JP2012100652A - Apparatus for destruction of cell membrane - Google Patents

Abstract

PROBLEM TO BE SOLVED: To extract cell contents, while controlling their damage.SOLUTION: The apparatus includes an energizing part 6, in which a plate electrode 2A and a needle electrode 3A, the latter of which has a smaller surface area than the former, are arranged to face each other in the solution containing a predetermined concentration of an electrolyte and a predetermined concentration of cells. In the energizing part 6, an electric current is generated by the movement of electrolyte ions made by ionizing the electrolyte in the solution between the plate electrode 2A and the needle electrode 3A, and the area between the plate electrode 2A and the needle electrode 3A becomes energized with an electric current.

Description

  The present invention relates to a cell membrane disruption apparatus that destroys cell membranes of cells contained in a solution.

  Conventionally, high voltage pulses, underwater discharge, or shock waves generated in water have been used as a technology to destroy (sterilize) cell membranes of cells contained in solutions using electrical methods without using chemicals or ultrasonic waves. There is something to use.

  Moreover, as a technique for destroying a cell membrane using an electric technique and extracting a biological substance (content) in a cell, there is a method for extracting a biological substance disclosed in Patent Document 1.

  In this method, in order to extract the biological material in the cell, an electrode is provided outside the chamber having a volume of about μl (micro liter), and an alternating electric field of about 1 V (volt) to 100 V is applied for a relatively long time. The sample is continuously applied.

  In addition, as a technique for destroying cell membranes of cells using an electric technique, there is a flow path substrate disclosed in Patent Document 2.

  In this flow path substrate, a part of the flow path is locally a narrowed portion narrower than the size of one cell, and the cell is supplemented by the narrowed portion and a cell disruption voltage is applied.

International Publication No. 2005/083078 (International 09/09 International Publication) JP 2008-259491 A (released on October 30, 2008) JP2007-174901 (released July 12, 2007)

  However, in the technology using the high voltage pulse, the underwater discharge, or the shock wave generated in the water, the contents in the cell are damaged due to the application of the high voltage or the physical shock caused by the discharge or the shock wave. There was a problem.

  In the method disclosed in Patent Document 1, since only a solution corresponding to the volume of a small chamber having a volume of about 1 μl can be processed by one voltage application process, a large amount of solution cannot be processed quickly in one chamber. There was a problem. In addition, since an AC electric field of about 1 V to 100 V is continuously applied to the sample for a relatively long time, the contents in the cell may be damaged.

  Furthermore, in the flow path substrate disclosed in Patent Document 2, each cell is supplemented and destroyed by a constriction narrower than the size of one cell, so that the contents in the cell can be quickly removed. However, there was a problem that extraction could not be performed efficiently.

  The present invention has been made in view of the above-described conventional problems, and an object of the present invention is to provide a cell membrane disruptor capable of extracting contents in cells while suppressing damage.

  In order to solve the above problems, the cell membrane disruption apparatus of the present invention comprises an anode disposed opposite to each other in a solution containing an electrolyte having a predetermined concentration and cells having a predetermined concentration, and more than the anode. A cathode that is a needle-like electrode having a small surface area, and in the solution existing between the anode and the cathode, an electric current is generated by movement of electrolyte ions ionized by the electrolyte, and the anode and the cathode are energized. It is characterized by comprising energization means for making a state.

  According to the above configuration, the energization means generates a current (non-Faraday current) due to the migration of the electrolyte ions ionized by the electrolyte in the solution existing between the anode and the cathode, and instantaneously (between the anode and the cathode). B) Energized.

  Here, the time during which the non-Faraday current flows is the time from the start of the movement of the electrolyte ions to the formation of the electric double layer around the anode and the cathode, and may be about 10 ms (milliseconds). Are known. Therefore, at this time, a local potential difference (voltage) is instantaneously generated everywhere in the solution existing between the anode and the cathode (strictly, where the flow of electrolyte ions exists).

  Further, if the anode and the cathode are made of the same constituent material, the elution of the anode and the cathode can be suppressed, and the solution between the anode and the cathode is electrically intermediate after the non-Faraday current flows. Thus, an electrolyte bulk with little potential difference is formed.

  As a result, cells existing everywhere in the solution existing between the anode and the cathode are exposed to the instantaneous voltage only for about 10 ms.

  In addition, if the instantaneous voltage is always about 1 V or more during the time when the non-Faraday current flows, the cell membrane of the cell is irreversibly destroyed. For this reason, it is possible to kill (sterilize) a microorganism composed of a single cell or a plurality of cells, that is, a single cell microorganism or a multicellular microorganism. This also makes it possible to extract the contents in the cell.

  Therefore, according to the above configuration, since the cells are not exposed to voltage for a long time as compared with the method of Patent Document 1, the cell membrane is irreversibly while suppressing damage to the contents in the cells. Can be disrupted and the contents in the cell can be extracted.

  As described above, the contents in the cell can be extracted while suppressing the damage.

  In the above configuration, the cathode is a needle electrode having a smaller surface area than the anode. Thereby, even if the magnitude | size of the voltage applied between an anode and a cathode is somewhat small, a solution can be energized over a wide range. Therefore, the cell membrane of the cell can be destroyed quickly and efficiently. This makes it possible to kill (sterilize) single-cell microorganisms and multi-cellular microorganisms quickly and efficiently. In addition, the contents in the cell can be extracted quickly and efficiently.

  As described above, according to the cell membrane disruption apparatus of the present invention, cells in a solution or a microorganism composed of a single cell or a plurality of cells are quickly and efficiently killed, and the contents in the cells are suppressed from being damaged. It can be extracted quickly and efficiently.

  In addition, in order to solve the above-described problem, the cell membrane disruption apparatus of the present invention includes an anode, a cathode, and a cathode, which are disposed to face each other in a solution containing an electrolyte having a predetermined concentration and cells having a predetermined concentration. And a current-carrying means for generating a current due to movement of electrolyte ions ionized by the electrolyte in a solution existing between the anode and the cathode to bring the anode and the cathode into a current-carrying state. And

  According to the said structure, an electricity supply means generates a non-Faraday current in the solution which exists between an anode and a cathode, and makes an anode and the said cathode into an electricity supply state (instantly).

  Here, as described above, the time during which the non-Faraday current flows is the time from the start of the movement of the electrolyte ions until the electric double layer is formed around the anode and the cathode, and is about 10 ms. Therefore, at this time, a local potential difference is instantaneously generated everywhere in the solution existing between the anode and the cathode.

  Further, if the anode and the cathode are made of the same constituent material, the elution of the anode and the cathode can be suppressed, and the solution between the anode and the cathode is electrically intermediate after the non-Faraday current flows. Thus, an electrolyte bulk with little potential difference is formed.

  As a result, cells existing everywhere in the solution existing between the anode and the cathode are exposed to the instantaneous voltage only for about 10 ms.

  In addition, if the instantaneous voltage is always about 1 V or more during the time when the non-Faraday current flows, the cell membrane of the cell is irreversibly destroyed. For this reason, it is possible to kill (sterilize) a microorganism composed of a single cell or a plurality of cells, that is, a single cell microorganism or a multicellular microorganism. This also makes it possible to extract the contents in the cell.

  Therefore, according to the above configuration, since the cells are not exposed to voltage for a long time as compared with the method of Patent Document 1, the cell membrane is irreversibly while suppressing damage to the contents in the cells. Can be disrupted and the contents in the cell can be extracted.

  As described above, the contents in the cell can be extracted while suppressing the damage.

  Moreover, in the cell membrane disruption apparatus of the present invention, in addition to the above configuration, the anode is a cylindrical tube electrode, and one opening of the tube electrode is opened in a direction in which the cathode exists. Also good.

  According to the said structure, one opening of a cylindrical electrode is open | released in the direction where a cathode exists. Thereby, the solution can be energized over a wide range between the cylindrical electrode and the needle electrode.

  Further, by making the direction in which the solution flows coincide with the direction of the central axis of the cylindrical electrode, the solution passes through the cylindrical electrode without being hindered by the flow. Therefore, it is possible to quickly and highly efficiently kill cells in a solution or a microorganism composed of a single cell or a plurality of cells, and extract the contents in the cells quickly and efficiently while suppressing damage. .

  In the cell membrane disruption apparatus of the present invention, in addition to the above configuration, the anode may be a flat plate electrode.

  According to the said structure, a solution can be energized over a wide range between a plate electrode and a needle-shaped electrode by adjusting arrangement | positioning of a plate electrode and a needle-shaped electrode. Therefore, it is possible to quickly and highly efficiently kill cells in a solution or a microorganism composed of a single cell or a plurality of cells, and extract the contents in the cells quickly and efficiently while suppressing damage. .

  Further, in the cell membrane disruption apparatus of the present invention, in addition to the above-described configuration, the energization unit applies the pulse voltage having an amplitude that does not cause discharge between the anode and the cathode at least once, A current (non-Faraday current) due to movement of electrolyte ions may be generated in the solution.

  Here, applying a pulse voltage without applying a constant voltage between the anode and the cathode prevents the current from flowing in the solution for a long time and suppresses damage to the contents in the cell. It is.

  In addition, the number of times of application of the pulse voltage may be one, but as the number of times of application of the pulse voltage is increased, single cell microorganisms or multicellular microorganisms are killed (sterilized) with high efficiency, and the contents in the cells are It can be extracted with high efficiency.

  In addition to the above configuration, the cell membrane disruption apparatus of the present invention further includes a capacitor capable of accumulating electric charge, and the energizing means generates the anode by discharging electric charge accumulated in the capacitor. And applying a transient voltage that is transiently reduced so that no discharge occurs between the cathodes at least once, thereby generating a current (non-Faraday current) in the solution due to the migration of the electrolyte ions. May be.

  Here, applying a transient voltage without applying a constant voltage between the anode and the cathode prevents the current from flowing in the solution for a long time and suppresses damage to the contents in the cell. It is.

  Moreover, the number of times of application of the transient voltage may be one, but as the number of times of application of the transient voltage is increased, single-cell microorganisms or multi-cell microorganisms are killed (sterilized) with high efficiency, and the contents in the cells are It can be extracted with high efficiency.

  In addition to the above-described configuration, the cell membrane disruption apparatus of the present invention has a narrowed portion in which the flow channel width is locally narrowed, and the flow channel width in the narrowed portion is a width that allows a plurality of cells to pass through. The anode and the cathode may be arranged in parallel in the channel along the direction of solution flow in the vicinity of the constriction.

  According to the above configuration, since the region to be energized can be in the vicinity of the constricted portion, even if the solution has a large electric resistance, the anode and the cathode can be energized.

  Further, in the above configuration, the flow path width in the narrowed portion is a width that allows a plurality of cells to pass through. Therefore, compared with the flow path substrate described in Patent Document 2, single-cell microorganisms, multi-cellular microorganisms, etc. It can be killed (sterilized) with high efficiency, and the contents in the cells can be extracted quickly and efficiently.

  As described above, the cell membrane disruptor of the present invention has an anode disposed opposite to each other in a solution containing an electrolyte having a predetermined concentration and cells having a predetermined concentration, and a surface area smaller than that of the anode. A cathode that is a needle-like electrode, and in the solution existing between the anode and the cathode, an electric current is generated by the movement of electrolyte ions ionized by the electrolyte, and the anode and the cathode are energized. It is a structure provided with an electricity supply means.

  As described above, the cell membrane disrupting device of the present invention includes an anode and a cathode, which are disposed to face each other in a solution containing an electrolyte having a predetermined concentration and cells having a predetermined concentration. In the solution existing between the cathodes, there is provided an energization means for generating a current due to movement of electrolyte ions ionized by the electrolyte so as to energize the anode and the cathode.

  Therefore, there is an effect that the contents in the cell can be extracted while suppressing the damage.

It is a mimetic diagram showing an outline composition of a cell membrane destruction device which is one embodiment of the present invention. (A) is a figure which shows the mode of the solution before the process by the said cell membrane destruction apparatus, (b) is a figure which shows the state of the solution after a process, (c) is the number of processes, It is a graph which shows the relationship with the survival rate of microorganisms in seawater. It is a schematic diagram which shows schematic structure of the cell membrane destruction apparatus which is other embodiment of this invention. It is a schematic diagram which shows an example of the arrangement | positioning method of a cylindrical electrode and a needle-shaped electrode regarding the said cell membrane destruction apparatus. (A) is a figure which shows the structure of one Example of the said cell membrane destruction apparatus, (b) is a graph which shows the time change of the electrical potential difference produced locally between probe electrodes regarding the said Example. . It is a figure which shows the observation result of the plankton in the solution before the process by the said Example (immediately after an experiment start). (A) is a figure which shows the observation result of the plankton in the solution before the process by the said Example (immediately after the start of experiment), (b) performs the process by the said Example prepared separately for control experiments. It is a figure which shows the observation result of the plankton (after completion | finish of experiment) in the solution which is not broken. It is a figure which shows experimentally a mode that the electric current is actually flowing in the solution between a plate electrode and a needle-like electrode (needle electrode). It is a circuit diagram which shows the structure of the measuring apparatus for performing a pulse process, and the graph which shows the pulse waveform (5Hz, 5V) of the pulse voltage applied between a plate electrode and a needle-like electrode (needle electrode). It is a figure which shows schematic structure of the processing container. When the pulse voltage is applied (5 Hz, 5 V, 1000 times) and when the pulse voltage is not applied, the wavelength and O.D. D. It is a graph which shows the relationship with (Optical Density). O.D at a wavelength of 615 nm. D. It is a graph which shows the frequency dependence of. O.D at a wavelength of 615 nm. D. It is a graph which shows the processing time dependence.

  An embodiment of the present invention will be described below with reference to FIGS. Configurations other than those described in the following specific items may be omitted as necessary, but are the same as the configurations described in other items. For convenience of explanation, members having the same functions as those shown in each item are given the same reference numerals, and the explanation thereof is omitted as appropriate.

[1. About Cell Membrane Breaker 10]
First, based on FIG. 1 and FIG. 2 (a)-(c), the cell membrane destruction apparatus 10 which is one Embodiment of this invention is demonstrated. FIG. 1 is a schematic diagram showing a schematic configuration of the cell membrane disrupting apparatus 10.

  As shown in FIG. 1, the cell membrane destruction apparatus 10 includes a water tank 1, a plate electrode (anode) 2 </ b> A, a needle electrode (cathode) 3 </ b> A, ceramic tubes 4 </ b> L and 4 </ b> R, connection wirings 5 </ b> L and 5 </ b> R Connection terminals 7L and R are provided.

  The cell membrane disrupting device 10 destroys the cell membrane of cells (or a plurality of cells contained in a microorganism) contained in a liquid sample (solution), and thereby a microorganism composed of a single cell or a plurality of cells, that is, a single-cell microorganism or a multi-cell. Cellular microorganisms are killed (sterilized), and the contents in the cells are extracted.

  Here, extraction is the release of at least one or more intracellular contents, by opening or rupturing the cell wall or cell barrier so that the cell contents are diffused into the surrounding liquid. is there.

  In the present embodiment, ballast water (seawater containing microorganisms) is injected into the water tank 1 as a liquid sample, but is not limited to such a liquid sample. In addition, the density | concentration of the salt (electrolyte) contained in seawater is about 0.7M (M is mol / l: mol / liter) normally.

  As described above, the microorganism may be a single-cell microorganism composed of a single cell or a multi-cellular microorganism composed of a plurality of cells. The microorganism may be an archaeal microorganism, an eubacterial microorganism, a eukaryotic microorganism, or the like, such as bacteria, bacterial spores, viruses, fungi, and fungal spores.

  Examples of eukaryotic cells are plant cells, plant spores, animal cells, and mammalian cells. Mammalian cells are, for example, human cells, white blood cells, and nucleated red blood cells.

  Cellular contents include organelles, genetic material, proteins, and the like. Genetic material includes chromosomes, deoxyribonucleic acid (DNA), plasmid DNA, any type of ribonucleic acid (RNA: eg, mRNA, tRNA), and the like.

  Proteins include enzymes, structural proteins, transport proteins, ion channels, toxins, hormones, receptors and the like.

  The liquid sample may also be a biological sample such as a skin sample collected with a cotton swab, cerebrospinal fluid, blood, saliva, bronchial-alveolar lavage, bronchial aspirate, lung tissue, and urine.

  In addition, the liquid sample may contain one or more kinds of reagents necessary for performing nucleic acid amplification.

  In addition, the liquid sample may contain reagents such as primers, nucleic acids, deoxynucleoside triphosphates, and nucleic acid polymerases.

  Next, as shown in FIG. 1, the plate electrode 2A and the needle electrode 3A are opposed to each other so that the longitudinal direction of the needle electrode 3A is perpendicular to the surface of the plate electrode 2A having the larger surface area. Arranged. The arrangement method of the plate electrode 2A and the needle electrode 3A is not limited to this. For example, the longitudinal direction of the needle electrode 3A is parallel to the surface having the larger surface area of the plate electrode 2A. They may be arranged opposite to each other.

  The plate electrode 2A is an anode, and in this embodiment, the surface having the larger surface area is a plate electrode having a rectangular shape with a length of 3 cm and a width of 3 cm. However, the size and shape of the plate electrode 2A are limited to this. I can't. Moreover, although the constituent material of 2 A of plate electrodes is tungsten in this embodiment, it is not limited to this.

  On the other hand, the needle-like electrode 3A of the present embodiment is a cathode and is a needle-like (or rod-like) electrode having a surface area smaller than that of the plate electrode 2A, and its constituent material is tungsten in this embodiment. The dimensions, shapes, and constituent materials are not limited to these. In addition, as a constituent material of the plate electrode 2A and the needle-like electrode 3A, platinum, gold, carbon, silver, copper, or the like may be used in addition to the above-described tungsten.

  Further, as described below, in order to suppress elution of the plate electrode 2A and the needle electrode 3A, it is preferable that the plate electrode 2A and the needle electrode 3A are made of the same constituent material.

  Moreover, the diameter of the cross section of the acicular electrode 3A in the short direction is about 1 mm. At this time, the facing surface area ratio between the needle-like electrode 3A and the plate electrode 2A is 1: 1146.

  In order to make the energized range as wide as possible, it is preferable that the opposing surface area ratio of the plate electrode 2A to the needle electrode 3A is designed to be as large as possible.

  Further, the distance between the needle-like electrode 3A and the plate electrode 2A is about 11 cm in the present embodiment, but is not limited thereto.

  The plate electrode 2A is inserted into a fitting insertion hole (not shown) provided in the water tank 1 via a ceramic tube 4L that is an insulator, and is electrically connected to the connection terminal 7L. On the other hand, the needle-like electrode 3A is inserted into a fitting insertion hole (not shown) provided in the water tank 1 via a ceramic tube 4R that is an insulator, and is electrically connected to the connection terminal 7R.

  According to the above configuration, by adjusting the arrangement of the plate electrode 2A and the needle-like electrode 3A, the energization unit 6 described later causes the solution to extend between the plate electrode 2A and the needle-like electrode 3A over a wide range. Can be energized (instantaneously).

  The energization unit 6 generates a non-Faraday current due to movement of electrolyte ions (sodium ions and chlorine ions in the case of seawater) ionized in the solution existing between the plate electrode 2A and the needle electrode 3A. Thus, the plate electrode 2A and the needle electrode 3A are energized (instantaneously).

  The energizing unit 6 includes a capacitor (not shown) having a capacitance of 750 μF, and is generated by discharging a stored charge by applying a voltage of about 50 V to the capacitor. Is applied at least once. Note that, due to the transient voltage, no discharge occurs between the plate electrode 2A and the needle-like electrode 3A.

  Note that the capacitance of the capacitor in the cell membrane destruction apparatus 10 of the present embodiment is about several μF to several thousand μF, which is overwhelming compared to about several nF which is the capacitance of the capacitor in the conventional method. Big. For example, the capacitance of the capacitor in the cell membrane disruption device 10 of the present embodiment is preferably 10 μF or more and 9000 μ or less.

  Here, the transient voltage is applied between the plate electrode 2A and the needle electrode 3A without applying a constant voltage, so that no current flows in the solution for a long time. This is to suppress damage.

  Next, the time during which the non-Faraday current flows is the time from the start of the movement of the electrolyte ions until the electric double layer is formed around the plate electrode 2A and the needle electrode 3A, and is approximately 10 ms (milliseconds). It is known to be a degree. Therefore, at this time, a local potential difference (voltage) is instantaneously generated everywhere in the solution existing between the plate electrode 2A and the needle-like electrode 3A (strictly, where the flow of electrolyte ions exists). Arise.

  Further, in this embodiment, if the plate electrode 2A and the needle-like electrode 3A are made of the same constituent material, the elution of the plate electrode 2A and the needle-like electrode 3A can be suppressed, and after the non-Faraday current flows. In the solution between the anode and the cathode, an electrolyte bulk is formed which is electrically neutral and has almost no potential difference.

  Thereby, cells existing everywhere in the solution existing between the plate electrode 2A and the needle-like electrode 3A are exposed to the instantaneous voltage only for about 10 ms.

  Here, it will be examined how long a cell membrane of a cell in a solution is irreversibly destroyed when exposed to a voltage of a certain magnitude.

  Temporarily, when a uniform electric field E is applied stepwise at time t = 0 to cells of radius a, the time change of the membrane voltage V (θ) applied to the cell membrane is

(See Patent Document 3).

  Where θ is the zenith angle in polar coordinates with the cell center as the origin when the direction of the electric field E is θ = 0, τ is the time constant, C is the capacitance per unit area of the cell membrane, ρin , Ρout are the resistivity of the intracellular fluid and the outer fluid, respectively.

  However, equation (1) gives the membrane voltage when a spherical complete membrane is present. When a pore is formed at one point of the membrane by the application of voltage, the distribution of membrane voltage is large from this equation. Will be different.

  That is, in the equation (1), the magnitude of the voltage applied to the cell membrane becomes maximum at the position on the most upstream side (north pole: θ = 0) and the position on the most downstream side (south pole: θ = π) of the electric field lines. Therefore, when a voltage is applied, it is expected that a pore is first formed at any one of these positions.

  Then, a current flows through this, and charging of the other pole further proceeds, and an excessive voltage higher than that given by Equation (1) is applied to this position, and this position is irreversibly destroyed. Conceivable. In this way, cells are destroyed by the phenomenon of overvoltage propagating to another position on the cell membrane, like a shogi.

Here, it is known that the capacitance C per unit area of the cell membrane is about 1 μF / cm ² (micro farad / square centimeter) regardless of the cell type. Therefore, the time constant τ for charging the cell membrane of the formula (2) is, for example, when the surrounding medium is a solution having a physiological saline concentration of about 1.54 mM for a cell having a radius a = 5 μm (micrometer). The time constant τ is about 40 ns (nanoseconds), and when the surrounding medium is a low concentration saline solution of about 1 mM, the time constant τ is about 2 μs (microseconds).

  Therefore, in order to cause irreversible destruction in the cell membrane, a voltage of about 1 V or more should be applied to the cells for a longer time.

  Both the above-described values of the time constant τ = about 40 ns and the time constant τ = 2 μs are much smaller than about 10 ms, which is the time during which the non-Faraday current flows.

  That is, it is considered that the cell membrane of the cell is irreversibly destroyed if the instantaneous voltage magnitude is always about 1 V or more during the time when the non-Faraday current flows.

  Therefore, according to the above configuration, since the cells are not exposed to voltage for a long time as compared with the method of Patent Document 1, the cell membrane is irreversibly while suppressing damage to the contents in the cells. Can be destroyed. That is, it is possible to kill (sterilize) single-cell microorganisms, multicellular microorganisms, and the like. This also makes it possible to extract the contents in the cell.

  For example, FIG. 2A shows a state when a part of the solution in the water tank 1 is taken out and observed before the treatment by the cell membrane disrupting apparatus 10. On the other hand, FIG. 2B shows a state when a part of the solution in the water tank 1 is taken out and observed after the treatment by the cell membrane disrupting device 10.

  As shown in FIG. 2B, it can be seen that the cell membrane of the cells in the solution is irreversibly destroyed after the treatment by the cell membrane destruction apparatus 10.

  In the above configuration, the needle-like electrode 3A is a needle-like electrode having a smaller surface area than the plate electrode 2A. Thereby, even if the magnitude | size of the voltage applied between the plate electrode 2A and the needle-like electrode 3A is somewhat small, the solution can be energized over a wide range. Therefore, cells in a solution or a microorganism composed of a single cell or a plurality of cells can be killed with high efficiency, and the contents in the cells can be extracted with high efficiency while suppressing damage.

  As described above, according to the cell membrane disrupting device 10, cells in a solution or a microorganism composed of a single cell or a plurality of cells are quickly and efficiently killed, and the contents in the cells can be quickly suppressed while suppressing damage. And it can extract with high efficiency.

  Moreover, since the cell membrane destruction apparatus 10 is a very simple structure, if it is used as a ballast water treatment apparatus, it can be mounted on both large and small ships. Further, microorganisms can be treated without adding treatment chemicals, and there is no need to perform secondary treatment during drainage. Moreover, a small-scale apparatus can be realized as a ballast water treatment apparatus, and it can be installed in a piping system.

  Next, the number of times of application of the transient voltage applied between the plate electrode 2A and the needle-like electrode 3A by the energization unit 6 described above may be one, but as the number of times of application of the transient voltage is increased, the contents in the cell are increased. Can be extracted with high efficiency.

  For example, as shown in the graph of FIG. 2 (c), when the number of treatments by the cell membrane destruction apparatus 10 is actually small, the cell survival rate is about 38%, but the number of treatments is increased. As a result, the survival rate became 0%.

  In the present embodiment, the energization unit 6 uses a capacitor discharge phenomenon, but the configuration of the energization unit 6 is not limited to this.

  For example, a pulse voltage generator (not shown) is used as the energization unit 6 and a pulse voltage having an amplitude that does not cause a discharge between the plate electrode 2A and the needle electrode 3A is applied at least once. A Faraday current may be generated in the solution.

  The pulse width (time) of the pulse voltage is preferably larger than the time constant τ, but it is preferably as short as possible in order to suppress damage to the contents of the cells.

  Further, the magnitude of the pulse voltage is considered to be about 50 V with reference to the discharge by the capacitor described above, but is not limited to this (see the following 4. Experimental results of pulse processing).

  The pulse current is preferably a so-called unipolar pulse that does not cause polarity reversal, but may be a bipolar pulse if necessary.

  Here, a pulse voltage is applied between the plate electrode 2A and the needle electrode 3A without applying a constant voltage so that no current flows in the solution for a long period of time. This is to suppress damage.

  In addition, the number of times of application of the pulse voltage may be one, but as the number of times of application of the pulse voltage is increased, single cell microorganisms or multicellular microorganisms are killed (sterilized) with high efficiency, and the contents in the cells are It can be extracted with high efficiency.

[2. About Cell Membrane Breaker 20]
Next, based on FIG. 3 and FIG. 4, the structure of the cell membrane destruction apparatus 20 which is other embodiment of this invention is demonstrated. FIG. 3 is a schematic diagram showing a schematic configuration of the cell membrane destruction apparatus 20.

  As shown in FIG. 3, the cell membrane disrupting device 20 is different from the cell membrane disrupting device 10 described above as follows.

  (1) Instead of the water tank 1, a flow path including a sample inflow path (flow path) 1L, a narrowed portion (flow path) 1C, and a sample outflow path (flow path) 1R is used.

  (2) The combination of the cylindrical electrode 2B (anode) and the needle electrode (cathode) 3B is used instead of the combination of the plate electrode 2A and the needle electrode 3A.

  Note that the configurations other than the above (1) and (2) are the same as those described above, and thus the description thereof is omitted here.

  First, as shown in FIG. 3, the cell membrane destruction apparatus 20 of the present embodiment has a stenosis part 1C in which the channel width is locally narrow, and a plurality of cells have a channel width in the stenosis part 1C. A flow path (a sample inflow path 1L, a narrowed portion 1C, and a sample outflow path 1R) having a width as much as possible is provided.

  The sample inflow path 1L is an inflow path through which a liquid sample (sample) flows. On the other hand, the sample outflow path 1R is an outflow path through which the liquid sample flows out.

  That is, the cell membrane disrupting apparatus 20 of the present embodiment rapidly and efficiently kills (sterilizes) single-cell microorganisms and multicellular microorganisms while flowing a sample through the flow path at a predetermined flow rate. The contents in the cell can be extracted.

  The width of the stenosis 1C may be at least 20 μm or more so that two cells can pass through because the diameter of one cell is about 10 μm.

  Further, the cylindrical electrode 2B and the needle-like electrode 3B are arranged in parallel in the flow path in the vicinity of the narrowed portion 1C along the direction in which the solution flows.

  Thereby, since the region to be energized by the non-Faraday current can be in the vicinity of the constriction 1C, it is possible to energize between the cylindrical electrode 2B and the needle electrode 3B even if the solution has a large electric resistance. it can.

  Moreover, in the said structure, since the flow-path width in the constriction part 1C is a width | variety which can pass a some cell, compared with the flow-path board | substrate described in the said patent document 2, the content in a cell is rapidly and can be carried out. It can be extracted with high efficiency.

  Next, an example of an arrangement method of the cylindrical electrode 2B and the needle electrode 3B will be described based on FIG.

  As shown in FIG. 4, the cylindrical electrode 2B (which is cylindrical in this embodiment, but is not limited to this) has a circular left opening OL and a circular right opening (one opening) OR. The right opening OR of the cylindrical electrode 2B is preferably opened in the direction in which the needle electrode 3B exists.

  Thereby, the solution can be energized over a wide range between the cylindrical electrode 2B and the needle electrode 3B.

  For example, the distance d1 between the left opening OL and the right opening OR of the cylindrical electrode 2B is 3 cm, the radius of the right opening OR or the left opening OL is 1.5 cm, and all the inner surfaces of the cylindrical electrode 2B are Assuming that the needle electrode 3B (substantially a cylinder and the diameter of the circle is 1 mm) is opposed (the surface of the semi-cylinder is opposite), the opposed surface area ratio of the needle electrode 3B and the cylindrical electrode 2B is 1 : 60.

  In addition, the arrangement | positioning method of the cylindrical electrode 2B and the needle-shaped electrode 3B is not restricted to this, For example, it arrange | positions so that the direction of the central axis of the cylindrical electrode 2B and the longitudinal direction of the needle-shaped electrode 3B may correspond. Also good.

  The distance d2 between the cylindrical electrode 2B and the needle electrode 3B (the distance between the opening center C of the left opening OL and the needle electrode 3B) is about 11 cm as in the combination of the plate electrode 2A and the needle electrode 3A. However, it is not limited to this.

  Further, as shown in FIG. 4, by making the direction in which the solution flows coincide with the direction of the central axis of the cylindrical electrode 2B (the axis passing through the opening center C of the right opening OR and the left opening OL), It passes through the cylindrical electrode 2B without being interrupted. Therefore, single-cell microorganisms or multi-cellular microorganisms can be killed (sterilized) quickly and efficiently, and the contents in the cells can be extracted quickly and efficiently.

  In the above description, the dimensions of the cylindrical electrode 2B and the needle-like electrode 3B of the cell membrane destruction apparatus 20 and the distance between the electrodes are approximately the same as the dimensions of the plate electrode 2A and the needle-like electrode 3A of the cell membrane destruction apparatus 10. did.

  However, the dimension may be very small as compared with the present embodiment. When the sample inflow path 1L, the constriction 1C, and the sample outflow path 1R are microchannels on the order of micrometers, the dimensions are described. Can be on the order of micrometers.

  According to the above configuration, single-cell microorganisms and multi-cellular microorganisms can be quickly and efficiently killed (sterilized), and the contents in the cells can be extracted quickly and efficiently while suppressing damage. . Further, in the inspection of food hygiene, environmental hygiene, etc., the contents in the cells of harmful fungi to be inspected can be taken out quickly and efficiently without affecting the subsequent inspection.

  In addition, by adjusting the amplitude of the voltage applied by the energization unit 6, denaturation of the contents in the cell can be suppressed. Furthermore, when the cell membrane is crushed, the treatment can be performed without adding a chemical or the like, and the contents of the cells can be denatured by the chemical and the influence on the subsequent inspection by the chemical can be suppressed.

[3. Regarding the effect test of the cell membrane destruction apparatus 10]
Next, based on FIG. 5 (a) to FIG. 7 (b), the results of trial manufacture of the above-described cell membrane disruption apparatus 10 (hereinafter simply referred to as “Example”) will be described.

  FIG. 5A is a diagram showing a configuration of the prototype embodiment. 5A, a probe electrode for observing a local potential difference generated near the center of the water tank 1 is installed. Next, the implementation procedure (1 to 4) of the effect test will be described.

(Procedure 1: Adjustment of plankton used)
First, 10 ml of phytoplankton (hereinafter referred to as plankton) culture solution (1 ml of liter) of seawater (the number of plankton contained in 10 ml is about 8.7 × 10 ⁵ ) and carefully stirred is used for the experiment. did. The microorganism in the solution is Heterosigma akashiwo <plankton>.

(Procedure 2: Adjustment of staining solution)
Neutral Red (C15H17ClN4) was used as a staining solution for easy observation of plankton. The feature of this stain is that live plankton is dyed red and dead plankton is not dyed. Neutral Red was previously dissolved in pure water to prepare a 1% (weight%) solution.

(Implementation procedure 3: application of transient voltage)
As the electrode, the plate electrode 2A and the needle electrode 3A described above were used. The transient voltage described above was applied between the plate electrode 2A and the needle electrode 3A. About 500 ml of seawater containing plankton was placed in the aquarium 1 for processing. The processing is performed multiple times. This time, processing was performed 40 times, and the time required for the processing was about 40 seconds.

(Procedure 4: Plankton staining operation and observation)
In the staining treatment, 5 μl of Neutral Red, 1% (weight%) solution was added to 10 ml of seawater containing plankton. Further, after staining, the mixture was centrifuged at 3000 rpm for 10 minutes, and 9 ml of the supernatant was discarded.

  After the dyeing treatment as described above, the plankton was observed with a microscope.

  Next, the results of the effect test will be described based on FIGS.

  First, FIG. 5B is a graph showing the temporal change of the potential difference locally generated between the probe electrodes in the example.

  As shown in FIG. 5B, a potential difference of about 30 V is instantaneously generated between the probe electrodes as a step function, and thereafter, the potential difference decreases exponentially (transiently).

  Further, FIG. 5B shows a process from a potential difference of about 30 V as a step function to a time of 9 ms (milliseconds).

  From the above, it was found that a potential difference of 1 V or more can be locally generated for about 10 ms.

  Next, FIG. 6 is a figure which shows the observation result of the plankton in the solution before the process by the said Example (immediately after an experiment start).

  As shown in FIG. 6, it can be seen that the plankton shape is clear before the treatment according to the example, and it is alive because it is dyed red by the staining solution.

  Next, Fig.7 (a) is a figure which shows the observation result of the plankton in the solution before the process by the said Example (just after an experiment start).

  As shown in FIG. 7A, the shape of plankton is not known, and a plankton remnant was observed. From this, it is considered that the plankton film was destroyed and the contents appeared.

  Next, FIG.7 (b) is a figure which shows the observation result of the plankton (after completion | finish of experiment) in the solution by which the process by the said Example prepared separately for the control experiment was not performed.

  As shown in FIG. 7B, the shape of plankton is clear and stained red with the staining liquid, so that the plankton is alive. Further, when compared with the plankton after the processing of the cell membrane disrupting apparatus 10, it can be seen in FIG. 7A that the plankton is not broken due to the change with time.

  Next, based on FIG. 8, the results of experiments on whether or not current actually flows in the solution between the plate electrode 2A and the needle-like electrode (needle electrode) 3A of the cell membrane destruction apparatus 10 will be described.

  FIG. 8 shows a state in which both ends of an LED (light emitting diode) electrode are submerged in seawater and a transient voltage of 50 V is applied between the plate electrode 2A and the needle electrode 3A in the cell membrane destruction apparatus 10. Yes.

  The LED was lit for a moment depending on the direction of the electrode of the LED. From the above, it can be seen that a current that does not cause plasma discharge or physical shock can be passed through seawater.

[4. (Experimental result of pulse processing)
Meanwhile, antibody drug production using cells has been performed, and a pretreatment method for extracting biomolecules simply and efficiently while maintaining the function from the cells has been conventionally demanded. As such a pretreatment method, use of a surfactant, an ultrasonic cell disruption method, a pulse electric field method, and the like are conceivable. In particular, the pulse electric field method has many parameters that can be controlled such as time and applied voltage.

  However, in the pulse electric field method, a high voltage of about 10 kV (several kV to several tens kV) is applied to the external voltage, and there is room for improvement in terms of ensuring safety and requiring expensive equipment.

  On the other hand, as described above, when a voltage exceeding about 1 V is applied to the cell membrane itself in prokaryotic cells such as Escherichia coli, the electroporation cannot be repaired and the membrane is destroyed.

  Therefore, the inventors of the present application conducted an experiment on the pretreatment method of this embodiment in which the cell membrane is disrupted by applying a voltage pulse of an extremely low voltage of about several volts for the purpose of enzyme extraction from the inside of E. coli. In addition, using the chromogenic enzyme substrate method which is a highly sensitive detection means utilizing the activities of β-glucuronidase and 5-Bromo-4-chloro-3-indolyl-β-D-glucuronide present in E. coli, its absorbance Then, the enzyme activity was evaluated, and the effectiveness of a series of processes of the pretreatment method of the present embodiment and the detection method was confirmed.

  In the experiment, Escherichia coli (DH5α, Toyobo) was used as a strain to be used, and shake culture was performed at 37 ° C. for 12 hours or more in an LB liquid medium (Difco: Tryptone 10 g / L, Yeast extract 5 g / L, NaCl 10 g / L). (TAITEC, BR-40LF).

The culture solution was measured using a culture colorimeter (WPA, CO8000 Biowave). D. , LB liquid medium was added and O.D. D. After adjusting to = 0.4 ± 0.05, a sample solution was obtained (about 10 ⁸ cfu / mL). “OD” is an abbreviation for Optical Density (optical density or optical density). D. That's it. “Cfu” is an abbreviation for Colony Forming Unit.

  Next, a pulse power supply is constructed using a capacitor capacity of 3000 μF (micro farad), a signal generator (NF, 1915), and a multi-output DC stabilized power supply (TEXICO, PW36-1.5AD), and the output pulse is a digital oscilloscope. (Tektronix, TDS1001B).

  FIG. 9 shows a schematic diagram (circuit diagram) of the pulse output system used in the experiment and an example of the pulse shape. A tungsten electrode was used for the treatment tank (cylindrical, inner diameter 20 mm), the needle electrode was negative, the plate electrode was positive, and the distance between the electrodes was fixed at 2 mm. 4 mL of the sample solution was dispensed into a treatment tank and treated with a pulse voltage while stirring with a stirrer placed under a flat plate electrode.

In addition, to adjust the test reagent, 5-Bromo-4-chloro-3-indolyl-β-D-glucuronide Cyclohexylammonium Salt was adjusted to 19.2 mM with N, N-dimethylformamide (DMF) solvent (both Wako Jun) Yakuhin Kogyo), 200 mM NaH ₂ PO ₄ aqueous solution and 200 mM Na ₂ HPO ₄ aqueous solution mixed with 3.3: 6.7 (volume: volume) phosphate buffer solution, diluted 9-fold amount, substrate It was set as the solution. Each sample after application of the pulse voltage was dispensed and mixed 1: 1 with the substrate solution, and then subjected to an enzyme reaction at 37 ° C. for 16 hours or longer, followed by an O.V. spectrophotometer (JASCO, V-550). D. The wavelength distribution of was measured.

  Conventionally, an enzyme substrate method or an antigen-antibody method that can be determined by fluorescence or color development has been proposed for the purpose of confirmation of E. coli and coliforms for the purpose of simplicity and speed. Focusing on β-glucuronidase or β-galactosidase, which is specifically present in E. coli and coliforms, 5-Bromo-4-chloro-3-indolyl-β-D-glucuronide (X-gluc) and 5-Bromo- 4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) is used.

  In addition, even when the concentration of the bacteria is low, O.D. D. It can be evaluated from. In this specification, in order to obtain a guideline (pulse condition) for more efficiently extracting the contents after voltage pulse treatment with a low voltage, evaluation was performed using a highly sensitive spectrophotometer rather than on an agar medium.

  FIG. 11 shows the E. coli culture solution and O.D. after pulse treatment (5 Hz, 5 V, 1000 pulses). D. Indicates. In the range of 500 to 750 nm, O.D. D. Increase was observed with or without pulse treatment. D. More greatly increased results were obtained.

  Next, for the pulse shape (frequency and voltage), the O.D. D. The dependency was confirmed. FIG. 12 shows the results of applying voltages of 1V, 5V, and 10V while fixing the number of pulse processings to 1000 at each frequency of 1 Hz, 5 Hz, and 10 Hz.

  At this time, the O.D. D. Was corrected from the actually measured value as a background (Abs615-Abs750).

  Compared with the unprocessed case, the O.D. D. However, a significant difference was not observed at 1 V compared to when no pulse treatment was performed. Furthermore, 5 Hz is fixed as a measurement condition, and O.D. D. The measurement results are shown in FIG. 13 (pulse processing time 500, 1000, 1500 sec).

  Qualitatively, O.D. D. Increase as the processing time increases. D. After saturating, a trend of decreasing was observed at 5V. On the other hand, in the case of 10V, the same O.D. is obtained regardless of the processing time (500, 1000, 1500 sec). D. Under these conditions, it was difficult to detect β-glucuronidase by pulse treatment.

  From the above, it was shown that the enzyme contained in the cell can be taken out of the cell with high efficiency by a voltage pulse of a very low voltage of about several volts. Therefore, according to the pretreatment method (cell membrane disruption devices 10 and 20) of the present embodiment, it is possible to provide the cell membrane disruption device 10 having a low voltage and high cell membrane destruction efficiency. For example, the amplitude of the pulse voltage in the present embodiment is preferably 1 V or more and 100 V or less.

  The present invention can also be expressed as follows.

  That is, the cell membrane destruction apparatus of the present invention may efficiently flow a pulse current with a low voltage that does not generate an impact due to discharge in seawater over a wide range.

  Thereby, the intracellular contents can be efficiently extracted from the microorganisms in seawater while avoiding the damage.

  Further, since the processing speed of the extraction process is high and a small-scale device can be configured with a simple configuration, a cell membrane disrupting device that can be mounted on a ship can be provided.

  Further, the cell membrane destruction apparatus of the present invention may flow a pulse current to the sample at a low voltage that does not cause an impact due to discharge. Thereby, a cell membrane can be destroyed rapidly and highly efficiently.

  In the cell membrane disruption apparatus of the present invention, a pulse current may be passed through a flow path for cell membrane disruption, and the pulse current may be passed through a sample flowing through the flow path a plurality of times. Thereby, the cell membrane of harmful bacteria contained in the sample can be destroyed.

  Furthermore, according to the above configuration, in food hygiene and environmental hygiene inspections, a method for quickly and efficiently extracting the contents of harmful fungi in cells without affecting the subsequent inspection is provided. can do.

  The present invention can also be expressed as follows.

  That is, the cell membrane destruction apparatus of the present invention may flow a pulse current at a low voltage several times so as not to cause plasma discharge or physical shock. Thereby, the microorganisms in seawater can be processed efficiently.

  Moreover, the cell membrane destruction apparatus of the present invention may have a negative electrode structure having a smaller surface area than the positive electrode. Thereby, even a low voltage can be processed in a wide range and at a high speed.

  According to the above configuration, microbial treatment is possible without adding a treatment agent or the like, and there is no need to perform secondary treatment during drainage. Moreover, a small apparatus is anticipated and it can also be installed in a piping system.

  Moreover, the cell membrane destruction apparatus of the present invention may flow a pulse current at a low voltage several times so as not to cause plasma discharge or physical shock. Thereby, the cell membrane can be crushed and the contents in the cell can be taken out with high efficiency.

  In the cell membrane disruption apparatus of the present invention, the cell membrane disruption flow path may have a plurality of structures through which a pulse current flows.

  In the cell membrane disruption apparatus of the present invention, the flow path may be locally restricted in the portion where the pulse current flows. Thereby, even if it is a sample with large electrical resistance, a pulse current can be energized.

[Additional Notes]
Note that the present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims, and can be obtained by appropriately combining technical means disclosed in different embodiments. Embodiments are also included in the technical scope of the present invention.

  The present invention provides a bactericidal device that efficiently kills harmful bacteria such as Escherichia coli, Legionella, O-157, and Salmonella, an extraction device that efficiently extracts intracellular contents, and efficiently removes intracellular contents. It can be widely applied to analyzers that extract and analyze the extracted contents.

1 Water tank 1L Sample inlet (flow path)
1R Sample outflow channel
1C Constriction (flow path)
2A Plate electrode (anode)
2B Cylindrical electrode (anode)
3A, 3B Needle-like electrode (cathode)
4L, 4R Ceramic tube 5L, 5R Connection wiring 6 Current-carrying part (energization means)
7L, 7R Connection terminal 10 Cell membrane destruction device 20 Cell membrane destruction device C Opening center OL Left opening OR Right opening (one opening)
d1, d2 distance

Claims (7)

In a solution containing an electrolyte having a predetermined concentration and cells having a predetermined concentration, an anode disposed opposite to each other, and a cathode that is a needle-like electrode having a smaller surface area than the anode,
A cell membrane characterized by comprising an energization means for generating an electric current due to movement of electrolyte ions ionized by the electrolyte in a solution existing between the anode and the cathode so as to energize the anode and the cathode. Destruction device. The anode is a cylindrical cylindrical electrode,
The cell membrane destruction apparatus according to claim 1, wherein one opening of the cylindrical electrode is opened in a direction in which the cathode exists.   The cell membrane destruction apparatus according to claim 1, wherein the anode is a flat plate electrode.   The energizing means generates a current in the solution by moving the electrolyte ions by applying a pulse voltage having an amplitude that does not cause discharge between the anode and the cathode at least once. The cell membrane disruption device according to any one of claims 1 to 3. It has a capacitor that can store charge,
The energization means applies at least once a transient voltage that is generated by discharging the charge stored in the capacitor and transiently decreases with a magnitude that does not cause a discharge between the anode and the cathode. The cell membrane destruction apparatus according to any one of claims 1 to 3, wherein an electric current generated by movement of the electrolyte ions is generated in the solution. A channel having a narrowed portion where the channel width is locally narrowed, and the channel width in the narrowed portion is such that a plurality of cells can pass through,
The said anode and the said cathode are arrange | positioned in parallel with the inside of the said flow path along the flow direction of a solution in the vicinity of the said constriction part, The any one of Claim 1-5 characterized by the above-mentioned. The cell membrane destruction apparatus as described. In a solution containing an electrolyte having a predetermined concentration and cells having a predetermined concentration, the anode and the cathode are arranged opposite to each other, and
A cell membrane characterized by comprising an energization means for generating an electric current due to movement of electrolyte ions ionized by the electrolyte in a solution existing between the anode and the cathode so as to energize the anode and the cathode. Destruction device.