Electroporation method using an electroporation circuit, electroporation circuit and DC high-voltage power supply circuit

Abstract

To provide: an electroporation method using a circuit for electroporation, in which load on an element is reduced and fault of the element is therefore prevented; a circuit for electroporation; and a circuit for DC high-voltage power source.SOLUTION: An electroporation method using a circuit 1 for electroporation is provided. The electroporation circuit comprises at least two capacitors C1-Cm connected in series to each other, a plurality of time constant changing portion 2, switches S11-Smn, and resistors R11-Rmn connected in series to the switches.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The disclosure in the present application relates to an electroporation method using an electroporation circuit, an electroporation circuit, and a DC high-voltage power supply circuit.

Background Art

[0002] Many methods have been developed for introducing foreign substances such as nucleic acid molecules such as DNA and RNA, biological substances such as proteins, and compounds that are active ingredients of drugs into target cells. In particular, gene transfer technology for introducing nucleic acid molecules into cells is a basic technology of genetic engineering. Therefore, gene transfer technology is required in a wide range of fields such as genetically modified crops, gene therapy, genome analysis, and genome editing technology.

[0003] The methods of gene transfer technology can be classified into biological methods, chemical methods, and physical methods. Examples of biological methods include methods using viruses. Examples of chemical methods include the calcium phosphate method and the lipofection method. Examples of physical methods include the electroporation method (electrical perforation method), the particle gun method (gene gun method), the sonoporation method (method using ultrasonic waves), and the like.

[0004] Physical methods have the advantages that, compared with biological and chemical methods, there is no need to consider toxicity to cells and there is no limitation on applicable cells. In particular, as disclosed in Patent Document 1 and Patent Document 2, the electroporation method is the most versatile and widespread method among physical methods. The electroporation method is a method of applying an electric pulse to cells to transiently create small pores through which foreign substances can pass through the cell membrane and taking in DNA and the like. The electroporation method can obtain high introduction efficiency compared with chemical methods, is simple to operate, has high reproducibility and safety, and is a technology applicable to various biological species such as animal cells, plant cells, fungal cells such as yeast, and bacteria. In addition, it is also possible to apply to cells of various types of tissues.

Prior Art Documents

[0005] [Patent Document 1] International Publication No. 2016 / 017045 [Patent Document 2] Japanese Patent Application Publication No. 2-035071 [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] Patent Document 1 discloses an electric pulse generator for a multi-stage electroporator having means for generating high-voltage, short-duration electric pulses and means for generating low-voltage, long-pulse electric pulses. The electric pulse generator for a multi-stage electroporator disclosed in Patent Document 1 adjusts the high-voltage, short-duration electric pulses in the range of tens to thousands of volts according to the difference in impedance of the target object, and performs gene transfer on cells and organisms of both low-impedance and high-impedance targets. Patent Document 2 discloses an invention of an electric stimulation device in which the time constant when applying a DC voltage pulse via a capacitor is continuously variable.

[0007] The multi-stage electroporator electric pulse generator disclosed in Patent Document 1 includes a capacitor downstream of the high-voltage generation circuit that charges for generating electric pulses via a switch. Therefore, when charging the capacitor for generating electric pulses, the high voltage generated by the high-voltage generation circuit puts a load on the switch. Also, as disclosed in Patent Document 1, electroporation circuits require the generation of high-voltage pulses of several thousand volts. In such cases, the electroporation circuit needs to be designed for high voltages using elements such as switches with high voltage resistance. However, in conventional electroporation circuits, even if elements with high voltage resistance are used, if some elements fail for some reason, a load is placed on the other elements, causing them to fail as well.

[0008] To solve the above problem, the inventors, through diligent research, have newly discovered a way to reduce the load on the elements of the electroporation circuit that generates high-voltage pulses.

[0009] Therefore, the purpose of the disclosure in this application is to provide an electroporation method using an electroporation circuit that reduces the load on the element and prevents element failure, an electroporation circuit, and a DC high-voltage power supply circuit. Any other optional additional effects of the disclosure in this application will be made clear in the embodiments for carrying out the invention. [Means for solving the problem]

[0010] (1) An electroporation method using an electroporation circuit, The step includes supplying an electrical signal to the sample, The electroporation circuit is, At least two capacitors, each connected in series, It includes multiple time constant variations of an m x n (where m and n are natural numbers, m ≥ 2 and n ≥ 1) and Multiple time constant change sections are m rows and n columns, m time constant change units are connected in series in the first direction. If n is 2 or greater, it is connected in parallel to adjacent time constant change sections in a second direction different from the first direction. Each time constant change section is: Switch and Includes a resistor connected in series with the switch, Connect the capacitors connected in series and the time constant changing units connected in series in the first direction so that one time constant changing unit in the first direction is connected in parallel to one capacitor. When connected to the input circuit and sample, the series-connected capacitor and multiple time constant change units are connected in parallel to the input circuit and sample. Electroporation method. (2) In the step of supplying an electrical signal to the sample, Close the switch in the time constant change section so that the charge stored in two or more capacitors is discharged simultaneously. or Close the switch in the time constant change section so that the charge stored in two or more capacitors is discharged sequentially. The electroporation method described in (1) above. (3) In the step of supplying an electrical signal to the sample, At least one of the switches is controlled by PWM (Pulse Width Modulation). The electroporation method described in (1) or (2) above. (4) The electroporation circuit includes an input circuit, A DC high-voltage generation circuit is formed by an input circuit and at least two or more capacitors. The electroporation method described in any one of the above (1) to (3). (5) An electroporation circuit that supplies an electrical signal to a sample, The electroporation circuit is, At least two capacitors, each connected in series, It includes multiple time constant variations of an m x n (where m and n are natural numbers, m ≥ 2 and n ≥ 1) and Multiple time constant change sections are m rows and n columns, m time constant change units are connected in series in the first direction. If n is 2 or greater, it is connected in parallel to adjacent time constant change sections in a second direction different from the first direction. Each time constant change section is: Switch and Includes a resistor connected in series with the switch, Connect the capacitors connected in series and the time constant changing units connected in series in the first direction so that one time constant changing unit in the first direction is connected in parallel to one capacitor. When connected to the input circuit and the sample, the capacitor and the plurality of time constant changing units connected in series are connected in parallel to the input circuit and the sample. Circuit for electroporation. (6) Including a control unit that PWM-controls at least one of the switches. The electroporation circuit according to (5) above. (7) Including an input circuit. The input circuit and at least two or more capacitors constitute a DC high voltage generation circuit. The electroporation circuit according to (5) or (6) above. (8) A DC high voltage power supply circuit that outputs an electrical signal, The DC high voltage power supply circuit, Includes at least two or more capacitors connected in series, A plurality of time constant changing units of m rows and n columns (m and n are natural numbers, m ≥ 2, n ≥ 1). The plurality of time constant changing units of m rows and n columns, m time constant changing units are connected in series in the first direction, When n is 2 or more, it is connected in parallel to the time constant changing units adjacent in the second direction different from the first direction, Each time constant changing unit, Includes a switch, A resistor connected in series to the switch. Connect between the capacitors connected in series and between the time constant changing units connected in series in the first direction so that one time constant changing unit in the first direction is connected in parallel to one capacitor. When connected to the input circuit and the sample, the capacitor and the plurality of time constant changing units connected in series are connected in parallel to the input circuit and the sample. DC high voltage power supply circuit. (9) Including a control unit that PWM-controls at least one of the switches. The DC high voltage power supply circuit according to (8) above. (10) Including an input circuit. A DC high-voltage generation circuit is formed by an input circuit and at least two or more capacitors. A DC high-voltage power supply circuit as described in (8) or (9) above. [Effects of the Invention]

[0011] This reduces the load on the elements caused by high voltage and suppresses element failures caused by high voltage. [Brief explanation of the drawing]

[0012] [Figure 1] This figure shows an example of an electroporation circuit 1A according to the first embodiment, which comprises an m x n time constant change section 2. [Figure 2] This figure shows an example of an electroporation circuit 1B according to the first embodiment, which comprises a 2x1 time constant change section 2. [Figure 3] This figure shows an example of an electroporation circuit 1C according to the first embodiment, which comprises a 2x2 time constant change section 2. [Figure 4] Figure 4A shows an example of an electroporation circuit 1D according to a second embodiment, which comprises a 2x1 time constant change section 2. Figure 4B shows an example of PWM control of multiple switches by the control unit 4. [Figure 5] This figure shows an example of an electroporation circuit 1E according to a third embodiment, which comprises a 2x1 time constant change section 2. [Modes for carrying out the invention]

[0013] The electroporation method using the electroporation circuit, the electroporation circuit, and the DC high-voltage power supply circuit will be described in detail below with reference to the drawings. In this specification, components having the same or similar functions are denoted by the same or similar reference numerals. In some cases, repeated explanations of components denoted by the same or similar reference numerals may be omitted.

[0014] (First embodiment of an electroporation circuit) Referring to Figures 1 to 3, the electroporation circuit 1 according to the first embodiment will be described. Figure 1 is a diagram showing an example of the electroporation circuit 1A according to the first embodiment, which comprises an m x n (m and n are natural numbers, with m ≥ 2 and n ≥ 1) time constant change section 2. Figure 2 is a diagram showing an example of the electroporation circuit 1B according to the first embodiment, which comprises a 2 x 1 time constant change section 2. Figure 3 is a diagram showing an example of the electroporation circuit 1C according to the first embodiment, which comprises a 2 x 2 time constant change section 2.

[0015] The electroporation circuit 1A according to the first embodiment supplies an electrical signal to a sample connected to the electroporation circuit 1A. The electroporation circuit 1A according to the first embodiment comprises at least m capacitors C1 to Cm connected in series, and a plurality of time constant change units 2 arranged in m rows and n columns (m and n are natural numbers, with m≧2 and n≧1), as shown in Figure 1. When an input circuit 3 as input and a sample that supplies an electrical signal as output are connected to the electroporation circuit 1A according to the first embodiment, the series-connected capacitors C1 to Cm and the plurality of time constant change units 2 are connected in parallel to the input circuit 3 and the sample. The resistance Ra shown in Figure 1 represents the resistance of the sample when the sample is connected to the electroporation circuit 1A, and may be referred to as the sample resistance Ra below. In this specification, supplying an electrical signal to the sample resistance Ra has the same meaning as supplying an electrical signal to the sample. The electroporation circuit 1 according to the first embodiment optionally comprises an input circuit 3 and an output switch Sa.

[0016] The m capacitors C1 to Cm, connected in series, are connected in parallel to the input circuit 3 and the sample resistor Ra, as shown in Figure 1. Capacitors C1 to Cm are charged by the input circuit 3. When the charge stored in capacitors C1 to Cm is discharged, an electrical signal is supplied to the sample resistor Ra. The capacitances of capacitors C1 to Cm can be appropriately determined depending on how much electrical signal to supply to the sample resistor Ra. The capacitances of capacitors C1 to Cm may be the same or different. If the capacitances of capacitors C1 to Cm are different, the value of the resistance provided in the time constant change unit 2 may be appropriately adjusted so that the time constant τ, which will be described later, becomes the desired value.

[0017] Multiple time constant change units 2 arranged in m rows and n columns (where m and n are natural numbers, and m≧2 and n≧1) are connected in parallel to the input circuit 3 and the sample resistor Ra, as shown in Figure 1. The multiple time constant change units 2 are connected in series in a first direction, with m time constant change units 2 connected in series in a first direction. If n is 2 or greater, they are connected in parallel to adjacent time constant change units 2 in a second direction different from the first direction. In this specification, the direction in which rows increase or decrease is referred to as the first direction, and the direction in which columns increase or decrease is referred to as the second direction. Each time constant change unit 2 is equipped with a switch and a resistor connected in series. Specifically, the area enclosed by the dashed line in Figure 1 is a time constant change unit 2. The time constant change unit 2 in the first row and first column is equipped with a switch S11 and a resistor R11 connected in series, and the time constant change unit in the m row and n column is equipped with a switch Smn and a resistor Rmn connected in series. Furthermore, there is no particular limit to the number of time constant change units 2, as long as at least two are provided in the first direction. For example, the multiple time constant change units 2 arranged in m rows and n columns may be arranged in 2 rows and 1 column as shown in Figure 2, or in 2 rows and 2 columns as shown in Figure 3.

[0018] The time constant change unit 2 controls the electrical signal supplied to the sample resistor Ra. Specifically, this is done by opening and closing a switch provided in the time constant change unit 2. The time constant change unit 2 consists of a switch and a resistor connected in series. Therefore, opening and closing the switch changes the value of the resistance through which the current passes in the electroporation circuit 1. Incidentally, the electrical signal supplied to the sample resistor Ra can be an exponentially decaying pulse or a square wave that decays over time. For example, if the electrical signal supplied to the sample resistor Ra is an exponentially decaying pulse, the time constant τ of the pulse is defined as τ = CR. In this case, C is the capacitance of the capacitor in the electroporation circuit 1, and R is the value of the resistance in the electroporation circuit 1, including the sample resistor Ra. Therefore, by opening and closing the switch provided in the time constant change unit 2, the value of the resistance in the electroporation circuit 1 changes, and the time constant τ changes. Thus, by changing the time constant τ, the decay of an exponentially decaying pulse can be controlled. Furthermore, in the electroporation circuit 1, increasing the number of time constant changing units 2 increases the number of resistor combinations, thereby increasing the range over which the time constant τ can be controlled. Since the value of the time constant τ changes depending on the value of the resistors provided by the time constant changing unit 2, the values ​​of each resistor R11 to Rmn should be determined appropriately so that the value of the time constant τ becomes the desired value. Also, the resistors R11 to Rmn may have the same resistance value or may have different resistance values. If different resistance values ​​are used, although this is not a restriction, weighting may be applied to each column, for example. The weighting may be such that the values ​​of the resistors R11 to Rm1 in the first column are R[Ω], the values ​​of the resistors R12 to Rm2 in the second column are 2R[Ω], and the values ​​of the resistors R1n to Rmn in the nth column are nR[Ω]. In this case, since each resistor in rows 1 to m of columns 2 to n is connected in parallel with each resistor in rows 1 to m of column 1, the weight of column 2 is 1 / 2 and the weight of column n is 1 / n relative to column 1, so it is possible to assign weights to each column. Note that the weighting for each column is arbitrary; for example, column 2 could be 1 / 2, column 3 1 / 4, column 4 1 / 8, etc., and a person skilled in the art can decide how much weight to assign as appropriate.By weighting the time constants, it is possible not only to increase the number of time constant change units 2, but also to further increase the range over which the time constant τ can be controlled.

[0019] In the electroporation circuit 1A according to the first embodiment, connections are made between capacitors connected in series and between time constant change units 2 connected in series in the first direction, such that one time constant change unit 2 in the first direction is connected in parallel to each of capacitors C1 to Cm. For example, in the example shown in Figure 1, connections are made between capacitors C1 and C2, between R11 and S21, between R12 and S22, ... between R1n and S2n, so that the first row of time constant change units 2 is connected in parallel to capacitor C1. Similarly, the second row, third row, ... the mth row of time constant change units 2 are connected in parallel to capacitors C2 to Cm. In addition, in the electroporation circuit 1B equipped with a 2x1 time constant change unit 2 shown in Figure 2, connections are made between capacitors C1 and C2 and between R11 and S21. Therefore, the time constant change section 2 in the first row and first column is connected in parallel to capacitor C1, and the time constant change section 2 in the second row and first column is connected in parallel to capacitor C2. Furthermore, in the electroporation circuit equipped with a 2x2 time constant change section 2 shown in Figure 3, connections are made between capacitors C1 and C2, between R11 and S21, and between R12 and S22. Therefore, the time constant change sections 2 in the first row and first column and the first row and second column are connected in parallel to capacitor C1, and the time constant change sections 2 in the second row and first column and the second row and second column are connected in parallel to capacitor C2. Note that in the examples shown in Figures 1 to 3, one row of time constant change section 2 is connected in parallel to one capacitor, but one time constant change section 2 in the first direction may be connected in parallel to multiple capacitors. Furthermore, multiple time constant change sections 2 in the first direction may be connected in parallel to one capacitor.

[0020] As shown in Figure 1, by connecting the components, in electroporation circuit 1A, the charge stored in m capacitors C1 to Cm is divided among the parallel-connected time constant change units 2. Similarly, in electroporation circuits 1B and 1C shown in Figures 2 and 3, the charge stored in the two capacitors C1 and C2 is divided among the first row of time constant change units 2 and the second row of time constant change units 2. Therefore, the electrical signal supplied to the sample resistor Ra is divided, reducing the load on the multiple time constant change units 2. Thus, failure of each element of electroporation circuit 1 due to high voltage can be suppressed. Furthermore, the more connections there are between capacitors connected in series and between time constant change units 2 connected in series in the row direction, the more the charge stored in the capacitors is divided, reducing the load on the time constant change units 2.

[0021] The output switch Sa is an optional additional component in the electroporation circuit 1 according to the first embodiment. In the example shown in Figure 1, the output switch Sa switches between charging m capacitors C1 to Cm from the input circuit 3 and discharging them to supply an electrical signal to the sample resistor Ra. Therefore, the output switch Sa is placed downstream of the capacitors C1 to Cm and the time constant change unit 2, and the load on the output switch Sa due to large currents is also reduced.

[0022] The input circuit 3 is an optional additional component in the electroporation circuit 1A according to the first embodiment. The input circuit 3 is not particularly limited as long as it is connected to capacitors C1 to Cm and can charge capacitors C1 to Cm. An example of the input circuit 3 is a DC high voltage generation circuit such as a power supply device such as a constant power source. Furthermore, the input circuit 3 may constitute a DC high voltage generation circuit by itself, or it may be constituted by the input circuit 3 and capacitors C1 to Cm of the electroporation circuit 1A.

[0023] (First embodiment of an electroporation method using an electroporation circuit) A first embodiment of an electroporation method using an electroporation circuit will be described below. In the following description, "electroporation method using an electroporation circuit" may be referred to simply as "electroporation method."

[0024] The electroporation method according to the first embodiment comprises the step of supplying an electrical signal to a sample.

[0025] The step of supplying an electrical signal to the sample involves discharging the charge stored in the capacitor of the electroporation circuit 1 to supply an electrical signal to the sample resistor Ra. In the examples shown in Figures 1-3, the discharge of the charge from the charged capacitor is performed by closing (turning on) the output switch Sa. The electroporation circuit 1 comprises multiple capacitors as shown in Figures 1-3. Therefore, it is preferable to discharge all the charges stored in the multiple capacitors when supplying an electrical signal to the sample resistor Ra.

[0026] When controlling the time constant change unit 2 to supply an electrical signal to the sample resistor Ra, there are no particular restrictions on how the charges stored in the multiple capacitors are discharged. For example, the charges stored in the multiple capacitors may be discharged simultaneously, or the charges stored in the capacitors may be discharged sequentially until the charges stored in each capacitor are finally discharged.

[0027] To discharge the charge stored in multiple capacitors simultaneously, the switches in the time constant change section 2 in parallel with each capacitor in each row should be closed (opened) simultaneously. For example, to discharge the charge stored in multiple capacitors C1 to Cm simultaneously in the electroporation circuit 1A shown in Figure 1, either (1) close all switches in the time constant change section 2, or (2) simultaneously close at least one switch in any of the 1 to n columns in the time constant change section 2 for each row from 1 to m. Also, to discharge the charge stored in multiple capacitors simultaneously in the electroporation circuit 1B shown in Figure 2, switch S11 and switch S21 should be closed simultaneously. To discharge the charge stored in multiple capacitors simultaneously in the electroporation circuit 1C shown in Figure 3, either close all four switches in the time constant change section 2, or simultaneously close switch S11 or switch S12 and switch S21 or S22.

[0028] When sequentially discharging the charge stored in multiple capacitors, the switches provided in the time constant change unit 2 can be periodically closed (opened). For example, when discharging sequentially over two cycles, the switch of the time constant change unit 2 in parallel with the capacitor to be discharged is closed, and the switch of the time constant change unit 2 in parallel with the capacitor not to be discharged is opened (1st cycle). Then, the switch of the time constant change unit 2 in parallel with the capacitor that was not discharged is closed (2nd cycle). In the 2nd cycle, the switch that was closed in the 1st cycle may be opened or closed. The above describes the case where the discharge cycle is two, but the discharge from the capacitors may be performed sequentially at any desired cycle. For example, in the electrotoporation circuit 1A shown in Figure 1, if half of the m capacitors C1 to Cm are discharged sequentially, it will be two cycles, and if each of the m capacitors C1 to Cm is discharged sequentially, it will be m cycles. The electrotoporation circuits 1B and 1C shown in Figures 2 and 3 have two capacitors, so when they are discharged sequentially, it will be two cycles. In the electrotoporation circuit 1B shown in Figure 2, for example, when discharging C1 and C2 sequentially, switch S11 is closed in the first cycle to discharge capacitor C1. Then, switch S21 is closed in the second cycle to discharge capacitor C2. In the electrotoporation circuit 1C shown in Figure 3, for example, when discharging C1 and C2 sequentially, switch S11 and / or switch S12 are closed in the first cycle to discharge capacitor C1. Then, switch S21 and / or S22 are closed in the second cycle to discharge capacitor C2. Note that in Figures 2 and 3, when discharging capacitors C2 and C1 sequentially, the order should be reversed.

[0029] The electroporation circuit and electroporation method according to the first embodiment provide the following effects. (1) The electrical signal supplied to the sample resistor Ra is divided, reducing the load on the multiple time constant change units 2. Therefore, failure of each element of the electroporation circuit 1 due to high voltage can be suppressed. Also, since the load is reduced, a switch used in low-voltage circuits with low voltage tolerance can be used. Furthermore, by using the electroporation circuit 1, the load on the output switch Sa can be reduced. (2) Because it is equipped with multiple time constant changing units 2, there are many combinations of resistors through which the current passes, and the range over which the time constant τ of the electrical signal supplied to the sample resistor Ra can be controlled can be increased. (3) Since the time constant change unit 2 is equipped with a switch and a resistor, the charge stored in capacitors C1 to Cm can be discharged without closing the output switch Sa. Therefore, for example, if the wrong amount of charge is mistakenly stored in capacitors C1 to Cm, the charge stored in capacitors C1 to Cm can be safely discharged without supplying an electrical signal to the sample resistor Ra.

[0030] (Second embodiment of the electroporation circuit) Referring to Figure 4, the electroporation circuit 1D according to the second embodiment will be described. Figure 4 is a diagram showing an example of the electroporation circuit 1D according to the second embodiment, which comprises a 2x1 time constant change unit 2. Figure 4A is a diagram showing an example in which the control unit 4 controls multiple switches using PWM. Figure 4B is a diagram showing an example in which the control unit 4 controls one switch using PWM.

[0031] The electroporation circuit 1D according to the second embodiment differs from the electroporation circuit 1B according to the first embodiment in that it includes a control unit 4 that PWM (Pulse Width Modulation) controls at least one of the switches provided by the time constant change unit 2, but is otherwise the same as the electroporation circuit 1B. Therefore, the electroporation circuit 1D according to the second embodiment will be described mainly in terms of the differences from the first embodiment, and repetitive explanations of matters already explained in the first embodiment will be omitted. Thus, it goes without saying that even if not explicitly explained in the second embodiment, matters already explained in the first embodiment can be adopted.

[0032] The control unit 4 performs PWM control on the switch provided by the time constant change unit 2. PWM control controls the output by repeatedly opening and closing the switch. Furthermore, PWM control can control the output by changing the duty cycle, which is the ratio of the time the switch is closed in the cycle of opening and closing the switch.

[0033] This section describes an example of PWM control of a switch in an arbitrary time constant change unit 2 equipped with a resistor of resistance value R[Ω]. The switch is opened and closed by PWM control. In this way, the resistor in the time constant change unit 2 is treated as an apparent resistance value rather than its actual resistance value R[Ω]. When the duty cycle is set to 100%, the switch is always closed, and the apparent resistance value in the time constant change unit 2 is the same as the actual resistance value R[Ω]. When the duty cycle is set to 50%, the switch is closed 50% of the time in a given period, so the apparent resistance value in the time constant change unit 2 is 2×R[Ω]. Furthermore, when the duty cycle is set to 10%, the switch is closed 10% of the time in a given period, so the apparent resistance value in the time constant change unit 2 is 10×R[Ω]. Therefore, by PWM control of the switch in the time constant change unit 2, the resistance value of the time constant change unit 2 can be changed according to the duty cycle. Furthermore, without PWM control, the switch in the time constant change unit 2 simply opens and closes, and the resistance value changes discretely. However, when PWM control is performed, the resistance value changes continuously according to the duty cycle. Therefore, the time constant τ of the electrical signal supplied to the sample resistor Ra can be precisely controlled.

[0034] The time constant change unit 2, which is controlled by the control unit 4 using PWM, can be any switch and is not particularly limited. For example, as shown in Figure 4A, multiple switches may be controlled, or as shown in Figure 4B, only one switch may be controlled.

[0035] (Second embodiment of an electroporation method using an electroporation circuit) A second embodiment of the electroporation method will now be described. The electroporation method according to the second embodiment differs from the electroporation method according to the first embodiment in that, in the step of supplying an electrical signal to the sample resistor Ra, at least one of the switches provided by the time constant change unit 2 is PWM controlled, but otherwise it is the same. Therefore, the electroporation method according to the second embodiment will be described mainly in terms of the differences from the first embodiment, and repetitive explanations of matters already explained in the first embodiment will be omitted. Thus, it goes without saying that even if not explicitly explained in the second embodiment, matters already explained in the first embodiment can be adopted.

[0036] In the electroporation method according to the second embodiment, at least one of the switches provided by the time constant change unit 2 is PWM controlled during the step of supplying an electrical signal to the sample. As described above, by PWM controlling the switch, the value of the resistance can be continuously changed, so the time constant τ of the electrical signal supplied to the sample resistor Ra can be precisely controlled.

[0037] As described above, in the electroporation circuit 1D, any switch can be used for PWM control, and there are no particular restrictions. For example, as shown in Figure 4A, when switches S11 and S21 are simultaneously PWM controlled, the charges of capacitors C1 and C2 are discharged evenly, and an electrical signal is supplied to the sample resistor Ra. On the other hand, as shown in Figure 4B, when only one switch S21 is PWM controlled, although the charge of capacitor C2 is discharged by the PWM control, the charge of capacitor C1 is not PWM controlled, resulting in a difference in the amount of charge discharged. Therefore, in order to minimize the difference in the amount of charge discharged caused by the PWM control, it is preferable to switch the PWM control from switch S21 to switch S11. In the case of Figure 4B, in addition to the change in the apparent resistance value of resistor R21 due to the PWM control of switch S21, it is possible to select the connection of resistor R11 by opening and closing switch S11, which is not PWM controlled. Therefore, the apparent resistance value of resistor R21 and the presence or absence of resistor R11 connection have the effect of increasing the range over which the time constant τ of the electrical signal supplied to the sample resistor Ra can be controlled.

[0038] In the above explanation, in order to simplify the description of PWM control, an electroporation circuit 1D equipped with a 2x1 time constant change unit 2 was used. However, it is clear that electroporation circuits 1A and 1C, which have a different number of time constant change units 2, can also be equipped with a control unit 4 and the switch can be controlled by PWM.

[0039] In the case of electroporation circuits 1A and 1C, which have multiple rows of time constant change units 2, at least one of the switches in the time constant change unit 2 is PWM controlled, and the resistance values ​​of the time constant change unit 2 in each row may be weighted differently. By weighting and PWM controlling the time constant change unit 2 in each row, the electroporation circuit 1 can increase the range over which it can control the time constant τ of the electrical signal supplied to the sample resistor Ra, through the combination of weighted resistance values ​​and PWM control. In this case, it is preferable to perform the PWM control on the switch of the time constant change unit 2 with the lowest weighting, in other words, the switch of the time constant change unit 2 with the highest resistance value. Because the weighting is low, the apparent change in resistance due to the change in duty cycle can be made small, and the time constant τ can be controlled precisely.

[0040] The electroporation circuit 1D and electroporation method according to the second embodiment synergistically provide the following effects in addition to the effects of the electroporation circuits 1A to 1C and electroporation method according to the first embodiment. (1) By controlling the switch in the time constant change unit 2 with PWM, the apparent resistance value can be continuously changed by the duty cycle. Therefore, the time constant τ of the electrical signal supplied to the sample resistor Ra can be precisely controlled. (2) When switches equipped with multiple time constant change units 2 connected in parallel to each capacitor are simultaneously controlled by PWM, the charge stored in each capacitor can be discharged evenly. (3) When one of the switches provided by the multiple time constant change units 2 connected in parallel to each capacitor is PWM controlled, in addition to the apparent change in the resistance value of the time constant change unit 2 that is PWM controlled, it is possible to select the connection of the resistance in the time constant change unit 2 that is not PWM controlled. Therefore, the range over which the time constant τ of the electrical signal supplied to the sample resistor Ra can be controlled can be increased.

[0041] (Third embodiment of an electroporation circuit and an electroporation method using the electroporation circuit) Referring to Figure 5, the electroporation circuit 1E and the electroporation method according to the third embodiment will be described. Figure 5 is a diagram showing an example of the electroporation circuit 1E according to the third embodiment, which comprises a 2x1 time constant change section 2.

[0042] The electroporation circuit 1E according to the third embodiment differs from the electroporation circuit 1D according to the second embodiment in that it comprises an input circuit 3 and a DC high voltage generation circuit 5 is formed by at least two capacitors, each connected in series with the input circuit 3, but is otherwise the same as the electroporation circuit 1D. Therefore, the electroporation circuit 1E and the electroporation method according to the third embodiment will be described focusing on the differences from the above embodiments, and repetitive explanations of matters already explained in the above embodiments will be omitted. Thus, it goes without saying that even if not explicitly explained in the third embodiment, matters already explained in the above embodiments can be adopted.

[0043] The electroporation circuit 1E shown in Figure 5 comprises 12 capacitors C1 to C12, a 2x1 time constant change unit 2, a control unit 4, and an input circuit 3. The switches S11 and S21 on the time constant change unit 2 are PWM controlled by the control unit 4. In addition, the electroporation circuit 1E is connected between capacitors C6 and C7 and between the two time constant change units 2. Therefore, the 1x1 time constant change unit 2 is connected in parallel to capacitors C1 to C6, and the 2x1 time constant change unit 2 is connected in parallel to capacitors C7 to C12.

[0044] The electroporation circuit 1E includes an input circuit 3. The input circuit 3 shown in Figure 5 is the part enclosed by the dashed line. The input circuit 3 shown in Figure 5 does not generate high voltage on its own. However, the input circuit 3 is connected to capacitors C1 to C12, which are used to charge the electrical signal supplied to the sample resistor Sa, and the input circuit 3 and capacitors C1 to C12 constitute a DC high voltage generation circuit 5.

[0045] In the electroporation circuit 1E, when the switch S11 or switch S21, which is equipped with two time constant change units 2, is PWM controlled, a difference occurs between the discharge amount of capacitors C1-C6 and the discharge amount of capacitors C7-C12, as described above. In such cases, the switch that is PWM controlled is periodically switched to minimize the difference in the discharge amount of capacitors C1-C6 and capacitors C7-C12. However, if the duty cycle in the PWM control is changed or the timing of switching the PWM control differs, a difference in the discharge amount of the capacitors may occur even if the switch that is PWM controlled is periodically switched. Then, when charging is performed when there is a difference in the discharge amount of capacitors C1-C6 and capacitors C7-C12, a difference in the amount of charge charged to capacitors C1-C12 will occur, and depending on the amount of charge charged, the amount of charge required for the sample resistor Ra may be insufficient.

[0046] As shown in Figure 5, capacitors C1 to C12, which are charged by the electrical signal supplied to the sample resistor Ra, are connected to the input circuit 3 to form a DC high-voltage generation circuit 5. In this way, when a voltage is applied to the input circuit 3 at a predetermined frequency, charge is accumulated in capacitors C1 to C12 through repeated charging and discharging between the capacitor of the input circuit 3 and capacitors C1 to C12. As the charge is accumulated through repeated charging and discharging, the amount of charge accumulated in each of capacitors C1 to C12 can ultimately be made the same. Therefore, even if there is a difference in the amount of charge discharged, this difference can be eliminated when capacitors C1 to C12 are charged.

[0047] Furthermore, the capacitor used for charging and discharging the electrical signal supplied to the sample resistor Ra of the electroporation circuit according to the third embodiment also serves as the DC high voltage generation circuit 5. However, the discharge of charge to supply an electrical signal to the sample resistor Ra of the capacitor and the charging of charge by DC high voltage generation are carried out in different processes. Therefore, the circuit for discharging and the circuit for charging the capacitor can be viewed as separate circuits. Thus, the connection between the capacitor and the time constant change unit 2 and / or the connection between the capacitor and the input circuit 3 should be appropriately designed according to the number of capacitors, the number of time constant change units 2, and / or the circuit configuration of the input circuit 3.

[0048] The above description used an electroporation circuit 1E equipped with a 2x1 time constant change section 2. However, it is clear that even when the number of time constant change sections 2 differs, as shown in the electroporation circuits 1A and 1C in Figures 1 and 3, the capacitor supplying the electrical signal to the sample resistor Ra can be used to generate a high DC voltage.

[0049] The electroporation circuit 1E and electroporation method according to the third embodiment synergistically provide the following effects in addition to the effects of the electroporation circuits 1A to 1D and electroporation methods according to the above embodiments. (1) Capacitors C1 to C12 are used to generate a DC high voltage and to charge the electrical signal supplied to the sample resistor Ra. Therefore, even if a difference in charge amount occurs in capacitors C1 to C12 due to discharge, the difference in charge amount can be eliminated by charging capacitors C1 to C12. (2) Since capacitors C1 to C12 are used for generating a DC high voltage and charging the electrical signal supplied to the sample resistor Ra, the number of elements constituting the electroporation circuit 1E can be reduced.

[0050] (Embodiment of a DC high-voltage power supply circuit) The electroporation circuits 1A to 1E according to the above embodiment supply a high-voltage electrical signal to the sample. However, in addition to the sample, other devices such as circuits may be connected to the output side, and the electrical signal may be supplied to those circuits. Therefore, the electroporation circuit 1 according to the above embodiment can be used as a DC high-voltage power supply circuit.

[0051] It should be noted that the present invention is not limited to the embodiments described above. Within the scope of the present invention, any combination of the embodiments described above, any modification of any component of each embodiment, or any omission of any component is possible. Furthermore, any component may be added to each of the embodiments described above. [Industrial applicability]

[0052] The electroporation method, electroporation circuit, and DC high-voltage power supply circuit disclosed in this application can reduce the load on the elements due to high voltage. Therefore, it is useful for businesses that handle electroporation circuits and DC high-voltage power supply circuits. [Explanation of Symbols]

[0053] 1, 1A~1E…Electroporation circuit, 2…Time constant change section, 3…Input circuit, 4…Control section, 5…DC high voltage generation circuit, C1~Cm…Capacitor, Ra…Sampling resistor, R11~Rmn…Resistor, Sa…Output switch, S11~Smn…Switch

Claims

1. An electroporation method using an electroporation circuit, The step includes supplying an electrical signal to the sample, The electroporation circuit is, At least two capacitors, each connected in series, It includes multiple time constant change sections of m rows and n columns (where m and n are natural numbers, and m ≥ 2 and n ≥ 1), Multiple time constant change sections in m rows and n columns are, m time constant change units are connected in series in the first direction. If n is 2 or greater, it is connected in parallel to adjacent time constant change sections in a second direction different from the first direction. Each time constant change section is: Switch and Includes a resistor connected in series with the switch, Connect the capacitors connected in series and the time constant changing units connected in series in the first direction so that one time constant changing unit in the first direction is connected in parallel to one capacitor. When connected to the input circuit and sample, the series-connected capacitor and multiple time constant change units are connected in parallel to the input circuit and sample. Electroporation method.

2. In the step of supplying an electrical signal to the sample, Close the switch in the time constant change section so that the charge stored in two or more capacitors is discharged simultaneously. or Close the switch in the time constant change section so that the charge stored in two or more capacitors is discharged sequentially. The electroporation method according to claim 1.

3. In the step of supplying an electrical signal to the sample, At least one of the switches is controlled by PWM (Pulse Width Modulation). The electroporation method according to claim 1 or 2.

4. The electroporation circuit includes an input circuit. A DC high-voltage generation circuit is constructed using an input circuit and at least two or more capacitors. The electroporation method according to any one of claims 1 to 3.

5. An electroporation circuit that supplies an electrical signal to a sample, The electroporation circuit is, At least two capacitors, each connected in series, It includes multiple time constant change sections of m rows and n columns (where m and n are natural numbers, and m ≥ 2 and n ≥ 1), Multiple time constant change sections in m rows and n columns are, m time constant change units are connected in series in the first direction. If n is 2 or greater, it is connected in parallel to adjacent time constant change sections in a second direction different from the first direction. Each time constant change section is: Switch and Includes a resistor connected in series with the switch, Connect the capacitors connected in series and the time constant changing units connected in series in the first direction so that one time constant changing unit in the first direction is connected in parallel to one capacitor. When connected to the input circuit and sample, the series-connected capacitor and multiple time constant change units are connected in parallel to the input circuit and sample. Electroporation circuit.

6. Includes a control unit that PWM controls at least one of the switches, The electroporation circuit according to claim 5.

7. Includes an input circuit, A DC high-voltage generation circuit is constructed using an input circuit and at least two or more capacitors. The electroporation circuit according to claim 5 or 6.

8. A DC high-voltage power supply circuit that outputs an electrical signal, The DC high-voltage power supply circuit is, At least two capacitors, each connected in series, It includes multiple time constant change sections of m rows and n columns (where m and n are natural numbers, and m ≥ 2 and n ≥ 1), Multiple time constant change sections in m rows and n columns are, m time constant change units are connected in series in the first direction. If n is 2 or greater, it is connected in parallel to adjacent time constant change sections in a second direction different from the first direction. Each time constant change section is: Switch and Includes a resistor connected in series with the switch, Connect the capacitors connected in series and the time constant changing units connected in series in the first direction so that one time constant changing unit in the first direction is connected in parallel to one capacitor. When connected to the input circuit and sample, the series-connected capacitor and multiple time constant change units are connected in parallel to the input circuit and sample. A circuit for DC high-voltage power supplies.

9. Includes a control unit that PWM controls at least one of the switches, The DC high-voltage power supply circuit according to claim 8.

10. Includes an input circuit, A DC high-voltage generation circuit is constructed using an input circuit and at least two or more capacitors. A DC high-voltage power supply circuit according to claim 8 or 9.

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