell and having a charged portion” is referred to as an insertion object.

  When the introduction target substance has a charged portion, the introduction target substance itself may be used as an insertion target. When the introduction target substance does not have a charged portion, the insertion target substance can be produced by attracting the introduction target substance to the substance having the charged portion.

  The method for attracting the target substance to be introduced to the substance having a charged portion can be selected from any method such as adsorption, adhesion, chemical bond, physical bond, and chemical affinity utilization. Whether or not the introduction target substance is attracted to the substance having the charged portion can be known by analyzing the amount of the introduction target substance present in the insertion target adsorbed on the needle-like structure. In addition, when the target substance to be introduced has a fluorescence emission function, it can be visually confirmed whether or not it is attracted to the substance having a charged portion by its light emission / quenching behavior. Furthermore, if the target substance to be introduced can be introduced into the cell by inserting the insertion object into the cell, it can be considered that the “attraction” according to the present invention has been established.

  Since it is necessary to have an attractive force that does not hinder the release of the substance to be introduced into the cell, a method using chemical affinity is preferable as a method for attracting the substance to be introduced to the substance having a charged portion. Specifically, when the target substance to be introduced has a hydrophobic part, if the substance to be introduced coexists with another substance having a hydrophobic part and a charged part, the charged part is caused by the mutual affinity of the two hydrophobic parts. The target substance to be introduced can be attracted to the substance having, thereby forming an insertion object. The configuration of these two hydrophobic portions can be selected independently and arbitrarily. Examples of the hydrophobic part include an alkyl group, a nucleotide body, and a nucleoside hydrophobic part. Whether or not it has hydrophobicity can be confirmed by whether or not the attraction occurs.

  Next, the insertion object prepared in this way is adsorbed to a needle-like structure having a conductive portion. This adsorption method can be selected from any known methods for attaching substances to needles. Whether it is adsorbed or not can be determined by analyzing the amount of the object to be inserted on the conductive part. Moreover, if it can introduce | transduce into a cell by inserting a needle-like structure in a cell, it can also be considered that adsorption | suction which concerns on this invention was materialized.

  For example, a method of immersing a needle-like structure having a conductive part in a liquid containing an insertion object can be employed. At this time, it is also possible to adopt a method in which a voltage is applied to the conductive portion of the needle-shaped structure and the charged portion of the object to be inserted is attracted by the conductive portion of the needle-shaped structure using an electric attractive force. it can.

  FIG. 1 is a schematic diagram showing a state in which an insertion object 1 that is a target substance to be introduced having a charged portion is adsorbed to a needle-like structure 3 having a conductive portion 2. FIG. 2 is a schematic diagram showing a state in which an insertion object 1 formed by attracting an introduction target substance 5 to a substance 4 having a charged part is adsorbed to a needle-like structure 3 having a conductive part 2.

  Next, by inserting the needle-like structure into the cell and applying a voltage to the conductive part, the target substance to be introduced is released into the cell using the electrical repulsion between the conductive part and the charged part. Alternatively, the target substance to be introduced is released into the cell as the insertion object is released into the cell. FIG. 3 is a schematic diagram showing a state in which the insertion object 1 that is a target substance to be introduced having a charged portion is released into the cell 6.

  FIG. 4 is a schematic diagram showing a state in which the insertion object 1 formed by attracting the introduction target substance 5 to the substance 4 having a charged portion is released into the cell 6. The insertion target object 1 formed by attracting the introduction target substance 5 to the substance 4 having a charged part is directly attracted to the substance 4 having a charged part even after entering the cell. Therefore, in the case of FIG. 4, it is important to select a substance having a charged portion so as not to interfere with the action of the target substance after entering the cell. However, when DNA or the like is used as a substance having a charged portion, it is often decomposed and absorbed into cells to be rendered harmless.

  The voltage is applied to the conductive part between the conductive part and the liquid containing cells. The electric potential of the conductive part is selected so that the insertion object can be easily released. For example, when the charged portion has a negative charge, the conductive portion is also set to a negative potential so that electrical repulsion between the conductive portion and the charged portion can be used. 3 and 4 are examples in which a negative potential is applied to the conductive portion. It is preferable to employ a three-electrode method for accurately applying a potential to the conductive portion. The magnitude of the voltage and the application time can be appropriately selected. In the case of DNA, a condition in which a potential of about -0.001 to -1.5 V is applied to the conductive part for about 0.1 to 10,000 seconds with respect to the liquid containing cells can be exemplified.

  According to this method, unlike the conventional injection method, the introduction target substance can be introduced into the cell without using air pressure, so that the cell is less damaged. The fact that the structure portion inserted into the cell can be made thinner than the conventional capillary is also advantageous in reducing damage to the cell. Since no capillaries are used, the clogging problem is solved. The main operation is the insertion of a needle-like structure into the cell and the application of voltage, which is easy, so that the target substance can be introduced into the cell in a short time, and therefore there is little damage to the cell. Many introduction target substances can be introduced. It is also possible to introduce it multiple times into the same cell.

  The cells to be the subject of the present invention may be animal cells or plant cells. It may be a cell that has undergone some pretreatment. The cells may be independent of each other or may be a part of the tissue.

  The introduction target substance is not particularly limited as long as it is not contrary to the gist of the present invention. Examples include chemical substances, nucleotide bodies, proteins, physiologically active substances, substances extracted from biological tissues and cells. In the present invention, the nucleotide body means any of mononucleotide, oligonucleotide, and polynucleotide (ie, nucleic acid). When the target substance to be introduced has a charged portion, such as a base part or a phosphate group part of the nucleotide body, the target substance to be introduced itself can be handled as an insertion target.

  The substance having the charged portion may be selected from any substance that can attract the introduction target substance. In addition to substances that are ionized, substances that can be ionized, substances that are polarized in molecules, and substances that can be polarized in molecules can be selected. Whether or not it is suitable as a “substance having a charged part” according to the present invention can attract the target substance to be introduced and releases the insertion object into the cell by applying a voltage to the conductive part of the needle-like structure. It can be judged by whether it is done. In general, ionized substances are advantageous. Whether or not the insertion object has been released into the cell can be determined by analyzing the amount of the insertion object adsorbed on the conductive part of the needle-like structure before and after the insertion of the insertion object into the cell. . When the target substance to be introduced has a fluorescence emission function, it can be visually confirmed by its emission / quenching behavior.

  Among these, when the substance having the charged part has a hydrophobic part, the introduction target substance can be easily attracted as described above, which is preferable. For example, a hydrophobic drug such as Gleevec (target substance to be introduced) is inserted into a nucleotide body (substance having a charged part) that has a base or phosphate group that is a charged part and a sugar part that is a hydrophobic part. It is a thing. The nucleotide body thus has a base or phosphate group that is a charged portion and a sugar portion that is a hydrophobic portion, and thus is a preferable substance for use as an insertion target or as a part of an insertion target. . Among them, those having a small molecular weight are advantageous because they are easily released in cells and are easily decomposed after being introduced into cells.

  The needle-like structure according to the present invention has a sharp tip so that it can be easily inserted into a cell. There is no restriction | limiting in particular about the shape. FIG. 5 shows two examples. The size is not particularly limited as long as it can be inserted into cells, but unlike the injection needle used in the conventional injection method introduces the target substance through the inside of the needle, the surface of the conductive part Since the introduction target substance adsorbed on the cell is introduced into the cell, its cross-sectional area (the cross-sectional area of the XX cross section in FIG. 5) may be smaller than the injection needle used in the conventional injection method. In general, the degree of damaging the cells decreases, so the cross-sectional area is preferably small. For example, in order to introduce a substance to be introduced into a specific region of a cell having a size of about 10 μm with high positional accuracy, the maximum diameter of the cross section of the insertion portion can be exemplified to be about 1.0 μm or less.

  The needle-like structure may be entirely composed of a conductive part, but only a part of the needle-like structure may be a conductive part. The structure may be such that the tip of the conductive portion is sharp, the tip is a non-conductive portion, and the conductive portion is present behind the non-conductive portion. In the latter case, the structure is complicated, but since the selection range of the material is widened, it becomes easy to sharpen the tip and make it difficult to be dulled even after being used many times. In addition, when the needle-like structure has a portion that is not a conductive part, the insertion object adheres to that part, and the insertion object may be released when inserted into a cell. The impact should be taken into account. In this sense, it is preferable that at least a portion of the needle-like structure inserted into the cell is a conductive portion.

  The conductive part according to the present invention may have any shape. As the cross section, a circular shape is usually adopted, but other cross sections may be used. The surface may be flat, but it may be grooved or uneven to increase the surface area.

  The conductive part according to the present invention can be selected from conductive materials. Examples of the conductive material include metals such as silicon, silicide, gold, platinum, tungsten, and molybdenum, alloys, metal oxides, carbon, conductive plastics, and plating or vapor deposition of these materials on nonconductive materials. Can be applied or coated. 6A shows a case where the entire needle-like structure is made of a conductive material, FIG. 6B shows a case where a part of the needle-like structure is made of a conductive material including the tip, and FIG. The case where a part of the needle-like structure is made of a conductive material and the tip thereof is made of a non-conductive material is schematically illustrated. 6A to 6C, hatched portions are portions made of a conductive material. There is no particular limitation on the material that can be used for the needle-like structure other than the conductive portion.

  In the method for introducing a substance to be introduced into a cell according to the present invention, the substance to be introduced into the cell is controlled by controlling the amount of the insertion object to be adsorbed on the acicular structure or the amount released in the cell or both. Can be easily controlled. Therefore, introduction of a very small amount or quantitative introduction is possible.

  Specifically, in the liquid containing the insertion target for adsorbing the insertion target to the needle-like structure, the concentration of the insertion target, pH, the concentration of ions used, the concentration of the salt used, the temperature, Examples include a method of controlling a parameter selected from the group consisting of the type of solvent used, the concentration of a substance that changes the state of the target substance to be introduced, and the adsorption area on the needle-like structure.

  Also, the concentration of the insertion target, pH, the concentration of ions used, the concentration of the salt used, the temperature, the solvent used in the liquid containing the cells that are the target for releasing the insertion target adsorbed on the needle-like structure And a method of controlling a parameter selected from the group consisting of the type of the target substance, the concentration of the substance that changes the state of the target substance to be introduced, and the discharge area of the insertion object on the needle-like structure.

  In the above description, the adsorption area on the needle-like structure means the area of the portion where the insertion object is to be adsorbed, that is, the area of the conductive portion used for adsorption. Moreover, the insertion object discharge | release area on a needle-like structure means the area of the electroconductive part used in order to be inserted in a cell and to discharge | release an insertion target object.

Examples of the ion to be used include Na ⁺ and Cl ⁻ , the concentration of the salt to be used is 10 to 50 mM, and the solvent to be used is a HEPES buffer. Examples of the substance that changes the state of the target substance to be introduced include substances that can change the structure and association state of proteins such as surfactants.

  Further, there may be mentioned a method of controlling a parameter selected from the group consisting of a voltage applied to the conductive part, a voltage application time, a holding time of the conductive part in the cell, a surface potential of the conductive part, a material and a structure.

  By controlling at least one of these parameters, the amount of the target substance to be introduced into the cell can be controlled.

  In particular, the control of the discharge area of the insertion object on the needle-like structure is highly useful because it ultimately controls the release amount when the insertion object is discharged. More specifically, the insertion object discharge area on the needle-like structure can be controlled by inserting the needle-like structure into the cell by a desired length. In this case, if the position where the tip of the needle-like structure contacts the cell is taken as the insertion start point, the length of the needle-like structure to be inserted thereafter can be easily determined. The position at which the tip of the needle-shaped structure contacts the cell may be determined by visual observation under a microscope, but since changes in light reflection are often observed, it is more objective to capture this change. It becomes detection. Furthermore, the insertion start point may be detected by a force applied to the needle-like structure. Detection of a change in light reflection and a force applied to the needle-like structure can be easily performed mechanically. Depending on the detection means, depending on the deformation of the cell, the position in contact with the cell may have a slight width, but in such a case, from within that width unless it is contrary to the spirit of the present invention. A specific position can be determined as appropriate.

  In this way, by introducing the amount of the target substance to be introduced into the cell, a very small amount of the target substance can be introduced. In addition, the introduction amount of the introduction target substance can be accurately controlled.

  The introduction of the substance to be introduced into the cell is performed by inserting a needle-like structure having a charged part and a conductive part capable of adsorbing an insertion target containing the substance to be introduced, and the needle-like structure into the cell. A needle-like structure drive unit, a container containing a cell-containing liquid, and a voltage application unit for applying a voltage to the conductive unit and releasing the target substance into the cell. It can be carried out using a target substance introduction device.

  With this device, there is little damage to the cells, either introduction of a large number of introduction target substances, introduction of a plurality of introduction target substances into the same cell, introduction of a small amount of substances, precise control of the introduction amount or Introducing substances into cells that can do all of them can be realized with simple operations.

  The configuration of the present apparatus and the usage thereof will be exemplarily described with reference to FIGS. FIG. 7 is a diagram showing a configuration of the present apparatus, and FIG. 8 is a flowchart of an example of an operation for introducing a target substance into a cell using the present apparatus.

  First, according to step S1 of FIG. 8, a liquid is prepared in which an insertion object having a charged portion and containing a target substance to be introduced is dispersed or dissolved in, for example, water. Next, in accordance with step S2 in FIG. 8, necessary data such as the distance for inserting the needle-like structure into the cell, the magnitude of the applied voltage, and the application time are input to the control computer 71 in FIG. Next, according to step S3 of FIG. 8, the needle-like structure having the conductive portion is immersed in the liquid, and if necessary, a voltage is applied between the needle-like structure and the liquid and the needle-like structure is inserted into the needle-like structure. The object is adsorbed. Next, according to step S4 of FIG. 8, the needle-like structure 3 of FIG. 7 is attached to the needle-like structure attaching portion 74 at the tip of the needle-like structure driving unit 73, and according to step S5 of FIG. Using the display 76 that displays the image, the three-dimensional position of the target cell in the liquid 72 in which the cells in the container 78 are dispersed is designated by, for example, a light pen. Then, according to step S6 of FIG. 8, under the control of the computer 71, the needle-like structure driving unit 73 inserts the needle-like structure 3 into the cell. Thereafter, under the control of the computer 71 according to step S7 in FIG. 8, the voltage applying unit 77 applies a predetermined voltage between the conductive portion of the needle-like structure 3 and the liquid 72 for a predetermined time, and according to step S8 in FIG. Under the control of the computer 71, the needle-like structure driving unit 73 extracts the needle-like structure 3 from the cell, and the operation is completed. Note that it is preferable to use a three-electrode method for voltage application because the potential of the conductive portion can be more accurately controlled. The number of needle-like structures 3 may be two or more. In that case, each needle-like structure may have different introduction target substances. A structure in which a plurality of needle-like structures can be simultaneously inserted into one cell is also possible.

  Note that the meanings of the terms used in the above description of the method for introducing a substance to be introduced into cells have the same meaning in the description of the apparatus for introducing a substance to be introduced into cells unless otherwise specified. In addition, the preferred embodiments regarding the insertion target substance, the introduction target substance, and the substance having a charged portion in the description of the method of introducing the target substance into the cell are the same in the description of the target substance introduction apparatus for introduction into the cell. preferable.

  In the apparatus for introducing a substance to be introduced into a cell according to the present invention, it is preferable to have an insertion object amount control unit for controlling the amount of the insertion object to be adsorbed by the needle-like structure in the cell. Specifically, the insertion target amount control unit is configured to release the insertion target discharge area on the needle-like structure in the liquid containing cells, the voltage applied to the conductive part, the voltage application time, and the holding of the conductive part in the cell. It is preferable that at least one parameter of the time and the surface potential of the conductive portion can be controlled. In the above example, the computer 71 plays the role.

  The material and shape of the container containing the liquid in which the cells are dispersed can be arbitrarily selected. It has a flow path for flowing a liquid containing an insertion object to be adsorbed on the needle-shaped structure and a flow path for flowing a liquid containing a target cell, and the needle-shaped structure is inserted into a lid on the flow path. It is often preferable to use a plate-like body having a hole that can quickly change the type of the object to be inserted, facilitate the insertion of the needle-like structure into the cell, and accelerate the supply of the cell. For example, the plate-like body may have a size of several mm square in the vertical and horizontal directions.

  The needle-like structure driving unit has a function that allows the needle-like structure to be simply inserted into the cell by a predetermined length when accuracy is not required for the amount of the target substance to be introduced into the cell. However, when it is desired to control the amount of the target substance to be introduced, the needle-like structure driving unit has a function of adjusting the length for inserting the needle-like structure into the cell as shown in the above example. It is preferable to have.

  Moreover, it is preferable that the needle-like structure driving unit has a function of detecting the position where the tip of the needle-like structure is in contact with the cell as an insertion start point. The function of detecting the insertion start point by a change in light reflection or a force applied to the needle-like structure is particularly preferable because it can be easily realized by a known technique.

  The method and apparatus for introducing a substance to be introduced into cells according to the present invention can be used in a narrow sense to elucidate the function of a biological substance. In addition, there is an advantage that the introduction target substance can be instantaneously released only in the vicinity of the electrode by voltage application. Therefore, application to the medical field can be considered. In the future, as the functions of biological materials obtained by the Human Genome Project are elucidated, the scope of application can be expected to expand.

  More specifically, any of the following effects can be realized by the method and apparatus for introducing a target substance into cells according to the present invention.

  (1) Since air pressure by a hollow needle is not used, a fine needle-like structure can be produced to a possible level by a fine process, and damage to cells can be reduced.

  (2) Since only a very small amount of the target substance can be introduced, the field of application is wide, and a large number of target substances can be introduced or continuously introduced into the same cell.

  (3) Since the voltage can be accurately controlled by the three-electrode method, the introduction amount can be controlled by the voltage, and quantitative introduction is possible by controlling the voltage and the voltage application time. Further, since only the conductive part of the needle-like structure adsorbed with the introduction target substance needs to be inserted into the cell, damage to the cell can be reduced.

  The needle-like structure according to the present invention can be easily washed after releasing the target substance to be introduced into the cell and can be used repeatedly as long as it is not physically damaged. Therefore, the same or different introduction target substances can be continuously introduced into the cells. As cleaning in this case, cleaning with an acid, cleaning with an alkali, cleaning with an organic solvent, ultrasonic cleaning, cleaning with a voltage applied to the conductive portion, and a combination thereof can be employed. Cleaning in a state where a voltage is applied to the conductive portion and a combination of this and another cleaning method are preferable because the structure of the needle-like structure according to the present invention can be used. In this case, it is often sufficient to apply the voltage in an aqueous solution usually at 1 to 2 V for several seconds.

  In addition, when using the said plate-shaped body as a container containing the liquid with which the cell was disperse | distributed, work can be further facilitated by attaching the flow path for washing | cleaning.

  Next, an example comparative example of the present invention will be described in detail.

[Example 1]
As the needle-like structure, a tungsten wire with a tip diameter of 300 μm at the maximum and a gold plating was used. Therefore, the conductive part according to the present invention is an arbitrary part of the acicular structure. DNA was used as the introduction target substance. Therefore, in this case, the introduction target substance itself is an insertion object. HEK293 was used as the cell. The cells were dispersed in a culture dish containing a culture solution.

  DNA is dispersed in a buffer solution containing 50 mM NaCl (HEPES buffer, pH 7.4), the needle-like structure is immersed at room temperature for 10 seconds, and immediately immersed in the solution in which the cells are dispersed. The structure was inserted, and a voltage was applied so that the electric potential of the needle-like structure was −0.7 V with respect to the liquid.

  FIG. 9 shows the relationship between the time during which the needle-like structure is inserted into the cell and the amount of DNA introduced into the cell. A good linear relationship was obtained. The amount of DNA introduced into the cells was determined by real-time PCR. The minimum amount introduced was 100.

[Example 2]
The amount of DNA introduced into the cells was determined under the same conditions as in Example 1 except that the voltage bias was changed from -0.7 V to -1.0 V. Introduction was approved.

[Example 3]
When the amount of DNA introduced into the cell was determined under the same conditions as in Example 1 except that the length of the needle-like structure inserted into the cell was halved, it was approximately 1/4 in the same time. The introduction of quantity was allowed.

  In addition, the invention shown to the following additional remarks can be derived from the content disclosed above.

(Appendix 1)
An object to be inserted having a charged portion and containing a target substance to be introduced is adsorbed to a needle-like structure having a conductive portion,
Insert the needle-like structure into the cell,
By applying a voltage to the conductive part, the target substance to be introduced is released into the cell.
A method for introducing a target substance into a cell.

(Appendix 2)
The method of introducing a substance to be introduced into a cell according to appendix 1, wherein the insertion object is formed by attracting a substance to be introduced to a substance having a charged portion.

(Appendix 3)
The introduction target substance and the substance having the charged portion have a hydrophobic portion,
The target substance to be introduced is attracted to the substance having the charged part through the two hydrophobic parts to form an insertion object.
The method for introducing a substance to be introduced into cells according to appendix 2.

(Appendix 4)
The method for introducing a substance to be introduced into cells according to any one of appendices 1 to 3, wherein the insertion object comprises a nucleotide body.

(Appendix 5)
The method for introducing a substance to be introduced into cells according to any one of appendices 1 to 4, wherein the amount of adsorption of the insertion object to be adsorbed on the needle-like structure or the amount of release in the cells or both are controlled.

(Appendix 6)
In the liquid containing the insertion object, the concentration, pH, ion concentration, salt concentration, temperature, type of solvent, concentration of the substance that changes the state of the target substance to be introduced, and the adsorption area on the acicular structure ,
In the liquid containing the cells, the concentration, pH, ion concentration, salt concentration, temperature, type of solvent, concentration of the substance that changes the state of the target substance to be introduced, and release of the insertion target on the needle-like structure According to at least one parameter selected from the group consisting of area, voltage applied to the conductive part, voltage application time, retention time of the conductive part in the cell, surface potential of the conductive part, material and structure The method for introducing a substance to be introduced into cells according to any one of appendices 1 to 5, wherein the amount of adsorption or release or the amount of adsorption and release is controlled.

(Appendix 7)
7. The method for introducing a substance to be introduced into a cell according to appendix 6, wherein an insertion object release area on the needle-like structure is controlled by inserting the needle-like structure into the cell by a desired length.

(Appendix 8)
8. Introduction of a target substance to be introduced into a cell according to appendix 7, wherein the needle-like structure is inserted into the cell by a desired length using a position where the tip of the needle-like structure is in contact with the cell as an insertion start point. Method.

(Appendix 9)
9. The method for introducing a substance to be introduced into a cell according to appendix 8, wherein the insertion start point is detected by visual observation, a change in reflection of light, or a force applied to the needle-like structure.

(Appendix 10)
The needle-like structure after releasing the target substance to be introduced into the cell is washed with an acid, washed with an alkali, washed with an organic solvent, ultrasonic washed, washed with a voltage applied to the conductive part, and combinations thereof. The method for introducing a substance to be introduced into cells according to any one of appendices 1 to 9, comprising performing at least one washing selected from the group consisting of:

(Appendix 11)
A needle-like structure having a conductive part having a charged part and capable of adsorbing an insertion object including a target substance to be introduced;
A needle-like structure driving unit for inserting the needle-like structure into a cell;
A container containing a liquid containing cells;
A target substance introduction device for introduction into a cell, comprising: a voltage application unit for applying a voltage to the conductive part and releasing the introduction target substance into the cell.

(Appendix 12)
12. The introduction target substance introduction device into a cell according to appendix 11, wherein the insertion object is formed by attracting the introduction target substance to a substance having a charged portion.

(Appendix 13)
The introduction target substance and the substance having the charged portion have a hydrophobic portion,
The insertion object is prepared by attracting the introduction target substance to the substance having the charged portion through the two hydrophobic portions.
The apparatus for introducing a substance to be introduced into cells according to appendix 12.

(Appendix 14)
The introduction target substance introduction device for introduction into cells according to any one of appendices 11 to 13, wherein the insertion object includes a nucleotide body.

(Appendix 15)
15. The introduction target substance introduction device into a cell according to any one of appendices 11 to 14, further comprising an insertion object amount control unit that controls an amount of the insertion object to be adsorbed by the needle-like structure in the cell.

(Appendix 16)
The insertion object amount control unit includes a discharge area of the insertion object on the needle-like structure in the liquid containing the cells, a voltage applied to the conductive part, a voltage application time, a holding time of the conductive part in the cell, and The apparatus for introducing a substance to be introduced into cells according to any one of appendices 11 to 15, wherein the release amount is controlled by at least one parameter selected from the group consisting of a surface potential of the conductive portion.

(Appendix 17)
17. The apparatus for introducing a substance to be introduced into a cell according to appendix 16, wherein the needle-like structure driving unit has a function of adjusting a length for inserting the needle-like structure into the cell.

(Appendix 18)
The needle-like structure driving unit has a function of adjusting the length of insertion into a cell from the position where the tip of the needle-like structure is in contact with the cell as an insertion start point. A device for introducing a target substance into a cell.

(Appendix 19)
19. The apparatus for introducing a substance to be introduced into cells according to appendix 18, wherein the needle-like structure driving unit detects the insertion start point based on a change in light reflection or a force applied to the needle-like structure.

(Appendix 20)
The cell according to any one of appendices 11 to 19, wherein the conductive portion is made of a material selected from the group consisting of silicon, silicide, gold, platinum, tungsten, molybdenum and alloys thereof and those plated with them. Introduction target substance introduction device.

It is a schematic diagram which shows a mode that the insertion target object which is an introduction target substance which has a charging part was made to adsorb | suck to the acicular structure which has an electroconductive part. It is a schematic diagram which shows a mode that the insertion target object attracted to the substance which has a charging part was made to adsorb | suck to the acicular structure which has an electroconductive part. It is a schematic diagram which shows a mode that the insertion target object which is an introduction target substance which has a charging part is discharge | released in a cell. It is a schematic diagram which shows a mode that the insertion target object attracted | subjected the introduction | transduction target substance to the substance which has a charged part is discharge | released in a cell. It is a side view of the acicular structure which concerns on this invention. It is another side view of the acicular structure which concerns on this invention. It is another side view of the acicular structure which concerns on this invention. It is another side view of the acicular structure which concerns on this invention. It is a figure which shows the structural example of the apparatus which concerns on this invention. It is a flowchart which shows the example of the operation | work which introduce | transduces the introduction target substance into a cell using the apparatus which concerns on this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Insert object 2 Conductive part 3 Needle-like structure 4 Substance which has charged part 5 Introduction target substance 6 Cell 71 Control computer 72 Liquid in which cells are dispersed 73 Needle-like structure driving part 74 Needle-like structure attaching part 75 Microscope 76 Display 77 Voltage application unit 78 Container

Claims (7)

An object to be inserted having a charged portion and containing a target substance to be introduced is adsorbed to a needle-like structure having a conductive portion,
Insert the needle-like structure into the cell,
By applying a voltage to the conductive part, the target substance to be introduced is released into the cell.
A method for introducing a target substance into a cell.   The method of introducing a substance to be introduced into a cell according to claim 1, wherein the insertion object is formed by attracting a substance to be introduced to a substance having a charged portion. The introduction target substance and the substance having the charged portion have a hydrophobic portion,
The target substance to be introduced is attracted to the substance having the charged part through the two hydrophobic parts to form an insertion object.
The method for introducing a substance to be introduced into cells according to claim 2. In the liquid containing the insertion object, the concentration, pH, ion concentration, salt concentration, temperature, type of solvent, concentration of the substance that changes the state of the target substance to be introduced, and the adsorption area on the acicular structure ,
In the liquid containing the cells, the concentration, pH, ion concentration, salt concentration, temperature, type of solvent, concentration of the substance that changes the state of the target substance to be introduced, and release of the insertion object on the needle-like structure According to at least one parameter selected from the group consisting of area, voltage applied to the conductive part, voltage application time, retention time of the conductive part in the cell, surface potential of the conductive part, material, and structure The method for introducing a substance to be introduced into a cell according to any one of claims 1 to 3, wherein an adsorption amount or a release amount or an adsorption amount and a release amount are controlled.   The method for introducing a substance to be introduced into a cell according to claim 4, wherein the insertion target substance release area on the needle-like structure is controlled by inserting the needle-like structure into the cell by a desired length. A needle-like structure having a conductive part having a charged part and capable of adsorbing an insertion object including a target substance to be introduced;
A needle-like structure driving unit for inserting the needle-like structure into a cell;
A container containing a liquid containing cells;
A target substance introduction device for introduction into a cell, comprising: a voltage application unit for applying a voltage to the conductive part and releasing the introduction target substance into the cell.   The introduction target substance introduction device into a cell according to claim 6, wherein the insertion object is formed by attracting the introduction objective substance to a substance having a charged portion.

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