Cell membrane chip fixing mechanism

Abstract

The present invention makes it possible to make an electrode and a contact pin sufficiently electrically continuous on a chip provided with a well for forming a cell membrane. Specifically, a cell membrane chip fixing mechanism 1 comprises: a plate-shaped chip 10 having the vertical direction Z as the thickness direction; a contact pin 70 disposed in the downward direction Zb from the chip 10; and a pressing unit 90 for pressing an upper surface 11 of the chip 10. A well 20 for forming a cell membrane 64 is provided in the upper surface 11 of the chip 10. Electrodes 25 disposed on the well 20 are exposed on a lower surface 12 of the chip 10. The contact pin 70 contacts the electrodes 25 from the downward direction Zb. The pressing unit 90 presses an outer peripheral section 13 and a central section 14 on the upper surface 11 of the chip 10.

Description

Cell membrane chip fixing mechanism

[0001] The present disclosure relates to a cell membrane chip fixing mechanism.

[0002] For example, as shown in Patent Document 1, various techniques regarding cell membranes are disclosed. In Patent Document 1, an artificial cell membrane is formed in a well formed on the upper surface of a plate-like chip.

[0003] Japanese Patent Application Laid-Open No. 2007-029911

[0004] The chip is plate-shaped with the vertical direction being the thickness direction. On the upper surface of the chip, wells for forming cell membranes are provided. On the lower surface of the chip, electrodes arranged in the wells are exposed. Below the chip, contact pins are arranged. The contact pins contact the electrodes from below.

[0005] When the contact pins contact the electrodes, the contact pins push the chip upward from below, so the chip floats up.

[0006] Therefore, a method of holding the four corners of the chip is considered to suppress the floating of the chip. However, in this method, the central part of the chip warps upward, resulting in insufficient conduction between the contact pins and the electrodes.

[0007] This tendency becomes more prominent as the number of wells (as well as the corresponding electrodes and contact pins) increases.

[0008] An object of the present disclosure is to ensure sufficient conduction between an electrode and a contact pin in a chip provided with a well for forming a cell membrane.

[0009] The cell membrane chip fixing mechanism according to the present disclosure includes a plate-shaped chip with the vertical direction being the thickness direction, contact pins arranged below the chip, and a pressing unit for pressing the upper surface of the chip. On the upper surface of the chip, wells for forming cell membranes are provided. On the lower surface of the chip, electrodes arranged in the wells are exposed. The contact pins contact the electrodes from below. The pressing unit presses the outer peripheral part and the central part on the upper surface of the chip.

[0010] When the contact pins make contact with the electrodes (exposed on the underside of the tip) from below, the pressing unit presses down on the outer periphery and central part of the upper surface of the tip. This prevents the central part of the tip from warping upward. In tips provided with wells for forming cell membranes, sufficient electrical contact can be achieved between the electrodes and the contact pins.

[0011] In one embodiment, the pressing unit presses down on the entire upper surface of the chip.

[0012] This is advantageous in preventing the center of the tip from curling upwards.

[0013] In one embodiment, a support base is provided to support the chip from below, and the contact pins contact the electrode from below through a hole drilled in the support base.

[0014] The chip can be stably supported by the support base.

[0015] In one embodiment, the device comprises a case having an opening at the top and a lid covering the opening, the case housing the chip and the contact pin, the pressing unit being integrated with the lid, and the pressing unit pressing the upper surface of the chip in conjunction with the lid covering the opening.

[0016] The lid covers the opening of the case, and the pressing unit presses down on the top surface of the chip in conjunction with it, making operation easy.

[0017] In one embodiment, the cell membrane chip fixing mechanism includes a clamp that holds the lid downward.

[0018] The pressing force against the top surface of the tip can be increased with the help of a clamp.

[0019] In one embodiment, the well includes a first well and a second well, the electrode includes a first electrode disposed in the first well and a second electrode disposed in the second well, and the contact pin includes a first contact pin that contacts the first electrode from below and a second contact pin that contacts the second electrode from below.

[0020] Even when there are many wells (and corresponding electrodes and contact pins), sufficient electrical conductivity can be achieved between the electrodes and contact pins.

[0021] According to this disclosure, sufficient electrical conductivity can be achieved between the electrode and the contact pin in a chip provided with wells for forming a cell membrane.

[0022] Figure 1 shows a cross-sectional view of the artificial cell membrane chip fixation mechanism according to the first embodiment. Figure 2 shows a chip for forming an artificial cell membrane according to the first embodiment. Figure 3 shows an artificial cell membrane according to the first embodiment. Figure 4 shows a method for forming an artificial cell membrane according to the first embodiment. Figure 5 shows a cross-sectional view of the artificial cell membrane chip fixation mechanism according to the second embodiment. Figure 6 shows a cross-sectional view of the artificial cell membrane chip fixation mechanism according to the third embodiment.

[0023] Embodiments of the present disclosure will be described in detail below with reference to the drawings. The following description of preferred embodiments is illustrative in nature and is not intended to limit the present disclosure, its applications, or its uses in any way.

[0024] <First Embodiment> (Artificial Cell Membrane Chip Fixation Mechanism) The first embodiment will be described. Figure 1 shows the artificial cell membrane chip fixation mechanism (cell membrane chip fixation mechanism) 1 in a cross-sectional view (along line M in Figure 2). The artificial cell membrane chip fixation mechanism 1 comprises a chip 10, a current measuring device 30, a support base 50, contact pins 70, a substrate 76, a case 80, a lid 86, a pressing unit 90, and a support column 99.

[0025] (Artificial Cell Membrane Chip) Figure 2 shows a chip 10 (artificial cell membrane chip) for forming an artificial cell membrane 64, viewed from above Za. The chip 10 is plate-shaped with the vertical direction Z as the thickness direction and extending horizontally. In this example, the chip 10 is formed in a roughly rectangular shape. The upper surface 11 of the chip 10 includes an outer peripheral portion 13 and a central portion 14. The central portion 14 is located inside the outer peripheral portion 13 in the horizontal direction. The outer peripheral portion 13 is located outside the central portion 14 in the horizontal direction.

[0026] Multiple wells 20 are provided on the upper surface 11 of the chip 10. In this example, 96 wells 20 are arranged on the upper surface 11 of the chip 10, 12 in the front-to-back direction and 8 in the left-to-right direction (some figures are omitted from the illustration). The number of wells 20 in the chip 10 may be 1, 2 to 95, or 97 or more. The wells 20 are there to form an artificial cell membrane 64. The artificial cell membrane 64 is an artificially created cell membrane.

[0027] The well 20 is a recess formed on the upper surface 11 of the chip 10. The well 20 includes a first portion 21 and a second portion 22. The first portion 21 is substantially circular in shape when viewed from above Za. The second portion 22 is substantially circular in shape when viewed from above Za. The first portion 21 and the second portion 22 are connected so as to be adjacent to each other.

[0028] The first part 21 and the second part 22 are separated from each other by a partition wall 23. The partition wall 23 divides (partitions) the first part 21 and the second part 22. The partition wall 23 is provided with a through hole 24 (see Figure 2). The through hole 24 penetrates the partition wall 23. The through hole 24 connects the first part 21 and the second part 22.

[0029] As shown in Figures 1 and 2, electrodes 25 are positioned on the bottom surface of the well 20. The electrodes 25 penetrate the tip 10 in the vertical direction Z. The electrodes 25 are positioned on the bottom surface of the first portion 21 and the bottom surface of the second portion 22, respectively. In other words, there are two electrodes 25 in one well 20. The electrodes 25 are exposed on the bottom surface 12 of the tip 10.

[0030] (Artificial cell membrane) Figure 3 shows the artificial cell membrane 64 as viewed from above (Za). The artificial cell membrane 64 is formed within the well 20. The artificial cell membrane 64 consists of a lipid bilayer. The artificial cell membrane 64 is a replica of a living cell membrane and is artificially formed.

[0031] Lipid solution 61 and buffer solution 62 are injected into the first portion 21 and the second portion 22 of well 20. The lipid solution 61 is, for example, an organic solvent (oil) in which lipid molecules are dispersed. The buffer solution 62 is, for example, water or an aqueous solution. The buffer solution 62 does not mix with the lipid solution 61.

[0032] In the first section 21 and the second section 22, clumps of buffer solution 62 (e.g., water droplets) are formed. In the first section 21 and the second section 22, a lipid monolayer 63, in which lipid molecules are arranged, spontaneously forms around the clumps of buffer solution 62.

[0033] At the through-holes 24 in the partition wall 23 between the first part 21 and the second part 22, lipid monolayers 63 overlap to form a lipid bilayer, creating an artificial cell membrane 64. Of the first part 21 and the second part 22, one mimics the interior of a living cell, and the other mimics the exterior of a living cell.

[0034] (Ion Channels) Ions and proteins 65 are dispersed in the buffer solution 62. The proteins 65 are precursors to ion channels 66. When the proteins 65 attach to the artificial cell membrane 64, ion channels 66 are formed.

[0035] The ion channel 66 is a passage that penetrates the artificial cell membrane 64. The ion channel 66 connects the buffer solution 62 of the first part 21 and the buffer solution 62 of the second part 22. Ions move between the first part 21 and the second part 22 in the ion channel 66. The ion channel 66 is a passage for ions in the artificial cell membrane 64. Examples of ions include potassium ions and sodium ions. Ions have an electric charge.

[0036] The ion channel 66 opens and closes. When the ion channel 66 opens, ions move between the first part 21 and the second part 22. When the ion channel 66 opens, an ion current I flows due to the movement of ions.

[0037] When the ion channel 66 closes, ions do not move between the first part 21 and the second part 22. When the ion channel 66 closes, a small ion current I flows. This is because even when the ion channel 66 is closed, the ion current I does not become completely zero, but flows a small amount through the ion channel 66 by leaking through the artificial cell membrane 64. The ion current I when the ion channel 66 is closed is smaller than the ion current I when the ion channel 66 is open.

[0038] (Current measuring device) The ion current I is measured by the current measuring device 30. The current measuring device 30 comprises a measuring unit 31 and a processing unit 32. The measuring unit 31 is, for example, a known ammeter. The measuring unit 31 is connected in series to both ends (first portion 21 and second portion 22) of the ion channel 66. The measuring unit 31 is connected to the electrodes 25 of the well 20 via contact pins 70, which will be described later. The measuring unit 31 is connected to the electrodes 25 of the first portion 21 and the electrodes 25 of the second portion 22. The measuring unit 31 measures the ion current I flowing through the ion channel 66. The measuring unit 31 also has a function to apply a command voltage to the ion channel 66.

[0039] The processing unit 32 is, for example, a well-known computer. The processing unit 32 includes, for example, a processor mounted on a circuit board, a memory device for storing software to operate the processor, a data storage unit for storing data, and a transmission unit for sending the stored data to an external server.

[0040] The processing unit 32 is connected to the measurement unit 31. The processing unit 32 processes the ion current I measured by the measurement unit 31. For the ion current I measured by the measurement unit 31, the processing unit 32 performs, for example, amplification, noise removal, conversion to digital data, data storage, transmission of data to an external server, etc. The processing unit 32 analyzes, for example, the opening and closing of the ion channel 66. As an example, the processing unit 32 obtains the opening probability of the ion channel 66.

[0041] (Method for forming an artificial cell membrane) FIG. 4 shows a method for forming an artificial cell membrane 64. Note that FIG. 4 is a cross-sectional view taken along the L line in FIG. 2. The method for forming an artificial cell membrane 64 (artificial cell membrane forming method) includes an injection step S12 (first injection step S1 and second injection step S2).

[0042] In the injection step S12, the lipid solution 61 and the buffer solution 62 are injected into the well 20 (first part 21 and second part 22) using the pipette 40. The injection step S12 includes a first injection step S1 and a second injection step S2.

[0043] In the first injection step S1, the lipid solution 61 is injected into the well 20 (first part 21 and second part 22) using the pipette 40. In the second injection step S2, the buffer solution 62 is injected into the well 20 (first part 21 and second part 22) using the pipette 40. Note that the buffer solution 62 does not mix with the lipid solution 61.

[0044] When the injection step S12 (first injection step S1 and second injection step S2) ends, the artificial cell membrane 64 is formed in the well 20. Specifically, the artificial cell membrane 64 is formed as a lipid bilayer in the through-hole 24 of the partition wall 23 between the first part 21 and the second part 22 in the well 20.

[0045] The method for forming the artificial cell membrane 64 may be performed mechanically and automatically by a robot or the like, or may be performed manually by a human.

[0046] (Support base) As shown in FIG. 1, the support base 50 is disposed below the chip 10 in the Zb direction. The support base 50 is plate-shaped with the vertical direction Z as the thickness direction and extending in the horizontal direction. In this example, the support base 50 is substantially quadrangular. The horizontal size of the support base 50 is larger than the horizontal size of the chip 10. The support base 50 supports the chip 10 from below in the Zb direction.

[0047] A hole 51 is formed in the support base 50. The hole 51 penetrates the support base 50 in the vertical direction Z. The number of holes 51 is the same as the number of electrodes 25.

[0048] (Contact pin) As shown in FIG. 1, the contact pin 70 is disposed below the chip 10 in the Zb direction. Specifically, the contact pin 70 is disposed further below the support base 50 (disposed below the chip 10 in the Zb direction). The contact pin 70 extends in the vertical direction Z. The number of contact pins 70 is the same as the number of electrodes 25.

[0049] As described above, electrodes 25 are respectively disposed on the bottom surfaces of the first portion 21 and the second portion 22 in the well 20. The electrodes 25 are exposed on the lower surface 12 of the chip 10.

[0050] The contact pin 70 includes a fixed pin 71 and a movable pin 72. The lower end portion of the fixed pin 71 is connected to a substrate 76 described later. A part of the movable pin 72 is accommodated inside the fixed pin 71. The upper end portion of the movable pin 72 protrudes upward Za from the hole at the upper end portion of the fixed pin 71 or retracts downward Zb into the hole at the upper end portion of the fixed pin 71. The protrusion and retraction of the movable pin 72 with respect to the fixed pin 71 are performed by an actuator (not shown).

[0051] The fixed pin 71 of the contact pin 70 is located below the support base 50 in the Zb direction. The movable pin 72 of the contact pin 70 penetrates the hole 51 of the support base 50 from below in the Zb direction to above in the Za direction.

[0052] The contact pin 70 contacts the electrode 25 from below in the Zb direction. The contact pin 70 contacts the electrode 25 from below in the Zb direction through the hole 51 formed in the support base 50.

[0053] (Substrate) The substrate 76 is, for example, a printed circuit board. The substrate 76 is a plate-like structure with the vertical direction Z as the thickness direction and extending horizontally. In this example, the substrate 76 is approximately rectangular in shape. The substrate 76 is positioned below Zb below the contact pins 70. The upper surface of the substrate 76 is connected to the lower end of the fixing pin 71 of the contact pins 70. The substrate 76, the contact pins 70, and the electrode 25 are electrically connected to each other. The substrate 76 functions as the measurement section 31 of the current measuring device 30. Furthermore, the substrate 76 may also function as the processing section 32 of the current measuring device 30.

[0054] (Case) The case 80 is box-shaped. The case 80 is, for example, roughly rectangular. The case 80 has an opening 81 at the top Za. In this example, the opening 81 is roughly square in shape. The case 80 houses the chip 10 and the contact pins 70. In detail, the case 80 houses the chip 10 (well 20), the support base 50, the contact pins 70, the substrate 76 (current measuring device 30), the pressing unit 90 described later, and the support column 99 described later.

[0055] (Lid) The lid 86 is plate-shaped with the vertical direction Z as the thickness direction and extending horizontally. In this example, the lid 86 is approximately rectangular in shape. The lid 86 covers the opening 81 at the top Za of the case 80. By covering the opening 81 of the case 80, the lid 86 prevents noise from entering the case 80. A handle 87 is provided on the top surface of the lid 86. The handle 87 is grasped by the user.

[0056] (Pressing Unit) The pressing unit 90 has a pressing plate 91. The pressing plate 91 is plate-shaped with its thickness in the vertical direction Z and extending horizontally. In this example, the pressing plate 91 is approximately rectangular in shape. The horizontal size of the pressing plate 91 is larger than the horizontal size of the chip 10. The pressing plate 91 of the pressing unit 90 is located above the chip 10 Za and below the lid 86 Zb within the case 80.

[0057] The lower surface 91a of the pressing plate 91 of the pressing unit 90 faces the upper surface 11 of the chip 10. More specifically, the lower surface 91a of the pressing plate 91 of the pressing unit 90 faces the entire upper surface 11 of the chip 10. That is, the lower surface 91a of the pressing plate 91 of the pressing unit 90 faces the outer periphery 13 and the central portion 14 of the upper surface 11 of the chip 10.

[0058] The pressing unit 90 presses against the upper surface 11 of the chip 10. More specifically, the lower surface 91a of the pressing plate 91 of the pressing unit 90 presses against the entire upper surface 11 of the chip 10 from the upper part Za to the lower part Zb. That is, the lower surface 91a of the pressing plate 91 of the pressing unit 90 presses against the outer periphery 13 and the central part 14 of the upper surface 11 of the chip 10 from the upper part Za to the lower part Zb.

[0059] The pressing plate 91 of the pressing unit 90 may be moved manually in the vertical direction Z, or it may be moved mechanically in the vertical direction Z by an actuator.

[0060] (Support Columns) There are four support columns 99. The support columns 99 extend in the vertical direction Z. The support columns 99 pass through the four corners of the pressing plate 91 of the pressing unit 90, the four corners of the support base 50, and the four corners of the base plate 76. The support base 50 and the base plate 76 are fixed to the support columns 99. The pressing plate 91 of the pressing unit 90 moves in the vertical direction Z along the support columns 99.

[0061] (Wells, electrodes, contact pins) The wells 20 include a first well 20A, a second well 20B, a third well, and so on. The electrodes 25 include a first electrode 25A located in the first well 20A, a second electrode 25B located in the second well 20B, a third electrode located in the third well, and so on. The contact pins 70 include a first contact pin 70A that contacts the first electrode 25A from below Zb, a second contact pin 70B that contacts the second electrode 25B from below Zb, a third contact pin that contacts the third electrode from below Zb, and so on.

[0062] (Effects) When the contact pin 70 contacts the electrode 25 (exposed on the lower surface 12 of the tip 10) from below Zb, the pressing unit 90 presses the outer peripheral portion 13 and the central portion 14 on the upper surface 11 of the tip 10. This prevents the central portion 14 of the tip 10 from bending upward Za. In a tip 10 provided with a well 20 for forming an artificial cell membrane 64, sufficient electrical contact can be achieved between the electrode 25 and the contact pin 70.

[0063] The pressing unit 90 presses down on the entire upper surface 11 of the tip 10. This is advantageous in suppressing the central part 14 of the tip 10 from curving upward Za.

[0064] The chip 10 can be stably supported by the support base 50.

[0065] Even when there are many wells 20 (and corresponding electrodes 25 and contact pins 70), sufficient electrical conductivity can be achieved between the electrodes 25 and the contact pins 70.

[0066] <Second Embodiment> A second embodiment will now be described. In the following description, components similar to those in the above embodiment will be denoted by the same reference numerals, and detailed descriptions will be omitted. Figure 5 shows a cross-sectional view of the artificial cell membrane chip fixation mechanism 1.

[0067] The case 80 has an opening 81 at its upper side Za. The case 80 houses the chip 10 and the contact pins 70. The lid 86 covers the opening 81 at the upper side Za of the case 80.

[0068] The pressing unit 90 includes a pressing plate 91 and a plurality of connecting members 92. The pressing plate 91 is plate-shaped with its thickness in the vertical direction Z and extending horizontally. The connecting members 92 extend in the vertical direction Z. The connecting members 92 connect the upper surface of the pressing plate 91 to the lower surface of the lid 86. The plurality of connecting members 92 are arranged side by side with spacing between them in the horizontal direction.

[0069] The pressing unit 90 is integrated with the lid 86. When the lid 86 moves downward Zb, the pressing unit 90 also moves downward Zb in conjunction with it. The lower surface 91a of the pressing plate 91 of the pressing unit 90 presses against the upper surface 11 of the chip 10 in conjunction with the lid 86 covering the opening 81 of the case 80.

[0070] The artificial cell membrane chip fixing mechanism 1 includes a clamp 96. In this example, the clamp 96 is a toggle clamp 96a. The clamp 96 is positioned around the opening 81 in the upper wall of the case 80. The clamp 96 (toggle clamp 96a) presses the lid 86 downward Zb.

[0071] The other configurations are the same as in the first embodiment.

[0072] The operation is simple because the pressing unit 90 presses down on the upper surface 11 of the chip 10 in conjunction with the lid 86 covering the opening 81 of the case 80.

[0073] The pressing force applied to the upper surface 11 of the tip 10 can be increased with the help of the clamp 96 (toggle clamp 96a).

[0074] <Third Embodiment> A third embodiment will now be described. In the following description, components similar to those in the above embodiments will be denoted by the same reference numerals, and detailed descriptions will be omitted. Figure 6 shows a cross-sectional view of the artificial cell membrane chip fixation mechanism 1.

[0075] The clamp 96 is a handle clamp 96b. The clamp 96 (handle clamp 96b) presses the lid 86 downward Zb.

[0076] The other configurations are the same as in the second embodiment.

[0077] The operation is simple because the pressing unit 90 presses down on the upper surface 11 of the chip 10 in conjunction with the lid 86 covering the opening 81 of the case 80.

[0078] The pressing force applied to the upper surface 11 of the tip 10 can be increased with the help of the clamp 96 (handle clamp 96b).

[0079] <Other Embodiments> Although this disclosure has been described above with reference to preferred embodiments, this description is not limiting, and of course, various modifications, substitutions, or combinations are possible.

[0080] The pressing unit 90 may not press the entire upper surface 11 of the tip 10, but rather partially press the outer peripheral portion 13 and the central portion 14 of the upper surface 11 of the tip 10.

[0081] A cell membrane (biological cell membrane) 64 made of living tissue may be formed in well 20. In this case, it becomes a biological cell membrane chip fixation mechanism 1.

[0082] The well 20 may have any shape. The first portion 21, the second portion 22, the partition wall 23, and the through hole 24 are not required in the well 20.

[0083] The chip 10 is not limited to a roughly rectangular shape. For example, the chip 10 may have a polygonal shape other than a roughly rectangular shape, or a circular shape, etc.

[0084] This disclosure is extremely useful and has high potential for industrial application because it can be applied to cell membrane chip immobilization mechanisms.

[0085] Z Upward direction Za Upward Zb Downward I Ion current S12 Injection process S1 First injection process S2 Second injection process 1 Artificial cell membrane chip fixation mechanism (cell membrane chip fixation mechanism) 10 Chip 11 Top surface 12 Bottom surface 13 Outer periphery 14 Central part 20 Well 20A First well 20B Second well 21 First part 22 Second part 23 Separation wall 24 Through hole 25 Electrode 25A First electrode 25B Second electrode 30 Current measuring device 31 Measuring unit 32 Processing unit 40 Pipette 50 Support stand 51 Hole 61 Lipid solution 62 Buffer solution 63 Lipid monolayer 64 Artificial cell membrane (cell membrane) 65 Protein 66 Ion channel 70 Contact pin 70A First contact pin 70B Second contact pin 71 Fixing pin 72 Movable pin 76 Circuit board 80 Case 81 Opening 86 Lid 87 Handle 90 Pressing unit 91 Pressing plate 91a Bottom surface 92 Connecting material 96 Clamp 96a Toggle clamp 96b Handle clamp 99 Support column

Claims

1. A cell membrane chip fixing mechanism comprising: a plate-shaped chip with its thickness in the vertical direction; contact pins positioned below the chip; and a pressing unit that presses against the upper surface of the chip, wherein the upper surface of the chip is provided with a well for forming a cell membrane, electrodes positioned in the wells are exposed on the lower surface of the chip, the contact pins contact the electrodes from below, and the pressing unit presses against the outer periphery and central part of the upper surface of the chip.

2. The cell membrane chip fixing mechanism according to claim 1, wherein the pressing unit presses down on the entire upper surface of the chip.

3. The cell membrane chip fixation mechanism according to claim 1 or 2, comprising a support base that supports the chip from below, wherein the contact pins contact the electrode from below through a hole made in the support base.

4. A cell membrane chip fixing mechanism according to claim 1 or 2, comprising a case having an opening at the top, and a lid covering the opening, wherein the case houses the chip and the contact pin, the pressing unit is integrated with the lid, and the pressing unit presses the upper surface of the chip in conjunction with the lid covering the opening.

5. The cell membrane chip fixing mechanism according to claim 4, further comprising a clamp for pressing the lid downward.

6. The cell membrane chip fixation mechanism according to claim 1 or 2, wherein the well comprises a first well and a second well, the electrode comprises a first electrode disposed in the first well and a second electrode disposed in the second well, and the contact pin comprises a first contact pin that contacts the first electrode from below and a second contact pin that contacts the second electrode from below.

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