US20080199945A1 - Cantilever for measuring intra-cellular and inter-cellular microspaces - Google Patents
Cantilever for measuring intra-cellular and inter-cellular microspaces Download PDFInfo
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- US20080199945A1 US20080199945A1 US11/943,089 US94308907A US2008199945A1 US 20080199945 A1 US20080199945 A1 US 20080199945A1 US 94308907 A US94308907 A US 94308907A US 2008199945 A1 US2008199945 A1 US 2008199945A1
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- Prior art keywords
- cellular
- cantilever
- probe
- inter
- cell
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01Q—SCANNING-PROBE TECHNIQUES OR APPARATUS; APPLICATIONS OF SCANNING-PROBE TECHNIQUES, e.g. SCANNING PROBE MICROSCOPY [SPM]
- G01Q60/00—Particular types of SPM [Scanning Probe Microscopy] or microscopes; Essential components thereof
- G01Q60/24—AFM [Atomic Force Microscopy] or apparatus therefor, e.g. AFM probes
- G01Q60/38—Probes, their manufacture, or their related instrumentation, e.g. holders
- G01Q60/40—Conductive probes
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y35/00—Methods or apparatus for measurement or analysis of nanostructures
Definitions
- the present invention relates to a cantilever for measuring intra-cellular and inter-cellular microspaces suitable for measurement or operation in intra-cellular and inter-cellular microspaces.
- Such elucidation or control of cellular vital functions involves essential operations such as measuring electric potential difference in intra-cellular and inter-cellular microspaces and introducing and taking a gene or the like into and out of a cell.
- the Japanese Unexamined Patent Publication No. 2003-325161 describes a suggestion on a cell operating device and a cell operating method using the cell operating device.
- This publication discloses a technique on a configuration which is capable of detailed real-time observation of serial dynamics changes shown on the cell during an interval from introduction to manifestation of a gene, which is applicable even for elucidating or controlling cell differentiation, and which further includes a probe of an Atomic Force Microscope (AFM) which is provided, as cell operative means capable of minimizing cellular damage during introduction of a gene or the like, with a needle-shaped matter for inserting into a cell a substance relating to gene or gene manifestation which is fixed thereto.
- AFM Atomic Force Microscope
- the Japanese Unexamined Patent Publication No. 2006-166884 discloses a technique regarding a method for introducing into a cell a substance to be introduced, by binding the substance to be introduced to a needle-shaped material by means of an intermediate binding force such that the substance to be introduced is caused to remain in the cell when the needle-shaped material is punctured thereinto.
- This publication also discloses a technique relating to a configuration in which an atomic force microscope (AFM) is used for a method of detecting puncture of the needle-shaped material into the cell and as an introducing device for introducing the substance to be introduced into the cell.
- AFM atomic force microscope
- a probe 101 provided to an AFM cantilever 100 is etched by a focused ion beam so as to be configured as a needle-shaped material, and further, the probe 1001 as the needle-shaped material is used to perform a puncturing operation into a cell 103 and a nucleus 104 in the cell 103 .
- the Japanese Unexamined Patent Publication No. 2006-166884 also discloses a technique related to electrostatic coupling as a method to couple the substance to be introduced to the needle-shaped material by means of an intermediate binding force. Because such electrostatic coupling is a weak coupling, the substance to be introduced can be easily separated in the cell. In electrostatic coupling, cationic polymer is generally used.
- a thiol group is introduced into a needle-shaped material 105 , which is a silicon-made AFM probe etched to have a sharpened edge, to modify succinimide active ester on a surface of needle-shaped material to immobilize polylysine followed by electrostatic coupling of plasmid phrGFP as the substance to be introduced, before introducing the needle-shaped material 105 into the cell.
- the present invention has been made in view of the above-described problems, and an object of the present invention is to provide a cantilever for measuring intra-cellular and inter-cellular microspaces that allows stable intra-cellular measurement or operation to be easily performed, which is durable, reliable, and low cell-invasive.
- Another object of the present invention is to provide a cantilever for measuring intra-cellular and inter-cellular microspaces that allows performing, easily and stably with good reproducibility and minimum variation, nano-cell mapping, i.e., measurement in intra-cellular and inter-cellular microspaces and introducing and taking a substance to and out of the microspaces.
- a cantilever for measuring intra-cellular and inter-cellular microspaces includes: a support portion; a lever portion provided to the support portion so as to protrude therefrom; and a probe portion provided near a free end of the lever portion, wherein the probe portion includes a conductive probe made of a carbon-based material, and an insulating film to coat a periphery of the conductive probe.
- FIG. 1 is a perspective view showing an appearance configuration of a cantilever for measuring intra-cellular and inter-cellular microspaces according to a first embodiment of the present invention.
- FIG. 2 is a partially cut sectional view of a lever portion along A-A′ line of the cantilever for measuring intra-cellular and inter-cellular microspaces according to the first embodiment of the present invention of FIG. 1 .
- FIG. 3 is a sectional view of a probe portion according to the first embodiment, in a vertical direction with respect to a projecting direction.
- FIG. 4 is a sectional view of a distal end part in the projecting direction of the probe portion according to the first embodiment.
- FIG. 5 is a sectional view of a distal end part showing a first modification example of the probe portion.
- FIG. 6 is a sectional view of a distal end part showing a second modification example of the probe portion.
- FIG. 7 is a partially cut sectional view of a probe portion and a lever portion, showing a third modification example of the probe portion.
- FIG. 8 is a pattern view showing a schematic configuration of an entire system having the cantilever of the first embodiment of the present invention.
- FIG. 9 is an enlarged view of the probe portion of the cantilever and inside of a cell in measuring intra-cellular electric potential difference in the first embodiment.
- FIG. 10 is an illustrative view showing a state where a probe portion of a cantilever for measuring intra-cellular and inter-cellular microspaces according to a second embodiment of the present invention is applied with an electronegative potential to adsorb a substance to be introduced to the probe portion.
- FIG. 11 is an illustrative view showing a state where the probe portion, which is in an electrically floating state, is being inserted into the cell in the second embodiment.
- FIG. 12 is an illustrative view showing a state where an electropositive potential is applied to the probe portion of the cantilever to separate in the cell the substance to be introduced from the probe portion in the second embodiment.
- FIG. 13 is an illustrative view showing a state where, with the probe portion in the electrically floating state, only the probe portion is pulled out of the cell in the second embodiment.
- FIG. 14 is an illustrative view showing a state before the probe portion of the cantilever in the electrically floating state is inserted into the cell in the second embodiment.
- FIG. 15 is an illustrative view showing a state where the probe portion in the electrically floating state is inserted into the cell in the second embodiment.
- FIG. 16 is an illustrative view showing a state where the electronegative potential is applied to the probe portion of the cantilever to adsorb an intra-cellular substance in the cell to the probe portion in the second embodiment.
- FIG. 17 is an illustrative view showing a state where, with the electronegative potential remained applied to the probe portion, the probe portion is pulled out of the cell along with the intra-cellular substance adsorbed to the probe portion in the second embodiment.
- FIG. 18 is an illustrative view showing a state where a resistance meter is switched on to measure resistance between a conductive stage and the probe portion so as to detect contact between the probe portion and a cell membrane, according to a third embodiment of the present invention.
- FIG. 19 is an illustrative view showing a state where a pulse generating unit is switched on to apply a pulse wave electric potential when contact between the probe portion and the cell membrane is detected in the third embodiment.
- FIG. 20 is an illustrative view showing a state where the pulse generating unit is switched off to stop the application of the pulse wave electric potential after the probe portion is inserted into the cell in the third embodiment.
- FIG. 21 is an illustrative view showing a cell puncturing operation using a needle-shaped material in a prior art.
- FIG. 22 is a schematic view showing a state where electrostatic adsorption is conducted on a polylysine-modified needle in a prior art.
- FIGS. 1 to 7 relate to a first embodiment of a cantilever for measuring intra-cellular and inter-cellular microspaces of the present invention
- FIG. 1 is a perspective view showing an appearance configuration of the cantilever for measuring intra-cellular and inter-cellular microspaces according to the first embodiment
- FIG. 2 is a partially cut sectional view of a lever portion along A-A′ line of the cantilever for measuring intra-cellular and inter-cellular microspaces of FIG. 1
- FIG. 3 is a sectional view of a probe portion according to FIG. 1 , in a vertical direction with respect to a projecting direction
- FIG. 4 is a sectional view of a distal end part in the projecting direction of the probe portion of FIG. 1 ;
- FIG. 1 is a perspective view showing an appearance configuration of the cantilever for measuring intra-cellular and inter-cellular microspaces according to the first embodiment
- FIG. 2 is a partially cut sectional view of a lever portion along A-A′ line of the cantilever for measuring intra-cellular
- FIG. 5 is a sectional view of a distal end part showing a first modification example of the probe portion
- FIG. 6 is a sectional view of a distal end part showing a second modification example of the probe portion
- FIG. 7 is a partially cut sectional view of a probe portion and a lever portion, showing a third modification example of the probe portion.
- a cantilever for measuring intra-cellular and inter-cellular microspaces 1 (hereinafter referred to as cantilever 1 ) of the first embodiment, shown in FIG. 1 , is used for measurement or operation in intra-cellular and inter-cellular microspaces.
- the cantilever 1 includes a support portion 2 , a lever portion 3 protrudingly provided to the support portion 2 , a probe portion 4 provided near a free end of the lever portion 3 , an electrode pad 6 provided to a proximal end portion on the support portion 2 side of the lever portion 3 , and a wiring portion 5 to electrically connect a conductive probe 7 to be described below of the probe portion 4 and the electrode pad 6 via inside of the lever portion 3 .
- the support portion 2 is held to a cantilever holder or the like in a cell operating device not shown when performing measurement or operation in intra-cellular and inter-cellular microspaces.
- a proximal end side part of the lever portion 3 is supportedly fixed. Note that the manner in which the lever portion 3 is mounted to the support portion 2 is not specifically limited, i.e., the mounting may be in any manner.
- the lever portion 3 is configured of a material such as, for example, silicon oxide and a silicon nitride. Specifics of the configuration will be described later.
- the probe portion 4 is formed near the free end of the lever portion 3 .
- the probe portion 4 is provided so as to protrude in a vertical direction with respect to a longitudinal direction of the lever portion 3 .
- the first embodiment describes a configuration in which the probe portion 4 is provided in the vertical direction with respect to the lever portion 3 , no limitation is placed thereon.
- the probe portion 4 may be formed to be inclined toward any direction with respect to the lever portion 3 .
- the probe portion 4 includes a conductive probe 7 which is a conductive member made of a carbon-based material, and an insulating film 8 provided to coat the periphery of the conductive probe 7 , as shown in FIG. 2 .
- FIG. 3 shows a sectional configuration of the probe portion 4 , in the vertical direction with respect to the projecting direction of the probe portion 4 .
- the probe portion 4 is formed in, for example, a circular cylinder shape as shown in FIG. 3 .
- the conductive probe 7 configuring the probe portion 4 is also formed in, for example, a circular cylinder shape.
- the shapes of the probe portion 4 and the conductive probe 7 are not limited to a circular cylinder shape, and may be formed otherwise, for example, in a columnar shape such as of elliptic cylinder or prism, or in a conic shape such as of cone or pyramid. Nevertheless, the probe portion 4 and the conductive probe 7 are preferably formed in a circular cylinder shape in order to reduce cell damage, as described above.
- the insulating film 8 is provided to coat the periphery of the conductive probe 7 having such a shape. Accordingly, the shape of the insulating film 8 may be changed to change the shape of the probe portion 4 itself, while leaving as is the shape of the conductive probe 7 . Note that, as long as the insulating film 8 can ensure insulation when completely coating the periphery of the conductive probe 7 , the insulating film 8 is not specifically limited in shape, thickness and so on, and may be configured in any columnar or conic shape.
- a distal end surface 4 a of the probe portion 4 is formed in, for example, a planar shape, as shown in FIGS. 2 and 4 .
- a distal end surface 7 a of the conductive probe 7 is provided in the distal end surface 4 a .
- the distal end surface 7 a is provided in a manner to be in a same plane as that of the distal end surface of probe portion 4 .
- a proximal end surface 7 b is formed to a proximal end portion on a reverse side to the distal end surface 7 a of conductive probe 7 .
- an engaging portion 8 a to engage with and be fixed to the lever portion 3 .
- the conductive probe 7 is configured of a carbon-based material, which is at least one of single crystal diamond, polycrystalline diamond, nanodiamond, diamondlike carbon, and amorphous carbon, for example.
- a carbon-based material is doped with boron, Lin, or the like, to configure the conductive probe 7 .
- the conductive probe 7 which is configured using such a carbon-based material, has a high rigidity to improve durability and an oxidation-resistant characteristic, and therefore is reliable. Accordingly, the probe portion 4 configured using the conductive probe 7 , which is thus durable and reliable, allows an intra-cellular stable measurement or operation to be easily and stably performed.
- the insulating film 8 is configured of one layer of any of an oxide film, nitride film, and organic film, or by a laminated film including at least one of an oxide film, nitride film, and organic film. Such a laminated film can be formed by a commonly available method. Therefore, the insulating film 8 can be easily formed to cover the conductive probe 7 .
- the insulating film 8 may be formed using, instead of the laminated film, a carbon-based material including at least one of polycrystalline diamond, nanodiamond, diamondlike carbon, and amorphous carbon. Nevertheless, it is to be noted that, in the first embodiment, it is preferrable to use a carbon-based material to form the insulating film 8 , in view of adaptability to living body and capability in surface treatment to improve adhesion of the substance to be introduced (surface treatment to form a surface-modified portion to be described below).
- a distal end portion thereof has a diameter not greater than 1 ⁇ m, preferably not greater than 400 nm. It is further preferable to form the probe portion 4 to have a length not less than 2 ⁇ m, preferably not less than 5 ⁇ m, and more preferably not less than 10 ⁇ m.
- the conductive probe 7 of the probe portion 4 having such a configuration is electrically connected to the wiring portion 5 through contact of the proximal end surface 7 b with the wiring portion 5 in the lever portion 3 , as shown in FIG. 2 .
- the lever portion 3 engages with the engaging portion 8 a of the insulating film 8 of the probe portion 4 to fix the probe portion 4 itself.
- the lever portion 3 includes a protection film 9 to protect the wiring portion 5 in the lever portion, the wiring portion 5 arranged next to the protection film 9 , a lever main body portion 3 A to sandwichingly hold the wiring portion 5 with the protection film 9 , and an optical reflection film 10 which is coating treated on an outer surface of the lever main body portion 3 A.
- the protection film 9 is configured of an insulative member and provided to cover the entire surface on one side of the lever portion 3 except the part corresponding to the electrode pad 6 . Though not illustrated, the protection film 9 is also provided over the entire surface of the support portion 2 on a side oriented in the same direction as the one side of the lever portion 3 .
- an aperture 9 a is formed to expose the engaging portion 8 a of the insulating film 8 .
- the protection film 9 may be formed to entirely cover the circumference of the insulating film 8 of the probe portion 4 without providing the aperture 9 a . This permits firmly fixing the probe portion 4 to the lever portion 3 .
- the protection film 9 has an aperture at a part corresponding to the electrode pad 6 above the support portion 2 , for electric connection between the internal wiring portion 5 and the electrode pad 6 .
- the first embodiment describes a configuration in which the protection film 9 is provided, no limitation is placed thereon, i.e., there may be a configuration without the protection film 9 if so needed.
- the wiring portion 5 is electrically connected to the conductive probe 7 through contact with the proximal end surface 7 b of the conductive probe 7 .
- a proximal end portion of the wiring portion 5 on the support portion 2 side is electrically connected to the electrode pad 6 for electrical connection to an external device such as an electrometer, for example.
- a preferable material for the wiring portion 5 is platinum or iridium, which facilitates accumulation and growth of the conductive probe 7 of a carbon-based material. Yet, as long as adhesion with the conductive probe 7 can be assured, the wiring portion 5 may be configured of a material of a metal other than platinum or iridium. The wiring portion 5 may also be otherwise configured using a semiconductor material such as polysilicon.
- a conductive material such as titanium or chrome may be formed between the wiring portion 5 and the lever main body portion 3 A to improve adhesion therebetween.
- a conductive material such as titanium or chrome may be formed between the wiring portion 5 and the protection film 9 to improve adhesion therebetween.
- the electrode pad 6 may be formed with the wiring portion 5 itself, an electrode layer such as of gold, copper or aluminum may be formed on the pad to facilitate electric connection with an external terminal through solder connection, wire bonding, or the like.
- a conductive material such as titanium, chrome or the like may be formed between the electrode pad 6 and the electrode layer in order to improve adhesion between the electrode pad 6 and the electrode layer.
- the optical reflection film 10 provided on the outer surface of the lever main body portion 3 A is formed by being subjected to coating treatment with aluminum or gold. Note that the optical reflection film 10 may be configured not only limitedly by such a coating treatment but using another method. The optical reflection film 10 may also be configured provided with a multilayer film.
- the optical reflection film 10 serves to detect reflection angle or reflection amount of lights irradiated from the external optical system apparatus.
- the personal computer analyzes a detection result by the optical reflection film 10 to enable dynamically detecting whether or not the probe portion 4 is in contact with a cell membrane, which is effective for performing measurement or operation in the intra-cellular and inter-cellular microspaces.
- the cantilever 1 can be low cell-invasive, allowing various measurements or operations in the intra-cellular and inter-cellular microspaces to be performed easily and stably with good reproducibility and minimum variation.
- the protection film 9 covers the entire surface on one side of the lever portion 3 except the part corresponding to the electrode pad 6 above the support portion 2 , it is possible to easily perform stable measurement and operation in the intra-cellular and inter-cellular microspaces, in various environments such as one in which the cell is soaked in a water-system solution such as cultured liquid or isotonic sodium chloride solution.
- the use of the durable and reliable conductive probe 7 to configure the probe portion 4 can make the cantilever 1 to be highly durable and highly reliable.
- the probe portion 4 may be configured such that the distal end surface 4 a configures a distal end portion 4 a 1 in a shape with a distal end portion 7 a 1 of the conductive probe 7 protruding from the insulating film 8 , as shown with a probe portion 4 A in a first modification example of FIG. 5 , instead of providing the distal end surface 7 a of the conductive probe 7 in the same plane as that of the insulating film 8 .
- the distal end portion 4 a 1 of the probe portion 4 A and the distal end portion 7 a 1 of the conductive probe 7 are configured in a cone shape such as, for example, a circular cone or pyramid.
- the probe portion 4 may also be configured such that, in the case where, as shown by a probe portion 4 B in a second modification example of FIG. 6 , a distal end portion 4 a 2 is configured in a shape such that a distal end portion 7 a 2 of the conductive probe 7 protrudes from the insulating film 8 , the distal end portion 7 a 2 of the conductive probe 7 is formed in an arc shape such as a hemispheric or ellipse sphere shape, while also forming a distal end portion of the insulating film 8 in the periphery of the distal end portion 7 a 2 in an arc shape such as hemispheric or ellipse sphere shape.
- forming the distal end portion 4 a 2 of the probe portion 4 B in arc shape leads to an effect of allowing reducing cell damage.
- the probe portion 4 may be configured such that, in the case where, like the probe portion 4 C shown in the third modification example of FIG. 7 , the distal end portion 4 a 3 is configured in a shape such that the distal end surface 7 a 3 of the conductive probe 7 protrudes from the insulating film 8 , a protruding portion 7 c of the conductive probe 7 is provided, and the distal end surface 7 a 3 of the protruding portion 7 c and the distal end surface of the insulating film 8 are provided at different positions, not in a same plane.
- the provision of the protruding portion 7 c of the conductive probe 7 increases the exposing surface area of the conductive probe 7 , which is effective to improve the adhesion capability of the substance to be introduced and the intra-cellular substance.
- FIGS. 8 and 9 there are described configuration example and action in a system configuration to actually measure an electric potential difference of intra-cellular and inter-cellular microspaces using the cantilever 1 having the above configuration.
- FIG. 8 is a pattern view showing a schematic configuration of an entire system having the cantilever 1 of the first embodiment of the present invention.
- FIG. 9 is an enlarged view of the probe portion 4 of the cantilever 1 and the inside of the cell in measuring intra-cellular electric potential difference.
- the system includes: a cantilever 1 shown in FIG. 1 ; a connection line 11 , which is electrically connected to the electrode pad 6 of the cantilever 1 ; an electrometer 12 which is electrically connected to the connection line 11 and can measure an electric potential difference between a distal end portion of the probe portion 4 of the cantilever 1 and a conductive stage to be described below; and a conductive stage 13 which is electrically connected to the connection line 11 from the electrometer 12 and configured of a conductive member having a placement surface 13 a on which a cell 50 is placed.
- the cantilever 1 and the conductive stage 13 are electrically connected via the connection line 11 and the electrometer 12 .
- the cantilever holder for holding the support portion 2 of the cantilever 1 and other external devices such as a personal computer: configuration and action of only principal parts are described.
- the operator moves the probe portion 4 of the cantilever 1 toward the cell 50 as shown in FIG. 9 .
- the cell 50 has an intracellular nucleus 51 and a cell membrane 52 as is well-known.
- the contact of the probe portion 4 of the cantilever 1 with the cell membrane 52 may be detected either electrically by the electrometer 12 or dynamically by means of a reflected light from the optical reflection film 10 , or the like, as described above.
- the operator inserts the probe portion 4 of the cantilever 1 into the cell 50 , penetrating the cell membrane 52 , as shown in FIG. 9 .
- the operator measures an electric potential difference between the distal end surface 4 a of the probe portion 4 in the cell 50 and the conductive stage 13 , using the electrometer 12 . Measurement result at this time is recorded by a personal computer not shown.
- the operator here re-stabs the probe portion 4 into the cell 50 in the method mentioned above, measuring an electric potential difference for each re-stabbing of the probe portion. This allows obtaining a measurement result of electric potential distribution at a desired position in the cell 50 .
- the number of times of the re-stabbing preferably ranges from about dozens of times to 100 times, but no limitation is placed thereon, i.e., the re-stabbing may be performed by a number of times as needed. Even if the probe portion 4 is re-stabbed by dozens of times, the cell 50 will not die. The position of the re-stabbing into the cell 50 may be set as desired as needed.
- the nano-cell mapping maps a physical parameter such as of intra-cellular and inter-cellular substances, electric potentials or the like present in an organization/trachea, or maps subtle distribution of strength and weakness of a specific biological function, and is not limited to use for the electric potential distribution.
- the probe portion 4 and the lever portion 3 are covered by the insulating film 8 and the protection film 9 , respectively, it is possible to measure the electric potential difference and the electric potential distribution even if the cell 50 is soaked in a water system solution such as culture media or isotonic sodium chloride solution.
- the first embodiment it is enabled to easily and stably measure an electric potential difference between the inside of the cell and the cell membrane, as well as to easily and stably measure, in real-time, intra-cellular or inter-cellular electric potential distribution. It is therefore rendered possible to realize a cantilever (the cantilever 1 ) for measuring intra-cellular and inter-cellular microspaces, which is durable, reliable, and low cell-invasive, enabling stable intra-cellular measurement or operation to be easily performed.
- FIGS. 10 to 17 relate to a second embodiment of the cantilever for measuring intra-cellular and inter-cellularmicrospaces of the present invention.
- FIGS. 10 to 13 are illustrative views to describe a method to introduce into the cell the substance to be introduced using a system including the cantilever.
- FIGS. 14 to 17 are illustrative views to describe a method to take the intra-cellular substance out of the cell using a system including the cantilever.
- FIGS. 10 to 17 components same as the cantilever 1 and those of the system of the first embodiment are attached with the same symbols, omitting descriptions thereof: only different parts are described.
- At least distal end part of the conductive probe 7 has a surface-modified portion 40 subjected to a surface treatment to facilitate adhesion of the intra-cellular substance and the substance to be introduced into the cell.
- the surface-modified portion 40 is to be formed by being subjected to a surface treatment using any one of hydrogen, oxygen, fluorine, amino acid, silane, organic molecules, or biological molecules.
- the surface-modified portion 40 may be formed on at least one of, for example, the distal end surface 7 a or an exposed portion of the conductive probe 7 and the distal end surface or an exposed portion of the insulating film 8 forming the distal end surface 4 a.
- Formation of the surface-modified portion 40 is not limited to on the distal end portion of the probe portion 4 , but may be on a range from the distal end portion to circumference of the probe portion 4 , subjected to a surface treatment using the above-mentioned material.
- the method to form the surface-modified portion 40 is not limited to surface treatment, but any method including physical combination may be used to form the surface-modified portion.
- the surface-modified portion 40 is not necessarily a requirement, but as described below, it is effective to form the surface-modified portion 40 to introduce the substance to be introduced into the intra-cellular and inter-cellular microspaces or taking the intra-cellular substance out of the cell.
- the cantilever 1 which includes the surface-modified portion 40 subjected to a surface treatment near the distal end portion of the probe portion 4 , it is facilitated to adhere a substance to be introduced 53 described below to the distal end portion of the probe portion 4 , and to adhere an intra-cellular substance 54 in the intra-cellular and inter-cellular microspaces to the distal end portion of the probe portion 4 when taking the intra-cellular substance 54 out of the intra-cellular and inter-cellular microspaces.
- FIG. 10 shows a system configured using the cantilever 1 as mentioned above, for introducing the substance to be introduced into the cell or taking the intra-cellular substance out of the cell.
- this system includes the cantilever 1 including the surface-modified portion 40 , a power supply portion 14 including a first power supply V 1 and a second power supply V 2 , replacing the electrometer 12 of the first embodiment, and a switch SW for changing over between the first power supply V 1 and the second power supply V 2 .
- the conductive stage 13 is connected so as to be ground potential.
- Positive pole side of the first power supply V 1 of the power supply portion 14 is connected with one terminal of the switch SW, and negative pole side thereof with the connection line 11 from the conductive stage 13 .
- Negative pole side of the second power supply V 2 of the power supply portion 14 is connected with the other terminal of the switch SW, and positive pole side thereof with the connection line 11 from the conductive stage 13 .
- the switch SW is connected with the connection line 11 from the electrode pad 6 of the cantilever 1 .
- the switch SW is changed over to the one terminal on the first power supply V 1 side of the power supply portion 14 , the electric potential of the positive pole is applied to the cantilever 1 side.
- the switch SW is changed over to the other terminal on the second power supply V 2 side of the power supply portion 14 , an electric potential of the negative pole, having reversed polarity, is applied to the cantilever 1 side.
- the switch SW can be turned off to change over the cantilever 1 to a floating state where no electric potential is applied thereto from the first power supply V 1 or the second power supply V 2 .
- FIG. 10 shows a state where an electronegative potential is applied to the probe portion 4 of the cantilever 1 to adsorb to the probe portion the substance to be introduced 53 ;
- FIG. 11 shows a state where the probe portion 4 , placed in an electrically floating state, is being inserted into the cell 50 ;
- FIG. 12 shows state where the electropositive potential is applied to the probe portion 4 of the cantilever 1 to separate the substance to be introduced 53 in the cell 50 ;
- FIG. 13 shows a state where, only the probe portion 4 , placed in the electrically floating state, is pulled out of the cell 50 .
- the operator operates the switch SW to switch off the power supply portion 14 , as shown in FIG. 11 , so as to place the probe portion 4 of the cantilever 1 in the electrically floating state. Then, while retaining this state, the operator moves the cantilever 1 toward the cell 50 and inserts the former into the latter.
- the operator operates to change over the switch SW to the one terminal on the first power supply V 1 side, as shown in FIG. 12 .
- the operator operates the switch SW to switch off the power supply portion 14 so as to place the probe portion 4 of the cantilever 1 in the electrically floating state, as shown in FIG. 13 . Then, while retaining this state, the operator pulls the cantilever 1 out of the cell 50 , so as to leave the substance to be introduced 53 , such as a gene or protein, in the intra- and inter-cell 50 microspace.
- the substance to be introduced 53 such as a gene or protein
- FIG. 14 shows a state before the probe portion 4 of the cantilever 1 , which is remained in the electrically floating state, is inserted into the cell 50 .
- FIG. 15 shows a state where the probe portion 4 , which is remained in the electrically floating state, is inserted into the cell 50 .
- FIG. 16 shows a state where the electronegative potential is applied to the probe portion 4 of the cantilever 1 to adsorb the intra-cellular substance 54 in the cell 50 to the probe portion 4 .
- FIG. 17 shows a state where, with the electronegative potential remained applied to the probe portion 4 , the probe portion 4 is pulled out of the cell along with the intra-cellular substance 54 adsorbed to the probe portion.
- the operator now takes the intra-cellular substance 54 out of the cell 50 using a system configured as shown in FIG. 14 .
- the cell 50 including the intra-cellular substance 54 inside thereof is placed in advance on the placement surface 13 a of the conductive stage 13 .
- the operator operates the switch SW to switch off the power supply portion 14 so as to place the probe portion 4 of the cantilever 1 in the electrically floating state as shown in FIG. 14 .
- the operator operates to change over the switch SW to the other terminal on the second power supply V 2 side, as shown in FIG. 16 .
- the probe portion 4 of the cantilever 1 is applied with the negative potential from the second power supply V 2 .
- This allows electrically promoting adsorption of the substance to be introduced 53 which is positively charged, such as a gene or protein, to the vicinity of the distal end portion of the probe portion 4 .
- the operator operates to pull the cantilever 1 out of the cell 50 while leaving the probe portion 4 of the cantilever 1 applied with the electronegative potential, as shown in FIG. 17 .
- This can facilitate taking the intra-cellular substance 54 such as a gene or protein out of the cell 50 .
- the operator only needs to operate the switch SW to switch off the power supply portion 14 so as to place the probe portion 4 of the cantilever 1 in the electrically floating state.
- the probe portion 4 of the cantilever 1 may be applied with an electric potential that is reverse to that described above in the second embodiment.
- the probe portion 4 was described to be placed in the electrically floating state when inserting or pulling the probe portion 4 of the cantilever 1 into or out of the intra-cellular and inter-cellular microspaces, the insertion or pulling out may be performed while applying an electric potential to the probe portion 4 .
- the second embodiment it is possible not only to obtain the same effect as in the first embodiment, but also to introduce the substance to be introduced 53 and taking the intra-cellular substance 54 into and out of the intra- and inter-cell 50 microspaces in an easy manner by controlling electric polarities and electric potentials of the conductive stage 13 on which the cell 50 is placed and the cantilever 1 .
- the surface-modified portion 40 in the vicinity of the distal end portion of the probe portion 4 of the cantilever 1 , it becomes possible to surely introduce the target substance to be introduced 53 into the intra- and inter-cell 50 microspaces, as well as to selectively and surely take out the target intra-cellular substance 54 in the intra- and inter-cell 50 microspaces.
- FIGS. 18 to 20 relate to a third embodiment of the cantilever for measuring intra-cellular and inter-cellular microspaces of the present invention, and are illustrative views to describe a method to facilitate inserting the probe portion into the cell using a system including the cantilever.
- FIG. 18 shows a state where a resistance meter is switched on to measure the resistance between a conductive stage and the probe portion so as to detect contact between the probe portion and a cell membrane.
- FIG. 19 shows a state where a pulse generating unit is switched on to apply a pulse-like electric potential when contact between the probe portion and the cell membrane is detected.
- FIG. 20 shows a state where the pulse generating unit is switched off to stop the application of the pulse wave electric potential after the probe portion is inserted into the cell.
- FIGS. 18 to 20 components same as the cantilever 1 and those of the system of the first and second embodiments are attached with the same symbols, omitting descriptions thereof: only different parts are described.
- the cantilever 1 and a system of the third embodiment which are configured in generally the same manner as in the second embodiment, includes a resistance meter 15 and a pulse generating unit 16 , replacing the power supply portion 14 , and a switch SW 1 for changing over between the resistance meter 15 and the pulse generating unit 16 .
- this configuration is provided such that contact between the probe portion 4 and the cell membrane can be detected through a measurement result by the resistance meter 15 , and when such detection is made, the pulse generating unit 16 applies a pulse wave electric potential so as to better facilitate inserting the probe portion 4 into the cell 50 .
- the switch SW 1 is connected with the connection line 11 from the electrode pad 6 of the cantilever 1 , such that, when the switch SW 1 is changed over to the one terminal on the resistance meter 15 side, resistance between the conductive stage 13 and the probe portion 4 can be measured, while on the other hand, when changed over to the other terminal on the pulse generating unit 16 side, a pulse wave electric potential is applied between the conductive stage 13 and the probe portion 4 .
- switching off the switch SW 1 can stop the application of the pulse wave electric potential from the pulse generating unit 16 .
- FIGS. 18 to 20 specifics of the method for inserting the probe portion 4 into the cell are described.
- the operator operates to change over the switch SW 1 to one terminal on the resistance meter 15 side, as shown in FIG. 18 .
- the operator measures the resistance between the conductive stage 13 and the probe portion 4 to detect contact between the probe portion 4 and the cell 50 (cell membrane 52 ).
- the resistance meter 15 measures a predetermined resistance value between the conductive stage 13 and the probe portion 4 , which allows detecting that the probe portion 4 is in contact with the cell 50 .
- the operator then operates to change over the switch SW 1 to the other terminal on the pulse generating unit 16 side, as shown in FIG. 19 .
- the pulse generating unit 16 is switched on, causing application of a pulse wave electric potential between the conductive stage 13 and the probe portion 4 .
- the operator moves the probe portion 4 of the cantilever 1 toward the cell 50 to insert the former into the latter.
- the application of the pulse wave electric potential to the conductive probe 7 of the probe portion 4 allows easily opening the cell membrane 52 (not shown) of the cell 50 to insert the conductive probe 7 into the cell 50 .
- the operator switches off the switch SW 1 to stop the application of the pulse wave electric potential from the pulse generating unit 16 , as shown in FIG. 20 .
- the method for detecting the contact between the probe portion 4 and the cell 50 may employ a dynamic detection method to be conducted using the optical reflection film 10 of the cantilever 1 , in stead of measuring the resistance between the conductive stage 13 and the probe portion 4 of the cantilever 1 .
- the electric potential to be applied between the conductive stage 13 and the probe portion 4 of the cantilever 1 may be of either alternating current (high frequency) or direct current, and also may include both components of alternating and direct currents.
- the electric potential to be applied between the conductive stage 13 and the probe portion 4 of the cantilever 1 may be a pulse potential containing a bias component of alternating or direct current.
- the pulse generating unit 16 may be switched on to apply the pulse wave electric potential even since before causing the probe portion 4 of the cantilever 1 to contact the cell 50 .
- the third embodiment it is possible not only to obtain the same effect as in the second embodiment, but also to easily insert the probe portion 4 of the cantilever 1 into the intra- and inter-cell 50 microspaces by applying an electric stimulation to the cell membrane 52 in addition to dynamically operating the cantilever 1 when inserting the probe portion 4 of the cantilever 1 into the cell 50 .
- an electric stimulation to the cell membrane 52 in addition to dynamically operating the cantilever 1 when inserting the probe portion 4 of the cantilever 1 into the cell 50 .
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Abstract
Description
- This application claims benefit of Japanese Application No. 2007-38446 filed in Japan on Feb. 19, 2007, the contents of which are incorporated by this reference.
- 1. Field of the Invention
- The present invention relates to a cantilever for measuring intra-cellular and inter-cellular microspaces suitable for measurement or operation in intra-cellular and inter-cellular microspaces.
- 2. Related Art Statement
- In recent years of bio-field, there has been focused on elucidating or controlling cellular vital functions.
- Such elucidation or control of cellular vital functions involves essential operations such as measuring electric potential difference in intra-cellular and inter-cellular microspaces and introducing and taking a gene or the like into and out of a cell.
- A number of suggestions have been made on method or device for introducing into a cell a substance to be introduced such as a gene. Known examples of such suggestions include disclosures in Japanese Unexamined Patent Publication Nos. 2003-325161 and 2006-166884.
- The Japanese Unexamined Patent Publication No. 2003-325161 describes a suggestion on a cell operating device and a cell operating method using the cell operating device. This publication discloses a technique on a configuration which is capable of detailed real-time observation of serial dynamics changes shown on the cell during an interval from introduction to manifestation of a gene, which is applicable even for elucidating or controlling cell differentiation, and which further includes a probe of an Atomic Force Microscope (AFM) which is provided, as cell operative means capable of minimizing cellular damage during introduction of a gene or the like, with a needle-shaped matter for inserting into a cell a substance relating to gene or gene manifestation which is fixed thereto.
- However, the technique described in the Japanese Unexamined Patent Publication No. 2003-325161 has a problem in that the substance relating to gene or gene manifestation remains fixed to the needle-shaped matter, so that the gene or the like is not constantly held in the cell when the needle is pulled out, resulting in incapability of producing a gene recombinant.
- A proposal in view of such problem is made in Japanese Unexamined Patent Publication No. 2006-166884, which describes a proposal on a method to introduce a substance into a cell.
- The Japanese Unexamined Patent Publication No. 2006-166884 discloses a technique regarding a method for introducing into a cell a substance to be introduced, by binding the substance to be introduced to a needle-shaped material by means of an intermediate binding force such that the substance to be introduced is caused to remain in the cell when the needle-shaped material is punctured thereinto. This publication also discloses a technique relating to a configuration in which an atomic force microscope (AFM) is used for a method of detecting puncture of the needle-shaped material into the cell and as an introducing device for introducing the substance to be introduced into the cell.
- Specifically, in the method described in the Japanese Unexamined Patent Publication No. 2006-166884, as shown in
FIG. 21 , aprobe 101 provided to anAFM cantilever 100 is etched by a focused ion beam so as to be configured as a needle-shaped material, and further, the probe 1001 as the needle-shaped material is used to perform a puncturing operation into acell 103 and anucleus 104 in thecell 103. - The Japanese Unexamined Patent Publication No. 2006-166884 also discloses a technique related to electrostatic coupling as a method to couple the substance to be introduced to the needle-shaped material by means of an intermediate binding force. Because such electrostatic coupling is a weak coupling, the substance to be introduced can be easily separated in the cell. In electrostatic coupling, cationic polymer is generally used.
- Specifically, in the method described in the Japanese Unexamined Patent Publication No. 2006-166884, as shown in a schematic block diagram of
FIG. 22 , a thiol group is introduced into a needle-shaped material 105, which is a silicon-made AFM probe etched to have a sharpened edge, to modify succinimide active ester on a surface of needle-shaped material to immobilize polylysine followed by electrostatic coupling of plasmid phrGFP as the substance to be introduced, before introducing the needle-shaped material 105 into the cell. - However, the technique described in the Japanese Unexamined Patent Publication No. 2006-166884 has a problem in that the electrostatic coupling, which is weak, can be canceled, resulting in the substance to be introduced to be detached before introduction into the cell, thus making it impossible to introduce a desired material into the cell.
- Further, the techniques described in the Japanese Unexamined Patent Publication No. 2006-166884 suggests or discloses nothing in terms of taking a substance out of a cell and measuring intra-cellular electric potential distribution. Accordingly, there is desired a specific device that allows stable intra-cellular measurement or operation to be easily performed.
- The present invention has been made in view of the above-described problems, and an object of the present invention is to provide a cantilever for measuring intra-cellular and inter-cellular microspaces that allows stable intra-cellular measurement or operation to be easily performed, which is durable, reliable, and low cell-invasive.
- Another object of the present invention is to provide a cantilever for measuring intra-cellular and inter-cellular microspaces that allows performing, easily and stably with good reproducibility and minimum variation, nano-cell mapping, i.e., measurement in intra-cellular and inter-cellular microspaces and introducing and taking a substance to and out of the microspaces.
- Briefly, a cantilever for measuring intra-cellular and inter-cellular microspaces according to the present invention includes: a support portion; a lever portion provided to the support portion so as to protrude therefrom; and a probe portion provided near a free end of the lever portion, wherein the probe portion includes a conductive probe made of a carbon-based material, and an insulating film to coat a periphery of the conductive probe.
- Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The features and advantages of the invention may be realized and obtained by means of the instrumentalities and combinations particularly pointed out hereinafter.
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FIG. 1 is a perspective view showing an appearance configuration of a cantilever for measuring intra-cellular and inter-cellular microspaces according to a first embodiment of the present invention. -
FIG. 2 is a partially cut sectional view of a lever portion along A-A′ line of the cantilever for measuring intra-cellular and inter-cellular microspaces according to the first embodiment of the present invention ofFIG. 1 . -
FIG. 3 is a sectional view of a probe portion according to the first embodiment, in a vertical direction with respect to a projecting direction. -
FIG. 4 is a sectional view of a distal end part in the projecting direction of the probe portion according to the first embodiment. -
FIG. 5 is a sectional view of a distal end part showing a first modification example of the probe portion. -
FIG. 6 is a sectional view of a distal end part showing a second modification example of the probe portion. -
FIG. 7 is a partially cut sectional view of a probe portion and a lever portion, showing a third modification example of the probe portion. -
FIG. 8 is a pattern view showing a schematic configuration of an entire system having the cantilever of the first embodiment of the present invention. -
FIG. 9 is an enlarged view of the probe portion of the cantilever and inside of a cell in measuring intra-cellular electric potential difference in the first embodiment. -
FIG. 10 is an illustrative view showing a state where a probe portion of a cantilever for measuring intra-cellular and inter-cellular microspaces according to a second embodiment of the present invention is applied with an electronegative potential to adsorb a substance to be introduced to the probe portion. -
FIG. 11 is an illustrative view showing a state where the probe portion, which is in an electrically floating state, is being inserted into the cell in the second embodiment. -
FIG. 12 is an illustrative view showing a state where an electropositive potential is applied to the probe portion of the cantilever to separate in the cell the substance to be introduced from the probe portion in the second embodiment. -
FIG. 13 is an illustrative view showing a state where, with the probe portion in the electrically floating state, only the probe portion is pulled out of the cell in the second embodiment. -
FIG. 14 is an illustrative view showing a state before the probe portion of the cantilever in the electrically floating state is inserted into the cell in the second embodiment. -
FIG. 15 is an illustrative view showing a state where the probe portion in the electrically floating state is inserted into the cell in the second embodiment. -
FIG. 16 is an illustrative view showing a state where the electronegative potential is applied to the probe portion of the cantilever to adsorb an intra-cellular substance in the cell to the probe portion in the second embodiment. -
FIG. 17 is an illustrative view showing a state where, with the electronegative potential remained applied to the probe portion, the probe portion is pulled out of the cell along with the intra-cellular substance adsorbed to the probe portion in the second embodiment. -
FIG. 18 is an illustrative view showing a state where a resistance meter is switched on to measure resistance between a conductive stage and the probe portion so as to detect contact between the probe portion and a cell membrane, according to a third embodiment of the present invention. -
FIG. 19 is an illustrative view showing a state where a pulse generating unit is switched on to apply a pulse wave electric potential when contact between the probe portion and the cell membrane is detected in the third embodiment. -
FIG. 20 is an illustrative view showing a state where the pulse generating unit is switched off to stop the application of the pulse wave electric potential after the probe portion is inserted into the cell in the third embodiment. -
FIG. 21 is an illustrative view showing a cell puncturing operation using a needle-shaped material in a prior art. -
FIG. 22 is a schematic view showing a state where electrostatic adsorption is conducted on a polylysine-modified needle in a prior art. - Preferred embodiments of the present invention are described below referring to the drawings.
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FIGS. 1 to 7 relate to a first embodiment of a cantilever for measuring intra-cellular and inter-cellular microspaces of the present invention, whereinFIG. 1 is a perspective view showing an appearance configuration of the cantilever for measuring intra-cellular and inter-cellular microspaces according to the first embodiment;FIG. 2 is a partially cut sectional view of a lever portion along A-A′ line of the cantilever for measuring intra-cellular and inter-cellular microspaces ofFIG. 1 ;FIG. 3 is a sectional view of a probe portion according toFIG. 1 , in a vertical direction with respect to a projecting direction;FIG. 4 is a sectional view of a distal end part in the projecting direction of the probe portion ofFIG. 1 ;FIG. 5 is a sectional view of a distal end part showing a first modification example of the probe portion;FIG. 6 is a sectional view of a distal end part showing a second modification example of the probe portion; andFIG. 7 is a partially cut sectional view of a probe portion and a lever portion, showing a third modification example of the probe portion. - A cantilever for measuring intra-cellular and inter-cellular microspaces 1 (hereinafter referred to as cantilever 1) of the first embodiment, shown in
FIG. 1 , is used for measurement or operation in intra-cellular and inter-cellular microspaces. - Specifically, the
cantilever 1 includes asupport portion 2, alever portion 3 protrudingly provided to thesupport portion 2, aprobe portion 4 provided near a free end of thelever portion 3, anelectrode pad 6 provided to a proximal end portion on thesupport portion 2 side of thelever portion 3, and awiring portion 5 to electrically connect aconductive probe 7 to be described below of theprobe portion 4 and theelectrode pad 6 via inside of thelever portion 3. - The
support portion 2 is held to a cantilever holder or the like in a cell operating device not shown when performing measurement or operation in intra-cellular and inter-cellular microspaces. Onto, for example, a bottom face portion of thesupport portion 2, a proximal end side part of thelever portion 3 is supportedly fixed. Note that the manner in which thelever portion 3 is mounted to thesupport portion 2 is not specifically limited, i.e., the mounting may be in any manner. - The
lever portion 3 is configured of a material such as, for example, silicon oxide and a silicon nitride. Specifics of the configuration will be described later. - The
probe portion 4 is formed near the free end of thelever portion 3. Theprobe portion 4 is provided so as to protrude in a vertical direction with respect to a longitudinal direction of thelever portion 3. - Note that, although the first embodiment describes a configuration in which the
probe portion 4 is provided in the vertical direction with respect to thelever portion 3, no limitation is placed thereon. Theprobe portion 4 may be formed to be inclined toward any direction with respect to thelever portion 3. - Next, referring to
FIGS. 2 to 7 , there are described specific configurations of principal parts of the cantilever 1: thelever portion 3 and theprobe portion 4. - In this embodiment, the
probe portion 4 includes aconductive probe 7 which is a conductive member made of a carbon-based material, and an insulatingfilm 8 provided to coat the periphery of theconductive probe 7, as shown inFIG. 2 . -
FIG. 3 shows a sectional configuration of theprobe portion 4, in the vertical direction with respect to the projecting direction of theprobe portion 4. In other words, in the first embodiment, theprobe portion 4 is formed in, for example, a circular cylinder shape as shown inFIG. 3 . Theconductive probe 7 configuring theprobe portion 4 is also formed in, for example, a circular cylinder shape. - Note that the shapes of the
probe portion 4 and theconductive probe 7 are not limited to a circular cylinder shape, and may be formed otherwise, for example, in a columnar shape such as of elliptic cylinder or prism, or in a conic shape such as of cone or pyramid. Nevertheless, theprobe portion 4 and theconductive probe 7 are preferably formed in a circular cylinder shape in order to reduce cell damage, as described above. - The insulating
film 8 is provided to coat the periphery of theconductive probe 7 having such a shape. Accordingly, the shape of the insulatingfilm 8 may be changed to change the shape of theprobe portion 4 itself, while leaving as is the shape of theconductive probe 7. Note that, as long as the insulatingfilm 8 can ensure insulation when completely coating the periphery of theconductive probe 7, the insulatingfilm 8 is not specifically limited in shape, thickness and so on, and may be configured in any columnar or conic shape. - A
distal end surface 4 a of theprobe portion 4 is formed in, for example, a planar shape, as shown inFIGS. 2 and 4 . In thedistal end surface 4 a, adistal end surface 7 a of theconductive probe 7 is provided. Thedistal end surface 7 a is provided in a manner to be in a same plane as that of the distal end surface ofprobe portion 4. To a proximal end portion on a reverse side to thedistal end surface 7 a ofconductive probe 7, aproximal end surface 7 b is formed. - At a lower portion of the insulating
film 8, there is provided anengaging portion 8 a to engage with and be fixed to thelever portion 3. - Here, materials of the
conductive probe 7 and the insulatingfilm 8 configuring theprobe portion 4 are described. - Specifically, the
conductive probe 7 is configured of a carbon-based material, which is at least one of single crystal diamond, polycrystalline diamond, nanodiamond, diamondlike carbon, and amorphous carbon, for example. Such a carbon-based material is doped with boron, Lin, or the like, to configure theconductive probe 7. - Thus, the
conductive probe 7, which is configured using such a carbon-based material, has a high rigidity to improve durability and an oxidation-resistant characteristic, and therefore is reliable. Accordingly, theprobe portion 4 configured using theconductive probe 7, which is thus durable and reliable, allows an intra-cellular stable measurement or operation to be easily and stably performed. - The insulating
film 8 is configured of one layer of any of an oxide film, nitride film, and organic film, or by a laminated film including at least one of an oxide film, nitride film, and organic film. Such a laminated film can be formed by a commonly available method. Therefore, the insulatingfilm 8 can be easily formed to cover theconductive probe 7. - The insulating
film 8 may be formed using, instead of the laminated film, a carbon-based material including at least one of polycrystalline diamond, nanodiamond, diamondlike carbon, and amorphous carbon. Nevertheless, it is to be noted that, in the first embodiment, it is preferrable to use a carbon-based material to form the insulatingfilm 8, in view of adaptability to living body and capability in surface treatment to improve adhesion of the substance to be introduced (surface treatment to form a surface-modified portion to be described below). - Thus, by covering the periphery of the
conductive probe 7 with the insulatingfilm 8, there is provided a configuration preferable to precisely measure electric potential in intra-cellular and inter-cellular microspaces, as described below. This configuration is also preferable to allow easily introducing the substance to be introduced to a desired position in the intra-cellular and inter-cellular microspaces and taking a substance out from a desired position. - Note that it is preferable to form the
probe portion 4 such that a distal end portion thereof has a diameter not greater than 1 μm, preferably not greater than 400 nm. It is further preferable to form theprobe portion 4 to have a length not less than 2 μm, preferably not less than 5 μm, and more preferably not less than 10 μm. - The
conductive probe 7 of theprobe portion 4 having such a configuration is electrically connected to thewiring portion 5 through contact of theproximal end surface 7 b with thewiring portion 5 in thelever portion 3, as shown inFIG. 2 . - Here, configuration of the
lever portion 3 is described. As shown inFIG. 2 , thelever portion 3 engages with the engagingportion 8 a of the insulatingfilm 8 of theprobe portion 4 to fix theprobe portion 4 itself. Further, thelever portion 3 includes aprotection film 9 to protect thewiring portion 5 in the lever portion, thewiring portion 5 arranged next to theprotection film 9, a levermain body portion 3A to sandwichingly hold thewiring portion 5 with theprotection film 9, and anoptical reflection film 10 which is coating treated on an outer surface of the levermain body portion 3A. - The
protection film 9 is configured of an insulative member and provided to cover the entire surface on one side of thelever portion 3 except the part corresponding to theelectrode pad 6. Though not illustrated, theprotection film 9 is also provided over the entire surface of thesupport portion 2 on a side oriented in the same direction as the one side of thelever portion 3. - In the vicinity of the
probe portion 4 on theprotection film 9, anaperture 9 a is formed to expose the engagingportion 8 a of the insulatingfilm 8. Note that, although the first embodiment describes a configuration in which theaperture 9 a is provided in the vicinity of theprobe portion 4 on theprotection film 9, no limitation is placed thereon. Theprotection film 9 may be formed to entirely cover the circumference of the insulatingfilm 8 of theprobe portion 4 without providing theaperture 9 a. This permits firmly fixing theprobe portion 4 to thelever portion 3. - Though not illustrated, the
protection film 9 has an aperture at a part corresponding to theelectrode pad 6 above thesupport portion 2, for electric connection between theinternal wiring portion 5 and theelectrode pad 6. - Note that, although the first embodiment describes a configuration in which the
protection film 9 is provided, no limitation is placed thereon, i.e., there may be a configuration without theprotection film 9 if so needed. - The
wiring portion 5 is electrically connected to theconductive probe 7 through contact with theproximal end surface 7 b of theconductive probe 7. A proximal end portion of thewiring portion 5 on thesupport portion 2 side is electrically connected to theelectrode pad 6 for electrical connection to an external device such as an electrometer, for example. - Note that a preferable material for the
wiring portion 5 is platinum or iridium, which facilitates accumulation and growth of theconductive probe 7 of a carbon-based material. Yet, as long as adhesion with theconductive probe 7 can be assured, thewiring portion 5 may be configured of a material of a metal other than platinum or iridium. Thewiring portion 5 may also be otherwise configured using a semiconductor material such as polysilicon. - In addition, a conductive material such as titanium or chrome may be formed between the
wiring portion 5 and the levermain body portion 3A to improve adhesion therebetween. Likewise, a conductive material such as titanium or chrome may be formed between thewiring portion 5 and theprotection film 9 to improve adhesion therebetween. - Although the
electrode pad 6 may be formed with thewiring portion 5 itself, an electrode layer such as of gold, copper or aluminum may be formed on the pad to facilitate electric connection with an external terminal through solder connection, wire bonding, or the like. In this case, a conductive material such as titanium, chrome or the like may be formed between theelectrode pad 6 and the electrode layer in order to improve adhesion between theelectrode pad 6 and the electrode layer. - The
optical reflection film 10 provided on the outer surface of the levermain body portion 3A is formed by being subjected to coating treatment with aluminum or gold. Note that theoptical reflection film 10 may be configured not only limitedly by such a coating treatment but using another method. Theoptical reflection film 10 may also be configured provided with a multilayer film. - In a system configuration comprised, for example, of the
cantilever 1 and external devices such as an external optical system apparatus and a personal computer, theoptical reflection film 10 serves to detect reflection angle or reflection amount of lights irradiated from the external optical system apparatus. In such a system, the personal computer analyzes a detection result by theoptical reflection film 10 to enable dynamically detecting whether or not theprobe portion 4 is in contact with a cell membrane, which is effective for performing measurement or operation in the intra-cellular and inter-cellular microspaces. - Thus, with the above-described configuration, the
cantilever 1 can be low cell-invasive, allowing various measurements or operations in the intra-cellular and inter-cellular microspaces to be performed easily and stably with good reproducibility and minimum variation. - In addition, since the
protection film 9 covers the entire surface on one side of thelever portion 3 except the part corresponding to theelectrode pad 6 above thesupport portion 2, it is possible to easily perform stable measurement and operation in the intra-cellular and inter-cellular microspaces, in various environments such as one in which the cell is soaked in a water-system solution such as cultured liquid or isotonic sodium chloride solution. - Moreover, the use of the durable and reliable
conductive probe 7 to configure theprobe portion 4 can make thecantilever 1 to be highly durable and highly reliable. - Note that, in the first embodiment, the
probe portion 4 may be configured such that thedistal end surface 4 a configures adistal end portion 4 a 1 in a shape with adistal end portion 7 a 1 of theconductive probe 7 protruding from the insulatingfilm 8, as shown with aprobe portion 4A in a first modification example ofFIG. 5 , instead of providing thedistal end surface 7 a of theconductive probe 7 in the same plane as that of the insulatingfilm 8. - In this case, the
distal end portion 4 a 1 of theprobe portion 4A and thedistal end portion 7 a 1 of theconductive probe 7 are configured in a cone shape such as, for example, a circular cone or pyramid. - The
probe portion 4 may also be configured such that, in the case where, as shown by aprobe portion 4B in a second modification example ofFIG. 6 , adistal end portion 4 a 2 is configured in a shape such that adistal end portion 7 a 2 of theconductive probe 7 protrudes from the insulatingfilm 8, thedistal end portion 7 a 2 of theconductive probe 7 is formed in an arc shape such as a hemispheric or ellipse sphere shape, while also forming a distal end portion of the insulatingfilm 8 in the periphery of thedistal end portion 7 a 2 in an arc shape such as hemispheric or ellipse sphere shape. In other words, forming thedistal end portion 4 a 2 of theprobe portion 4B in arc shape leads to an effect of allowing reducing cell damage. - Furthermore, the
probe portion 4 may be configured such that, in the case where, like theprobe portion 4C shown in the third modification example ofFIG. 7 , thedistal end portion 4 a 3 is configured in a shape such that thedistal end surface 7 a 3 of theconductive probe 7 protrudes from the insulatingfilm 8, a protrudingportion 7 c of theconductive probe 7 is provided, and thedistal end surface 7 a 3 of the protrudingportion 7 c and the distal end surface of the insulatingfilm 8 are provided at different positions, not in a same plane. In other words, the provision of the protrudingportion 7 c of theconductive probe 7 increases the exposing surface area of theconductive probe 7, which is effective to improve the adhesion capability of the substance to be introduced and the intra-cellular substance. - Next, referring to
FIGS. 8 and 9 , there are described configuration example and action in a system configuration to actually measure an electric potential difference of intra-cellular and inter-cellular microspaces using thecantilever 1 having the above configuration. -
FIG. 8 is a pattern view showing a schematic configuration of an entire system having thecantilever 1 of the first embodiment of the present invention.FIG. 9 is an enlarged view of theprobe portion 4 of thecantilever 1 and the inside of the cell in measuring intra-cellular electric potential difference. - As shown in
FIG. 8 , the system includes: acantilever 1 shown inFIG. 1 ; aconnection line 11, which is electrically connected to theelectrode pad 6 of thecantilever 1; anelectrometer 12 which is electrically connected to theconnection line 11 and can measure an electric potential difference between a distal end portion of theprobe portion 4 of thecantilever 1 and a conductive stage to be described below; and aconductive stage 13 which is electrically connected to theconnection line 11 from theelectrometer 12 and configured of a conductive member having aplacement surface 13 a on which acell 50 is placed. - With such a configuration, the
cantilever 1 and theconductive stage 13 are electrically connected via theconnection line 11 and theelectrometer 12. Note that, for the sake of simplicity, descriptions will be omitted of the cantilever holder for holding thesupport portion 2 of thecantilever 1 and other external devices such as a personal computer: configuration and action of only principal parts are described. - It is now supposed that an operator measures an intra-cellular electric potential difference using the system configured as shown in
FIG. 8 . In this case, the operator places thecell 50 on theplacement surface 13 a of theconductive stage 13. - With the power of the system turned on, the operator moves the
probe portion 4 of thecantilever 1 toward thecell 50 as shown inFIG. 9 . Note that thecell 50 has anintracellular nucleus 51 and acell membrane 52 as is well-known. - Then, the
distal end surface 4 a of theprobe portion 4 ofcantilever 1 contacts thecell membrane 52 of thecell 50. - In the first embodiment, the contact of the
probe portion 4 of thecantilever 1 with thecell membrane 52 may be detected either electrically by theelectrometer 12 or dynamically by means of a reflected light from theoptical reflection film 10, or the like, as described above. - Thereafter, the operator inserts the
probe portion 4 of thecantilever 1 into thecell 50, penetrating thecell membrane 52, as shown inFIG. 9 . - Next, the operator measures an electric potential difference between the
distal end surface 4 a of theprobe portion 4 in thecell 50 and theconductive stage 13, using theelectrometer 12. Measurement result at this time is recorded by a personal computer not shown. - In the first embodiment, the operator here re-stabs the
probe portion 4 into thecell 50 in the method mentioned above, measuring an electric potential difference for each re-stabbing of the probe portion. This allows obtaining a measurement result of electric potential distribution at a desired position in thecell 50. - For example, the number of times of the re-stabbing preferably ranges from about dozens of times to 100 times, but no limitation is placed thereon, i.e., the re-stabbing may be performed by a number of times as needed. Even if the
probe portion 4 is re-stabbed by dozens of times, thecell 50 will not die. The position of the re-stabbing into thecell 50 may be set as desired as needed. - With the use of the above-mentioned system, by thus re-stabbing the
probe portion 4 into thecell 50 several times to measure respective electric potential differences, it is enabled to measure a minute electric potential distribution in a desired position in thecell 50, and therefore to perform nano-cell mapping by the electric potential distribution. - Note that the nano-cell mapping maps a physical parameter such as of intra-cellular and inter-cellular substances, electric potentials or the like present in an organization/trachea, or maps subtle distribution of strength and weakness of a specific biological function, and is not limited to use for the electric potential distribution.
- Also, since the
probe portion 4 and thelever portion 3 are covered by the insulatingfilm 8 and theprotection film 9, respectively, it is possible to measure the electric potential difference and the electric potential distribution even if thecell 50 is soaked in a water system solution such as culture media or isotonic sodium chloride solution. - Thus, according to the first embodiment, it is enabled to easily and stably measure an electric potential difference between the inside of the cell and the cell membrane, as well as to easily and stably measure, in real-time, intra-cellular or inter-cellular electric potential distribution. It is therefore rendered possible to realize a cantilever (the cantilever 1) for measuring intra-cellular and inter-cellular microspaces, which is durable, reliable, and low cell-invasive, enabling stable intra-cellular measurement or operation to be easily performed.
- Furthermore, by using a system including the
cantilever 1 to measure an intra-cellular electric potential distribution, it is made possible to perform nano-cell mapping of intra-cellular and inter-cellular electric potential. -
FIGS. 10 to 17 relate to a second embodiment of the cantilever for measuring intra-cellular and inter-cellularmicrospaces of the present invention.FIGS. 10 to 13 are illustrative views to describe a method to introduce into the cell the substance to be introduced using a system including the cantilever.FIGS. 14 to 17 are illustrative views to describe a method to take the intra-cellular substance out of the cell using a system including the cantilever. - Note that, in
FIGS. 10 to 17 , components same as thecantilever 1 and those of the system of the first embodiment are attached with the same symbols, omitting descriptions thereof: only different parts are described. - In the case of the
cantilever 1 of the second embodiment, which is configured in generally the same manner as thecantilever 1 of the first embodiment, at least distal end part of theconductive probe 7 has a surface-modifiedportion 40 subjected to a surface treatment to facilitate adhesion of the intra-cellular substance and the substance to be introduced into the cell. - The surface-modified
portion 40 is to be formed by being subjected to a surface treatment using any one of hydrogen, oxygen, fluorine, amino acid, silane, organic molecules, or biological molecules. - Note that the surface-modified
portion 40 may be formed on at least one of, for example, thedistal end surface 7 a or an exposed portion of theconductive probe 7 and the distal end surface or an exposed portion of the insulatingfilm 8 forming thedistal end surface 4 a. - Formation of the surface-modified
portion 40 is not limited to on the distal end portion of theprobe portion 4, but may be on a range from the distal end portion to circumference of theprobe portion 4, subjected to a surface treatment using the above-mentioned material. - Further, the method to form the surface-modified
portion 40 is not limited to surface treatment, but any method including physical combination may be used to form the surface-modified portion. - Furthermore, the surface-modified
portion 40 is not necessarily a requirement, but as described below, it is effective to form the surface-modifiedportion 40 to introduce the substance to be introduced into the intra-cellular and inter-cellular microspaces or taking the intra-cellular substance out of the cell. - Therefore, with the
cantilever 1 thus configured, which includes the surface-modifiedportion 40 subjected to a surface treatment near the distal end portion of theprobe portion 4, it is facilitated to adhere a substance to be introduced 53 described below to the distal end portion of theprobe portion 4, and to adhere anintra-cellular substance 54 in the intra-cellular and inter-cellular microspaces to the distal end portion of theprobe portion 4 when taking theintra-cellular substance 54 out of the intra-cellular and inter-cellular microspaces. -
FIG. 10 shows a system configured using thecantilever 1 as mentioned above, for introducing the substance to be introduced into the cell or taking the intra-cellular substance out of the cell. - As shown in
FIG. 10 , this system includes thecantilever 1 including the surface-modifiedportion 40, apower supply portion 14 including a first power supply V1 and a second power supply V2, replacing theelectrometer 12 of the first embodiment, and a switch SW for changing over between the first power supply V1 and the second power supply V2. In this system, theconductive stage 13 is connected so as to be ground potential. - Positive pole side of the first power supply V1 of the
power supply portion 14 is connected with one terminal of the switch SW, and negative pole side thereof with theconnection line 11 from theconductive stage 13. - Negative pole side of the second power supply V2 of the
power supply portion 14 is connected with the other terminal of the switch SW, and positive pole side thereof with theconnection line 11 from theconductive stage 13. - The switch SW is connected with the
connection line 11 from theelectrode pad 6 of thecantilever 1. When the switch SW is changed over to the one terminal on the first power supply V1 side of thepower supply portion 14, the electric potential of the positive pole is applied to thecantilever 1 side. On the other hand, when the switch SW is changed over to the other terminal on the second power supply V2 side of thepower supply portion 14, an electric potential of the negative pole, having reversed polarity, is applied to thecantilever 1 side. - Note that the switch SW can be turned off to change over the
cantilever 1 to a floating state where no electric potential is applied thereto from the first power supply V1 or the second power supply V2. - Next, there are described a method to introduce the substance to be introduced into the cell, and a method to take the intra-cellular substance out of the cell, using a system including the
cantilever 1 configured as mentioned above. - First, the method to introduce into the cell the substance to be introduced is described referring to
FIGS. 10 to 14 . - Note that
FIG. 10 shows a state where an electronegative potential is applied to theprobe portion 4 of thecantilever 1 to adsorb to the probe portion the substance to be introduced 53;FIG. 11 shows a state where theprobe portion 4, placed in an electrically floating state, is being inserted into thecell 50;FIG. 12 shows state where the electropositive potential is applied to theprobe portion 4 of thecantilever 1 to separate the substance to be introduced 53 in thecell 50; andFIG. 13 shows a state where, only theprobe portion 4, placed in the electrically floating state, is pulled out of thecell 50. - It is supposed that an operator now introduces the substance to be introduced 53 into the
cell 50 using the system configured as shown inFIG. 10 . In this case, the operator places in advance thecell 50 on theplacement surface 13 a of theconductive stage 13. - Then, with the power supply of the system turned on, the operator operates to change over the switch SW to the other terminal on the second power supply V2 side, as shown in
FIG. 10 . - This results in the
probe portion 4 of thecantilever 1 to be applied with a negative potential from the second power supply V2. This allows electrically promoting the adsorption of the substance to be introduced 53 which is positively charged, such as a gene or protein, to the vicinity of the distal end portion of theprobe portion 4. - Thereafter, the operator operates the switch SW to switch off the
power supply portion 14, as shown inFIG. 11 , so as to place theprobe portion 4 of thecantilever 1 in the electrically floating state. Then, while retaining this state, the operator moves thecantilever 1 toward thecell 50 and inserts the former into the latter. - Next, after confirming that the
probe portion 4 of thecantilever 1 has reached the desired position in the cell, the operator operates to change over the switch SW to the one terminal on the first power supply V1 side, as shown inFIG. 12 . - This results in the
probe portion 4 of thecantilever 1 to be applied with the electropositive potential from the first power supply V2. In other words, by changing over the polarity of the electric potential to be applied to theprobe portion 4 so that the electropositive potential is applied thereto, it is enabled to promote separation of the substance to be introduced 53 adhering to the distal end portion of theprobe portion 4 in the intra- andinter-cell 50 microspaces. - Thereafter, the operator operates the switch SW to switch off the
power supply portion 14 so as to place theprobe portion 4 of thecantilever 1 in the electrically floating state, as shown inFIG. 13 . Then, while retaining this state, the operator pulls thecantilever 1 out of thecell 50, so as to leave the substance to be introduced 53, such as a gene or protein, in the intra- andinter-cell 50 microspace. - Next, a method to take the intra-cellular substance out of the cell is described referring to
FIGS. 14 to 17 . -
FIG. 14 shows a state before theprobe portion 4 of thecantilever 1, which is remained in the electrically floating state, is inserted into thecell 50.FIG. 15 shows a state where theprobe portion 4, which is remained in the electrically floating state, is inserted into thecell 50.FIG. 16 shows a state where the electronegative potential is applied to theprobe portion 4 of thecantilever 1 to adsorb theintra-cellular substance 54 in thecell 50 to theprobe portion 4.FIG. 17 shows a state where, with the electronegative potential remained applied to theprobe portion 4, theprobe portion 4 is pulled out of the cell along with theintra-cellular substance 54 adsorbed to the probe portion. - It supposed that the operator now takes the
intra-cellular substance 54 out of thecell 50 using a system configured as shown inFIG. 14 . Note that thecell 50 including theintra-cellular substance 54 inside thereof is placed in advance on theplacement surface 13 a of theconductive stage 13. - Then, with the power supply of the system turned on, the operator operates the switch SW to switch off the
power supply portion 14 so as to place theprobe portion 4 of thecantilever 1 in the electrically floating state as shown inFIG. 14 . - While retaining this floating state, the operator moves the
cantilever 1 toward thecell 50 to insert the former into the latter, as shown inFIG. 15 . - Next, after confirming that the
probe portion 4 of thecantilever 1 has reached a desired position in thecell 50, specifically, a position preferable to take out theintra-cellular substance 54, the operator operates to change over the switch SW to the other terminal on the second power supply V2 side, as shown inFIG. 16 . - As a result, the
probe portion 4 of thecantilever 1 is applied with the negative potential from the second power supply V2. This allows electrically promoting adsorption of the substance to be introduced 53 which is positively charged, such as a gene or protein, to the vicinity of the distal end portion of theprobe portion 4. - Thereafter, after confirming that the
intra-cellular substance 54 is adsorbed to theprobe portion 4, the operator operates to pull thecantilever 1 out of thecell 50 while leaving theprobe portion 4 of thecantilever 1 applied with the electronegative potential, as shown inFIG. 17 . This can facilitate taking theintra-cellular substance 54 such as a gene or protein out of thecell 50. - Note that, to separate the taken out
intra-cellular substance 54 from theprobe portion 4, the operator only needs to operate the switch SW to switch off thepower supply portion 14 so as to place theprobe portion 4 of thecantilever 1 in the electrically floating state. - By the afore-mentioned method, it is made possible to easily and stably introduce and take a gene, protein or the like into and out of the intra-cellular and inter-cellular microspaces.
- Note that, in the second embodiment, the
probe portion 4 of thecantilever 1 may be applied with an electric potential that is reverse to that described above in the second embodiment. - Furthermore, although the
probe portion 4 was described to be placed in the electrically floating state when inserting or pulling theprobe portion 4 of thecantilever 1 into or out of the intra-cellular and inter-cellular microspaces, the insertion or pulling out may be performed while applying an electric potential to theprobe portion 4. - Thus, according to the second embodiment, it is possible not only to obtain the same effect as in the first embodiment, but also to introduce the substance to be introduced 53 and taking the
intra-cellular substance 54 into and out of the intra- andinter-cell 50 microspaces in an easy manner by controlling electric polarities and electric potentials of theconductive stage 13 on which thecell 50 is placed and thecantilever 1. - Also, in this case, by forming the surface-modified
portion 40 in the vicinity of the distal end portion of theprobe portion 4 of thecantilever 1, it becomes possible to surely introduce the target substance to be introduced 53 into the intra- andinter-cell 50 microspaces, as well as to selectively and surely take out the targetintra-cellular substance 54 in the intra- andinter-cell 50 microspaces. -
FIGS. 18 to 20 relate to a third embodiment of the cantilever for measuring intra-cellular and inter-cellular microspaces of the present invention, and are illustrative views to describe a method to facilitate inserting the probe portion into the cell using a system including the cantilever.FIG. 18 shows a state where a resistance meter is switched on to measure the resistance between a conductive stage and the probe portion so as to detect contact between the probe portion and a cell membrane.FIG. 19 shows a state where a pulse generating unit is switched on to apply a pulse-like electric potential when contact between the probe portion and the cell membrane is detected.FIG. 20 shows a state where the pulse generating unit is switched off to stop the application of the pulse wave electric potential after the probe portion is inserted into the cell. - Note that, in
FIGS. 18 to 20 , components same as thecantilever 1 and those of the system of the first and second embodiments are attached with the same symbols, omitting descriptions thereof: only different parts are described. - The
cantilever 1 and a system of the third embodiment, which are configured in generally the same manner as in the second embodiment, includes aresistance meter 15 and apulse generating unit 16, replacing thepower supply portion 14, and a switch SW1 for changing over between theresistance meter 15 and thepulse generating unit 16. - In other words, this configuration is provided such that contact between the
probe portion 4 and the cell membrane can be detected through a measurement result by theresistance meter 15, and when such detection is made, thepulse generating unit 16 applies a pulse wave electric potential so as to better facilitate inserting theprobe portion 4 into thecell 50. - Note that the switch SW1 is connected with the
connection line 11 from theelectrode pad 6 of thecantilever 1, such that, when the switch SW1 is changed over to the one terminal on theresistance meter 15 side, resistance between theconductive stage 13 and theprobe portion 4 can be measured, while on the other hand, when changed over to the other terminal on thepulse generating unit 16 side, a pulse wave electric potential is applied between theconductive stage 13 and theprobe portion 4. - Also note that switching off the switch SW1 can stop the application of the pulse wave electric potential from the
pulse generating unit 16. - Now, referring to
FIGS. 18 to 20 , specifics of the method for inserting theprobe portion 4 into the cell are described. - It is supposed that the operator now inserts the
probe portion 4 of thecantilever 1 into thecell 50 using a system configured as shown inFIG. 18 . The operator places in advance thecell 50 on theplacement surface 13 a of theconductive stage 13. - Then, with the power supply of the system turned on, the operator operates to change over the switch SW1 to one terminal on the
resistance meter 15 side, as shown inFIG. 18 . With resultant electrical connection between theconductive stage 13 and theprobe portion 4 via theresistance meter 15, the operator measures the resistance between theconductive stage 13 and theprobe portion 4 to detect contact between theprobe portion 4 and the cell 50 (cell membrane 52). - Here, it is supposed that the
probe portion 4 is brought into contact with thecell 50 by the operator's operation as shown inFIG. 19 . In this case, theresistance meter 15 measures a predetermined resistance value between theconductive stage 13 and theprobe portion 4, which allows detecting that theprobe portion 4 is in contact with thecell 50. - If the
resistance meter 15 detects that theprobe portion 4 is in contact with thecell 50, the operator then operates to change over the switch SW1 to the other terminal on thepulse generating unit 16 side, as shown inFIG. 19 . - As a result, the
pulse generating unit 16 is switched on, causing application of a pulse wave electric potential between theconductive stage 13 and theprobe portion 4. - With the pulse wave electric potential being applied, the operator moves the
probe portion 4 of thecantilever 1 toward thecell 50 to insert the former into the latter. At this time, the application of the pulse wave electric potential to theconductive probe 7 of theprobe portion 4 allows easily opening the cell membrane 52 (not shown) of thecell 50 to insert theconductive probe 7 into thecell 50. - Then, after confirming that the
probe portion 4 of thecantilever 1 has reached the desired position in thecell 50, the operator switches off the switch SW1 to stop the application of the pulse wave electric potential from thepulse generating unit 16, as shown inFIG. 20 . - Note that, in the third embodiment, the method for detecting the contact between the
probe portion 4 and thecell 50 may employ a dynamic detection method to be conducted using theoptical reflection film 10 of thecantilever 1, in stead of measuring the resistance between theconductive stage 13 and theprobe portion 4 of thecantilever 1. - Further, the electric potential to be applied between the
conductive stage 13 and theprobe portion 4 of thecantilever 1 may be of either alternating current (high frequency) or direct current, and also may include both components of alternating and direct currents. Moreover, the electric potential to be applied between theconductive stage 13 and theprobe portion 4 of thecantilever 1 may be a pulse potential containing a bias component of alternating or direct current. - Moreover, the
pulse generating unit 16 may be switched on to apply the pulse wave electric potential even since before causing theprobe portion 4 of thecantilever 1 to contact thecell 50. - Therefore, according to the third embodiment, it is possible not only to obtain the same effect as in the second embodiment, but also to easily insert the
probe portion 4 of thecantilever 1 into the intra- andinter-cell 50 microspaces by applying an electric stimulation to thecell membrane 52 in addition to dynamically operating thecantilever 1 when inserting theprobe portion 4 of thecantilever 1 into thecell 50. As a result, it is made possible to easily insert theprobe portion 4 into thecell 50 even if the distal end portion of theprobe portion 4 of the cantilever 1A has a relatively large diameter. - The present invention is not limited to the above-mentioned embodiments, but may be modified in various ways without departing from the spirit of the present invention.
- Furthermore, an embodiment configured of appropriately combining parts of the above-mentioned embodiments also belongs to the present invention.
- In this invention, it is apparent that working modes different in a wide range can be formed on this basis of this invention without departing from the spirit and scope of the invention. This invention is not restricted by any specific embodiment except being limited by the appended claims.
Claims (10)
Priority Applications (1)
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US12/902,544 US8349603B2 (en) | 2007-02-19 | 2010-10-12 | Cantilever for measuring intra-cellular and inter-cellular microspaces |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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JP2007-038446 | 2007-02-19 | ||
JP2007038446A JP4798454B2 (en) | 2007-02-19 | 2007-02-19 | Cantilever system for microspace measurement in and between cells |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
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US12/902,544 Continuation US8349603B2 (en) | 2007-02-19 | 2010-10-12 | Cantilever for measuring intra-cellular and inter-cellular microspaces |
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US20080199945A1 true US20080199945A1 (en) | 2008-08-21 |
Family
ID=39707013
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US11/943,089 Abandoned US20080199945A1 (en) | 2007-02-19 | 2007-11-20 | Cantilever for measuring intra-cellular and inter-cellular microspaces |
US12/902,544 Active 2028-05-05 US8349603B2 (en) | 2007-02-19 | 2010-10-12 | Cantilever for measuring intra-cellular and inter-cellular microspaces |
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US12/902,544 Active 2028-05-05 US8349603B2 (en) | 2007-02-19 | 2010-10-12 | Cantilever for measuring intra-cellular and inter-cellular microspaces |
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US (2) | US20080199945A1 (en) |
JP (1) | JP4798454B2 (en) |
Cited By (3)
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US20110256577A1 (en) * | 2008-11-05 | 2011-10-20 | Fujirebio Inc. | Method for sensing a biochemical and/or biomechanical process of a biological material and method for analyzing biological materials |
EP2835654A1 (en) * | 2013-08-09 | 2015-02-11 | Université de Genève | Insulator coated conductive probe and method of production thereof |
CN110108905A (en) * | 2019-05-22 | 2019-08-09 | 长春理工大学 | A kind of nervous cell membrane potential and neuron membrane repair behavioral value method and device |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2017159062A1 (en) * | 2016-03-17 | 2017-09-21 | 国立大学法人名古屋工業大学 | Cantilever and method for manufacturing cantilever |
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JP2006058255A (en) * | 2004-08-24 | 2006-03-02 | Hitachi Ltd | Small-sized high-speed sample screening apparatus |
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JP4517074B2 (en) * | 2005-03-09 | 2010-08-04 | 独立行政法人産業技術総合研究所 | Detection method of live cell protein by immunodynamic measurement |
JP5172331B2 (en) * | 2005-03-31 | 2013-03-27 | 独立行政法人科学技術振興機構 | Cantilever for scanning probe microscope and scanning probe microscope having the same |
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- 2007-11-20 US US11/943,089 patent/US20080199945A1/en not_active Abandoned
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US5838005A (en) * | 1995-05-11 | 1998-11-17 | The Regents Of The University Of California | Use of focused ion and electron beams for fabricating a sensor on a probe tip used for scanning multiprobe microscopy and the like |
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WO2015018674A1 (en) * | 2013-08-09 | 2015-02-12 | Université De Genève | Insulator coated conductive probe and method of production thereof |
CN110108905A (en) * | 2019-05-22 | 2019-08-09 | 长春理工大学 | A kind of nervous cell membrane potential and neuron membrane repair behavioral value method and device |
Also Published As
Publication number | Publication date |
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JP4798454B2 (en) | 2011-10-19 |
JP2008199936A (en) | 2008-09-04 |
US8349603B2 (en) | 2013-01-08 |
US20110027872A1 (en) | 2011-02-03 |
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